Search tips
Search criteria 


Immunol Rev. 2013 July; 254(1): 305–325.
Published online 2013 June 16. doi:  10.1111/imr.12080
PMCID: PMC3707302

The immunological synapse: the gateway to the HIV reservoir


A major challenge in the development of a cure for human immunodeficiency virus (HIV) has been the incomplete understanding of the basic mechanisms underlying HIV persistence during antiretroviral therapy. It is now realized that the establishment of a latently infected reservoir refractory to immune system recognition has thus far hindered eradication efforts. Recent investigation into the innate immune response has shed light on signaling pathways downstream of the immunological synapse critical for T-cell activation and establishment of T-cell memory. This has led to the understanding that the cell-to-cell contacts observed in an immunological synapse that involve the CD4+ T cell and antigen-presenting cell or T-cell–T-cell interactions enhance efficient viral spread and facilitate the induction and maintenance of latency in HIV-infected memory T cells. This review focuses on recent work characterizing the immunological synapse and the signaling pathways involved in T-cell activation and gene regulation in the context of HIV persistence.

Keywords: immunological synapse, virological synapse, HIV latency, HIV reservoir, asymmetric cell division, Wnt/Notch

This article is part of a series of reviews covering HIV Immunology appearing in Volume 254 of Immunological Reviews.


Lymph nodes (LNs) were identified as major sites of viral replication in HIV-infected subjects 1, 2. Both solid and diffused secondary lymphoid organs (SLOs), such as the gut-associated lymphoid tissues (GALT), were also shown to be the primary sites of viral replication in human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infections 3, 4. A large number of studies in humans and non-human primates have reported higher frequencies of HIV/SIV-infected cells as well as higher copy numbers of viral transcripts in CD4+ T cells isolated from lymphoid tissues (GALT, LN) when compared with the peripheral blood 1, 58. This enrichment in HIV-infected cells can be attributed to several important characteristics of lymphoid tissues. These include (i) a privileged tissue architecture that favors close cellular contact between immune cells, thereby promoting cell-to-cell transmission of HIV and ensuring viral dissemination; (ii) a significant enrichment in the frequency of cells that are highly permissive to HIV infection, such as activated CD4+ T cells that can produce large numbers of viral particles; and (iii) a proinflammatory environment that enhances viral production from infected cells and promotes new infections. These factors contribute to the high levels of HIV replication observed in lymphoid organs from HIV- infected subjects and provide an explanation of the major role played by these compartments in the pathophysiology of HIV infection.

High frequencies of HIV-infected cells have also been reported in lymphoid tissues from subjects who have received suppressive antiretroviral therapy (ART) for prolonged periods of time 912, indicating that lymphoid organs are not only important sites of HIV production in untreated disease, but also play a major role in HIV persistence during therapy. The three aforementioned characteristics of lymphoid organs that contribute to HIV replication in untreated disease are likely to play a similar role during ART by promoting residual levels of viral replication 13. In addition to the close T-cell–T-cell and dendritic cell (DC)–T-cell contacts that favor viral transmission (the ‘virological synapses’), recent observations suggest that contact and crosstalk between immune cells (the ‘immunological synapse’) may play a critical role in HIV persistence by promoting the establishment and maintenance of viral latency 14. Therefore, although the immunological synapse and virological synapse share several common features, they may result in very distinct outcomes when their impact on viral persistence is examined (Fig. 1).1). Whereas the virological synapse functions to promote viral dissemination, the immunological synapse may result in the inhibition of viral production and in the establishment of HIV latency, thereby generating and maintaining a long-lived cellular reservoir for the virus. Details of the mechanisms by which the immunological synapse and virological synapse contribute to HIV persistence are still largely unknown. Our current understanding of these mechanisms and their downstream signaling pathways are detailed in this review. Identifying the cell types and receptors at play in these interactions will pave the way for the rational design of novel therapeutic approaches aimed at abrogating HIV persistence during ART.

Fig. 1
Model of virological and immunological synapse formation in the contribution to HIV persistence

The HIV reservoir

Current antiretroviral regimens dramatically suppress HIV replication resulting in a major reduction in HIV-related mortality and morbidity; however, these treatments do not eradicate HIV. Interruption of ART almost invariably leads to the reemergence of detectable viral replication even after years of continuous optimal suppressive therapy, thereby demonstrating the presence of a long-lived viral reservoir constituted of a pool of cells capable of producing replication-competent HIV 15. Two non-mutually exclusive mechanisms underlie HIV persistence during ART: (i) incomplete suppression of viral replication could allow the continuous replenishment of a small pool of infected cells, particularly in sites with suboptimal drug penetration or sites in which control by the host immune system is inefficient and (ii) persistence of a small pool of resting memory CD4+ T cells in which HIV is maintained as a transcriptionally silent provirus through epigenetic mechanisms.

HIV persists in a latent form through immunologically driven mechanisms

Fifteen years ago, several groups described a subset of resting memory CD4+ T cells with integrated HIV genomes that released infectious particles only upon cellular activation 7, 16. This latently infected reservoir was later shown to include mostly CD4+ T cells of central and transitional memory phenotype 17 and persisted in patients on ART who have no clinically detectable viremia 7, 18, 19. The kinetics of decay of the latently infected CD4+ T-cell subset has been shown to be approximately 60 years, as the mean half-life of CD4+ T cells was suggested to be 43.9 months 2022. The establishment of a latent reservoir in these subsets is a rare event that occurs during acute infection and is not fully prevented by the introduction of ART 23, 24. Two major forms of viral latency coexist in vivo 25. First, preintegration latency refers to unintegrated HIV DNA that is unstable and will either degrade or integrate into the host cell genome, usually upon cellular activation 2628. This form of latency is established after partial or complete block of the viral life cycle at steps prior to the integration of viral DNA. Second, postintegration latency refers to the presence of integrated HIV DNA in cells that are not actively producing viral particles. This latent state is extremely stable and is limited only by the lifespan of the infected cell and its progeny. Because the provirus is integrated in the host genome, its replication activity is significantly affected by the activation state of the host cell. Thus, CD4+ T cells may induce a latent state of the viral genome when transitioning from an activated effector phenotype to a quiescent memory cell.

Most mechanisms that lead to induction and maintenance of HIV latency operate at the transcriptional and epigenetic level. The first key factor is the site of provirus integration. Integrated HIV proviruses are typically found within actively transcribed genes in resting CD4+ T cells 29, 30. However, modifications such as histone acetylation, methylation, and adenosine triphosphate–dependent remodeling interfere with the transcription of viral genes by rendering the HIV LTR promoter region inaccessible to transcription factors 3134. In addition, the quiescent state of latently infected CD4+ T cells favors nuclear exclusion of the transcription factors nuclear-factor κB (NFκB) and nuclear factor of activated T cells (NFAT) that promote HIV expression 35, 36. In quiescent CD4+ T cells, elongation factors such as pTEFB can also be sequestered and thus negatively impact the generation of viral transcripts 37, 38. Many of these mechanisms have been elucidated using cell lines and the relative importance of each mechanism in vivo remains unclear. However, the block in HIV production in quiescent memory CD4+ T cells extends beyond transcription, as low levels of cell-associated viral RNA have been found in resting CD4+ T cells from virally suppressed subjects 39. A defect in nuclear export of RNA transcripts has been suggested to block HIV production in latently infected cells 40.

A critical unanswered question pertains to the nature of signals an HIV-infected cell receives to establish and ultimately maintain a latently infected reservoir. The immunological mechanisms involved in the generation and maintenance of memory CD4+ T cells have been suggested to regulate the induction of latency and the persistence of the HIV reservoir 41. Several lines of evidence suggest that the generation of memory T cells from effector T cells during HIV infection contributes to the establishment of a reservoir of long-lived latently infected cells. Latently infected memory T cells harboring replication-competent HIV can be isolated from viremic donors 16, indicating that the latent HIV reservoir is generated and maintained during the viremic phase of the disease. Negative signals, notably mediated by negative regulators of T-cell receptor (TCR) signaling 42, may initiate the transition from activated to quiescent phenotype by reducing the availability of cellular transcription factors essential for active viral gene expression, thereby establishing viral latency in long-lived memory CD4+ T cells harboring HIV-integrated DNA. Memory CD4+ T cells persist in response to prosurvival signals downstream of common γ chain (γc) cytokines [such as interleukin-7 (IL-7) and IL-15] and TCR stimulation 4345. We have demonstrated that these cytokines contribute to the persistence of HIV in this long-lived cellular compartment 17 by controlling homeostatic proliferation during ART 46, 47. Sequencing of HIV genomes in latently infected cells has revealed significant sequence homogeneity, which would support a model of homeostatic proliferation of a small number of latently infected cells 17. In contrast, a reservoir generated by ongoing viral replication and infection of new cells would be evidenced by an accumulation of mutations in the integrated HIV genomes 46, 47. Several immunological mechanisms could be responsible for proliferation-induced HIV persistence: (i) homeostatic proliferation driven by IL-7 and IL-15 48; (ii) inflammation-induced proliferation driven by proinflammatory cytokines such as IL-1, IL-6, and interferon-γ (IFN-γ) (49, discussed in this issue); (iii) antigen-induced proliferation; and (iv) self-renewal of stem cell memory T cells by Wnt/Notch signaling 50, 51. IL-7 or proinflammatory cytokines 5254 as well as TCR engagement 55 have been shown to induce HIV production in primary CD4+ T cells in vitro. These results suggest that memory CD4+ T cells harboring replication-competent HIV may be continuously exposed to reactivation signals. The maintenance of viral latency may be an active process whereby the positive signals conferred by TCR stimulation and/or cytokines could be counterbalanced by negative signals that would impede viral reactivation and subsequent elimination of these cells through cytopathic effect or cytotoxic killing. Negative signals could also lead to asymmetric division and the establishment of long-lived stem cell memory T cells.

Secondary lymphoid organs in HIV infection: exploring the battlefield

In addition to the persistence of HIV in a latent state within a small pool of long-lived CD4+ T cells, the incomplete suppression of viral replication by ART could allow the continuous replenishment of a small pool of infected cells 56, particularly in anatomical compartments in which drug penetration may not be optimal 10. Moreover, inflammatory cytokines such as IL-2, TNF (tumor necrosis factor), IL-6, and IL-18, and chemokines CC-chemokine ligand 19 (CCL19) and CCL21 are elevated during treated HIV disease; this provides the inflammatory environment that render CD4+ T cells more susceptible to infection in tissues of infected subjects 54, 5759 (Fig. 1).1). Viral replication during ART is well documented by studies showing residual viremia in the majority of ART-treated subjects 60, 61, or the presence of cell-associated viral RNA in tissues such as the GALT 12. However, as discussed above the absence of viral evolution 62, 63 and the lack of impact of treatment intensification on residual viremia 6466 argue against the role of viral production in HIV persistence. However, it cannot be excluded that specific microenvironments such as the immunological synapse could favor the continuous replenishment of a pool of HIV-infected cells. Indeed, evidence of cell-to-cell transmission of HIV in the presence of antiretroviral drugs has been recently described and indicates an important mechanism of HIV persistence 13 (Fig. 11).

SLOs provide an environment that enables lymphocytes to interact with antigen-presenting cells (APCs), resulting in the initiation of antigen-specific immune responses. SLOs are usually considered as sites of production of antigen-specific effector cells that have the ability to migrate to infected tissues and anatomical compartments in which infection by a pathogen takes place. This classical definition has to be revisited in the context of HIV infection, as the virus preferentially replicates in CD4+ T cells localized in SLOs. As a consequence, an effector response actively prevents viral dissemination in these SLOs when HIV-specific CTLs kill infected cells present locally 67. In the context of suboptimal adaptive immune responses characteristic of HIV infection, this duality is clearly beneficial to the virus by the continuous generation of new target cells through the interactions of APCs and CD4+ T cells via the immunological synapse. Of note, the innate immune response that includes natural killer (NK) cells, γδ T cells, NK T cells, and innate-like CD8+ T cells may also play an important role in limiting systemic pathogen spread in LNs 68, but whether these mechanisms may specifically impact HIV replication remains largely unknown.

Immunological synapse formation stimulates the differentiation of naive CD4+ T cells into effector helper cells through the recognition by the TCR of its cognate peptide–major histocompatibility complex class II (MHCII) complex on the surface of APCs 69. Two-photon imaging has revealed that naive T cells are in constant motion, scanning the lymph node in search of antigen by contacting 5000 DCs per hour 70, 71. The priming of naive T cells by a DC occurs in three distinct phases 72. During the first 8 h after entering the LN, T cells undergo multiple short encounters with DCs, decrease their motility, and upregulate activation markers such as the extended form of leukocyte function-associated antigen-1 (LFA-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) (stop signal). To establish an immunological synapse, a stop signal results in the arrest of CD4+ T-cell migration to a particular DC 73, 74. During the subsequent 12 h, T cells form long-lasting conjugates with DCs and begin to secrete IL-2 and IFN-γ. On the second day, coinciding with the onset of proliferation, T cells resume their rapid migration and short DC contacts. The priming of antigen-specific T cells is highly sensitive, as the formation of an immunological synapse requires only about 10 agonist peptides presented at the surface of the APC 75.

Effector CD4+ T cells secrete cytokines that modulate adaptive immune responses, with IFN-γ and IL-2 promoting Th1 responses, whereas secretion of IL-4 and IL-5 promotes Th2 responses. Some of these cytokines are endowed with antiviral activity such as IFN-γ. APCs can also induce the generation of regulatory T cells (Treg), which can downmodulate antigen-specific immune responses 76. Interestingly, Treg cells can exhibit their regulatory function at the immunological synapse by preventing the recruitment of signaling molecules on naive T cells such as PKCθ when both T cells have identical antigen specificity and are in contact with the same APC 77. It has become more and more evident that the interactions occurring at the T-cell–APC interface determine the nature of the T-cell response against a particular antigen. We now examine our current understanding of the structure and signaling pathways of the immunological synapse.

Spatial organization of the immunological synapse

The immunological synapse was originally characterized at the surface of T cells as concentric rings of membrane receptors 78. These three-dimensional contact domains, which are visible by confocal microscopy, are named supramolecular activation clusters (SMACs) and include the central SMAC (cSMAC), the peripheral SMAC (pSMAC), and the distal SMAC (dSMAC). During T-cell activation, TCRs accumulate into the cSMAC, surrounded in a bulls-eye manner by the pSMAC, which consists of a ring enriched in the adhesion molecule LFA-1 and its ligands, which are members of the intercellular adhesion molecule (ICAM) family and talin. Talin has been demonstrated to link the adhesion rings to the actin cytoskeleton 79. LFA-1 and ICAM-1 interactions act as a tether between the two cells, facilitating contact between the TCR and MHC and impeding T-cell mobility. Importantly, the stability provided by the pSMAC accounts for a 100-fold increase in the T-cell sensitivity to antigen 80. This organization (pSMAC and cSMAC) constitutes the mature immunological synapse. Finally, the most external ring or dSMAC is where proteins with large ectodomains such as CD43 and CD45 are located, far from the cSMAC 81. Importantly, the immunological synapse is dynamic: TCR signaling is sustained by TCR microclusters, made up of the TCR and peptide MHC complex that are continually forming in the dSMAC and moving into the cSMAC 82, 83. These newly formed microclusters are associated with signaling molecules such as ZAP-70 (ζ chain-associated protein kinase of 70 kDa), Lck (lymphocyte-specific protein tyrosine kinase), LAT (linker of activated T cells), and SLP-76 (SH2 domain–containing leukocyte protein of 76 kDa), but these associations are lost as the microclusters migrate toward the cSMAC. Therefore, TCR signaling is initiated and sustained in peripheral microclusters, and despite the large numbers of TCRs present in the cSMAC, signaling does not occur in the central part of the immunological synapse. Although the cSMAC may play several roles during the formation and termination of the immunological synapse, it may primarily serve to downregulate the TCR by endocytosis 84. The dSMAC also has CD45 and dynamic filamentous actin 8587 and has been implicated in T-cell sensitivity to antigen recognition 73, 8890. Significant for HIV infection, CD4 is initially recruited to the TCR–peptide–MHC microcluster in the cSMAC as well as the coreceptors CXCR4 and CCR5 91, 92.

The immunological synapse: fine tuning of the immune response

The highly stable and long-lived immunological synapse has been demonstrated to be required to completely activate T cells following TCR engagement and its triggering of a signaling cascade 93, 94. In addition to the TCR, a large number of proteins play essential roles in the formation of immunological synapse. They include coreceptors, adhesion molecules, and costimulatory and negative regulatory molecules. CD4, through its extracellular domain, acts as a coreceptor to the TCR and interacts directly with MHCII molecules on the surface of APCs. Although CD4+ T cells can respond with transient calcium signaling to a single agonist peptide–MHC ligand, a complete immunological synapse and productive calcium response require about 10 peptide–MHC complexes 75. This sensitivity is highly dependent on CD4, as anti-CD4 antibodies render T cells unable to detect less than about 30 ligands. Using its intracellular domain, CD4 amplifies the signal generated by the TCR by recruiting Lck 95. Lck phosphorylates the intracellular chains of the CD3 and ζ-chains of the TCR complex, allowing the recruitment of ZAP-70. Lck also phosphorylates and activates ZAP-70, which in turn phosphorylates LAT, a transmembrane protein that recruits a number of proteins essential for proper T-cell activation, including GRB2 (growth factor receptor-bound protein 2) and PLC-γ1 (phospholipase C γ1).

Adhesion receptors can be defined as receptors that promote adhesion beyond antigen recognition 96. The arrest of rolling leukocytes on the endothelium is nearly exclusively mediated by members of the integrin superfamily, such as the myeloid-specific integrin LFA-1, Mac-1, as well as the two α4 integrins, α4β1 (VLA-4) and α4β7 97. Soluble factors such as chemokines also have been implicated in T-cell arrest. For example, CCL19 and CCL21 are expressed by the vasculature during inflammation and signal through CCR7 on leukocytes, which results in the unfolding of LFA-1 into an extended conformation, enabling it to bind to ICAM-1 with intermediate affinity 98, 99. In addition, T cells polarize in response to CCL19 and become highly motile 100. As a result, the efficiency of naive T cells in scanning the surface of APCs is improved, increasing the probability of encountering a cognate peptide–MHC complex 69. Chemokines such as CCL19 and CCL21 were recently shown ex vivo to increase susceptibility of resting memory T cells to infection and establishment of latency 58, 101.

Regulatory molecules of the immunological synapse

Costimulatory and negative regulatory molecules can be defined as having a positive or a negative role in the regulation of TCR-mediated signals. Although some of these molecules may also have limited function outside the context of antigen recognition, costimulatory molecules play a critical role in the initiation of T-cell activation following the formation of the immunological synapse. For example, association of the TCR of a naive T cell with a peptide–MHC complex without interaction of the costimulatory receptor CD28 with its primary ligand CD80 (B7.1) results in an anergic T cell that produces very low amounts of IL-2 102. CD28 is highly enriched in TCR microclusters when engaged by CD80, and these CD28–CD80 complexes are transported to the center of the immunological synapse where they form a stable ring around the cSMAC 103. CD28 has a highly conserved short cytoplasmic tail that has no intrinsic enzymatic activity. However, phosphorylation of the tyrosine residues provides docking sites for SH2 domain–containing proteins, whereas the proline-rich motifs can bind SH3 domain–containing proteins. The role of CD28 costimulation on IL-2 production appears to have two stages: an initial phosphoinositol 3-kinase (PI3K)-dependent initial phase that acts on IL-2 transcription and a second phase which results in enhancement of IL-2 mRNA stability 104.

CD2 is also required for T-cell activation and cooperates with CD28 upon ligation to LFA-3/CD58 to induce the immunological synapse formation 105. CD2 binding with CD58 expressed on the surface of APCs augments and sustains antigen-induced Ca2+ increase in T cells 106. CD2 contributes to the generation of TCR triggered microdomains in the membrane that recruit signaling molecules like Lck and LAT 107 and play a direct role in T-cell signaling via multiple polyproline motifs that activate kinases such as Fyn. Like CD2, CD45 has been shown to be crucial for supporting signal transduction from the TCR. Through its intracellular region, CD45 associates with several intracellular protein tyrosine kinases essential for T-cell activation, including Lck, Fyn, and ZAP-70 108, 109. CD45 dephosphorylates Y505 on p56lck activating Lck kinase, followed by p56lck phosphorylation of TCR-ζ, and consequently recruitment of ZAP-70 108, 110. Interestingly, CD45 has been shown to negatively regulate the transcription factor NFAT, thereby reducing HIV LTR activation 111. Other costimulatory receptor–ligand pairs, including inducible costimulator (ICOS)–ICOS ligand (ICOS-L) 112, CD40–CD40L 113, CD6–CD166 114, and CD26–adenosine deaminase–adenosine receptor 115, were also reported to accumulate and function at the immunological synapse.

The formation of the immunological synapse and the subsequent T-cell activation can be inhibited by engagement of inhibitory receptors. The classic example is given by CTLA-4 (CD152), a negative regulator of T-cell activation that potently inhibits signaling through the TCR and reduces T-cell activation. HIV-specific CD4+ T cells upregulate CTLA-4 expression upon recognition of cognate peptide–MHC ligands leading to inhibition of effector function; blockade of this interaction augments HIV-specific CD4+ T-cell functions 116. Interestingly, CTLA-4 blockade augments viral replication in SIV-infected macaques 117, suggesting that this negative regulator may inhibit viral production in vivo, particularly in mucosal tissues where it is expressed at high levels (Fig. 1).1). This is consistent with data from our group that HIV Nef downregulates expression of CTLA-4, thereby maintaining CD4+ T-cell activation and viral replication 118. While T cells constitutively express CD28, CTLA-4 expression is induced by TCR stimulation 119. CTLA-4 has a much higher affinity for CD80 and CD86 than CD28. As a consequence, even low levels of CTLA-4 on the cell surface can compete for ligand binding with CD28. CTLA-4 forms microclusters, which directly accumulate in the cSMAC, exactly in the same region as CD28 120. Thus, CTLA-4 pushes CD28 away from the cSMAC, which results in the blockade of CD28-mediated costimulation 121. This is thought to be the main mechanism of CTLA-4–mediated inhibition of T-cell activation. CTLA-4 reverses the TCR-mediated stop signal needed for T-cell/APC interactions, thereby reducing the contact time between T cell and APC. This greatly reduces the contact area at the immunological synapse that leads to a major reduction in Ca2+ mobilization and IL-2 production 122, 123 and could also lead to significant inhibition of HIV replication. This signaling cascade results in cytoskeletal changes and microtubule-organizing center polarization toward the APC at the immunological synapse 124126, resulting in the redistribution of the secretory and recycling machineries of the target T cell and a polarized transport of cytokines and signaling molecules toward the immunological synapse in the absence of cell fusion 80, 127.

PD-1 (programmed death-1) is a relatively new member of the extended B7-CD28 family of T-cell regulatory molecules. PD-1 is not detectable on naive T cells, but its expression is induced in T cells, B cells, and myeloid cells after activation 128. PD-1 expression is also upregulated in T cells upon exposure to γc cytokines IL-2, IL-7, IL-15, and IL-21 129. The expression of PD-1 is particularly high on the surface of functionally exhausted T cells in multiple persistent viral infections in humans 130, 131, particularly during chronic HIV infection 132134, and is associated with various measures of viral persistence (ultrasensitive plasma HIV RNA levels, cell-associated HIV RNA levels, and proviral HIV DNA levels) 135. In addition, PD-1+ CD4+ T cells constitute a preferential reservoir for HIV 17. The interaction between PD-1 and its ligand PDL-1 has been shown to suppress HIV production in primary CD4+ T cells from viremic and virally suppressed subjects 136.

Signaling pathways initiated upon the interaction of PD-1 with its ligands (PDL-1/PDL-2) negatively regulate signals downstream of the TCR 137 and dampen cytokine production and proliferation 138. The level of PD-1 expression positively correlates with the degree of functional exhaustion, but this phenotype is actively maintained by signaling. PD-1 inhibits T-cell activation by pathways distinct from CTLA-4 139. PD-1 engagement leads to the inhibition of Akt phosphorylation by preventing CD28-mediated activation of PI3K. Using single-cell imaging, a recent study elucidated a molecular mechanism of PD-1–mediated suppression 140. Upon binding to PDL-1, PD-1 becomes clustered with TCRs and is transiently associated with the phosphatase SHP2 (Src homology 2 domain–containing tyrosine phosphatase 2). These negative costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling molecules, resulting in the suppression of T-cell activation. PD-1 ligation is more effective than CTLA-4 in suppressing CD3/CD28-induced changes in the T-cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct downstream cellular outcomes 139. Other negative regulators such as LAG-3 (lymphocyte-activation gene 3) 141 and BTLA (B- and T-lymphocyte attenuator) 142, which have been shown to localize at the immunological synapse, may exert a similar role. The engagement of negative regulators of T-cell activation such as PD-1, CTLA-4, and others induces epigenetic changes 143 and may regulate histone deacetylase (HDAC) activity induced by CD3/CD28 T-cell activation. These observations suggest a key role for multiple negative regulator molecules in the establishment and maintenance of the latent HIV reservoir (Fig. 11).

Molecular interactions that govern virological synapse formation

Along with co-opting the immunological synapse, another mechanism HIV employs to enhance viral spread is the direct transfer of virus between infected and uninfected CD4+ T cells (Fig. 1).1). Virological synapse formation is initiated via the interaction between surface HIV gp120 on the infected donor T cell and CD4 molecules on the surface of the uninfected target cell, in the absence of TCR–peptide–MHC interaction. Compared with the immunological synapse, the virological synapse is short lived, with a mean duration of 60 min 144 and completion of virus transfer within 3 h 145, 146. Gp120 and CD4 molecules are at the center of the microcluster and have been proposed to form a cSMAC-like structure similar to that observed for the TCR:MHC microclusters in immunological synapse formation 147149. Like the immunological synapse, LFA-1 and ICAM-1 have been shown to assemble into an adhesive ring in a pSMAC-like structure in the virological synapse 83, 150. Unlike the immunological synapse, however, other molecules are required for virological synapse formation; their localization and their role in HIV cell-to-cell transmission are not as clearly defined and are still the subject of investigation. The first descriptions of the virological synapse by the Sattentau group 146 indicated the involvement of HIV Env and Gag from the HIV-infected donor cell, and CD4, LFA-1, and CXCR4 enrichment in the target cell at the point of contact. Studies using inhibitors of CD4 and Env have confirmed the requirement for these molecules in cell-to-cell HIV transmission, but HIV coreceptor antagonists have failed to inhibit the process 145, 151. Additional data using antibodies which block gp120–CD4 binding suggest the complex between gp120 and CD4 initiates virological synapse formation without requiring either CXCR4 or CCR5 coreceptor molecules 145, 149; however, HIV coreceptors may be required for later steps, after synapse formation, in the target cell 152. Recent studies as to the role of Gag in virological synapse formation have determined that the matrix (MA) domain is required for cell-to-cell HIV transmission and that Gag is specifically and directionally recruited into a disk-shaped structure called a synaptic button 145, 153, 154. Accumulation of Gag in button structures is indicative of new particle assembly. However, Gag has also been reported to aggregate into ring-like structures 155. The significance of the different structures Gag can form during virological synapse formation is not yet completely understood.

Other cellular factors implicated in HIV virological synapse include the LFA-1 ligands ICAM-2 and ICAM-3, tetraspanins, lipid raft marker GM-1, and integrin α4β7 156159. For cell-free routes of HIV infection, LFA-1 binding to ICAM-1 has been demonstrated to enhance viral particle infectivity and decrease the effects of neutralizing antibodies by involvement in virus fusion 160. The role of the interaction of LFA-1 and its ICAM ligands during virological synapse formation remains unclear. It has been hypothesized that this complex may play a role in virological synapse formation similar to that observed in the immunological synapse that of pausing CD4+ T-cell migration to allow for the interaction of the target and donor cells 152. A study by Vasiliver-Shamis et al. 148 demonstrated that CD4+ T-cell interaction with gp120 and ICAM-1 in a virological synapse results in a TCR signaling cascade similar to that observed in an immunological synapse, such as the phosphorylation of Lck, CD3ζ, ZAP70, LAT, SLP-76 Itk, and PLCγ. In contrast, they did not observe recruitment of PKCθ or intracellular calcium mobilization, which may indicate that activation of the target CD4+ T cell through the virological synapse is incomplete 148. This is significant for HIV infection, as CD4+ T cells that are quiescent (G0) are highly resistant to infection; however, incomplete activation that results in progression to the G1b phase results in increased susceptibility to HIV infection 28, 161. Consequently, suboptimal stimulation of the target CD4+ T cell through the virological synapse may be all that is required for productive infection of an otherwise resistant T cell. This would be conducive to HIV infection of long-lived memory CD4+ T cells and may be one mechanism leading to the establishment of the HIV reservoir in HIV-infected subjects.

Another requirement for HIV cell-to-cell transmission is the presence of lipid raft domains and cholesterol in the virological synapse found by Jolly and Sattentau 157. During viral assembly in infected cells, HIV is focused in GM-1-rich lipid rafts at the plasma membrane 162169. In the virological synapse, lipid rafts in the infected donor cell are polarized to the site of the cell–cell contact 157. The role of lipid rafts in virological synapse formation in the recruitment of downstream signaling molecules or the stabilization of cell–cell contact remains to be determined.

Actin remodeling has also been shown to be required for virological synapse formation. Actin has been observed to accumulate at the synapse site in the infected donor CD4+ T cells 146, 170. Actin polymerization is induced by gp120 binding with CD4 and is required for CD4 and coreceptor recruitment to the virological synapse 171173. In the target CD4+ T cell, an opening in the actin structure has been observed 148. This opening has been hypothesized to be critical for successful cell-to-cell transmission, as polymerized actin can act as a physical barrier that reduces viral infectivity post entry 174, 175. The precise roles for the actin cytoskeleton in both donor and target cell in the virological synapse and the recruitment of necessary factors are still under investigation.

The virological synapse: a vehicle for HIV persistence

The potential significance of cell-to-cell HIV transmission is clear, as studies have demonstrated that cell-associated HIV can have 100–1000 times the infection efficiency of cell-free virus 144, 145, 176178. Given this efficiency, it may be hypothesized that a cell-to-cell infection route may result in the transfer of more than one virion per synapse. Indeed, several studies have indicated the generation of a high local MOI 179, 180, which could be visualized in time-lapse microscopy 154, 181, 182. This would arise from the presence of multiple virions potentially present at the site of transfer that may enter the target cell before mechanisms to suppress multiple infection, such as downregulation of CD4, can take place. Support for this mechanism in vivo comes from the observed higher HIV DNA copy number per infected cell derived from tissues, whereas peripheral blood CD4 may only have a single provirus per infected cell 183.

A new study performed in humanized BLT (bone marrow/liver/thymus) mice has offered additional insights into the significance of cell-to-cell transmission in vivo and provides an intriguing model in which to study this phenomenon. One of the advantages offered by humanized BLT mice is the fact that the system also allows both naive and memory CD4+ T-cell homing to SLOs, as indicated earlier are localized sites of high numbers of CD4+ T cells as potential target cells for viral replication and transmission. Indeed, the study by Murooka et al. 184 did find evidence for tethering interactions that indicated virological synapse formation. However, their study also reported approximately 10–20% of infected cells in the lymph node to be multinucleated syncytia 184. For most CD4+ T-cell models of virological synapse formation, syncytia are not normally observed 185. The short duration of the virological synapse compared with the immunological synapse may be a mechanism to reduce cell fusion events during cell-to-cell transmission of HIV. Another mechanism is the recruitment of cellular factors to the virological synapse that actively inhibits cell–cell fusion such as regulatory tetraspanins. Tetraspanins are a class of surface molecules that have a variety of functions in the regulation of cell signaling and adhesion. Tetraspanins such as CD9, CD63, and CD81 have been shown to inhibit cell–cell fusion 186. Several studies have demonstrated a requirement for CD63 and CD81 in the virological synapse specifically in cell-to-cell transmission 186188. High expression levels of tetraspanins were also demonstrated to reduce syncytium formation in a gag-dependent manner 187.

Whether syncytia as a common outcome of cell-to-cell HIV transmission in vivo are accurately recapitulated in the BLT mouse model or are a product of the experimental model background remains to be determined. Moreover, Murooka et al. 184 report a difference between the results of HIV infection in the BLT model system and another in vivo model system for studying HIV infection, SIV infection of Rhesus macaques. In the macaque model system, most SIV-infected CD4+ T cells are resting memory phenotype 189, whereas in the BLT mouse model the majority of the infected CD4+ T cells in the lymph node were resting, CD45RO+ cells of central or effector memory phenotype 184. In determining the role of cell-to-cell HIV transmission between CD4+ T cells in vivo, the phenotype of both the donor and target CD4+ T cell may provide important clues as to how HIV infection is initiated and ultimately how a latently infected reservoir is established. For example, it is well known that CD4+ immune cells at different activation states have varying rates of permissiveness for HIV infection, with the more activated CD4+ effector memory T-cell subset being the most permissive 27. If HIV infection was maintained solely in activated effector T cells, then the immune system may be more able to eradicate the virus due to a greater likelihood of ongoing viral replication in these cell types and recognition by effector T cells. However, it has been demonstrated previously that HIV is present in both central and effector memory CD4 subsets 17, and the mechanism of infection of these different subsets and establishment of the latent HIV reservoir in each is still poorly understood. The virological synapse may play a more significant role in establishment of the latent reservoir than previously thought, if it can be demonstrated in vivo that the interactions between CD4+ T cells themselves or between CD4+ T cells and APCs in the lymph nodes enhanced infection rates resting CD4+ T cells that have been shown to be more resistant to infection 26, 28, 190. If these CD4+ target cells that are more naturally resistant due to a lower activation state or quiescence become transiently, suboptimally activated during formation of the virological synapse, the target cell then may rapidly return to a quiescent state and be maintained as a long-lived memory cell carrying proviral DNA. What is more, the mechanism of cell-to-cell transmission may also allow HIV to spread without triggering immune system detection, as several studies have suggested that virological synapse transmission of virus limits the epitope exposure of HIV gp120 to broadly neutralizing antibodies 191, 192. Altogether, these data suggest a new paradigm for establishment of HIV infection that encompasses virological synapse formation and cell-to-cell HIV transmission as a key mediator of HIV persistence and defines new challenges on the road to eradication.

The role of the immune synapse in HIV latent reservoir maintenance

The immunological synapse represents a crucial point of communication between T cells and APCs through which the APC can direct the T cell to a number of different cell fates. Significantly, this interaction may contribute to the maintenance of the latent HIV reservoir through various mechanisms. For example, the context of the immune synapse can influence the effector function of HIV-specific T cells resulting in diminished anti-HIV responses. Likewise, the immune synapse may be able to alter the biology of HIV-infected cells rendering the HIV silent for long periods of time. This section seeks to identify ways in which this critical component of the immune response may be contributing to the maintenance of the latent HIV reservoir.

Immune synapse and induction of stemness as a mechanism of latency

As detailed above, one mechanism of HIV persistence during prolonged ART is the long-term survival of latently infected quiescent memory CD4+ T cells, which may serve as a reservoir that contributes to viral load rebound after cessation of ART. This mechanism can potentially be explained with the stem cell–like nature of memory T cells, which has become a point of recent interest 193. Memory CD4+ T cells share several characteristics with stem cells, most notably, they are long-lived, capable of self-renewal, and able to differentiate upon stimulation; latently HIV-infected memory CD4+ T cells share these traits. Indeed, a population of memory T cells was recently identified with stem cell–like properties 194. Using a mouse model, stem cell memory T cells (Tscm) were found to display a gene signature intermediate of naive T cells and central memory T cells (Tcm). Tscm are CD45RA+CD95+IL-2Rβ+ and demonstrated the robust reconstitution potential of memory T cells. In non-human primate models, this same population was observed during the acute phase of viral infection and was maintained long term, compared with other memory subsets, after the removal of antigen 195. Although HIV infection in this population has not been demonstrated, it stands to reason that a ‘reservoir of T-cell memory’ could also be a reservoir of HIV latency.

While little is yet known about how the mechanisms that lead to differentiation into stem cells T cells can be induced to become T-cell stem cells, observations of embryonic stem cells and inducible pluripotent stems can give some prediction as to what signals could be promoted by the immunological synapse. Negative regulators of T-cell signaling may be connected to signals that are known to provide critical stem cell functions. Indeed, several transcriptional coactivators have been described that change the outcome of TCR stimulation when present in the context of an immune synapse. One such example is the Yes-associated protein (Yap), a protein required for self-renewal of embryonic stem (ES) cells, which is responsible for a transcriptional profile of pluripotency genes that include Oct4, Nanog, and Sox2 196. Yap expression has recently been implicated in self-renewal and memory precursor maintenance in CD8+ T cells and is inhibited by activation of Akt and the Hippo pathway 197. Activation of PD-1 on T cells inhibits the activation of Akt, which may allow for sustained Yap expression and direct a more stem-like quality in the T cell 139. This observation suggests a mechanism by which the composition of molecules at the immune synapse may be able to shift an infected T cell toward a long-lived, stem-like memory cell. Yap expression in the context of Wnt signaling is another mechanism associated with stem cell self-renewal and regulation of differentiation. In colorectal cancers, the canonical Wnt signaling complex, β-catenin/TCF4 (T-cell factor 4), drives Yap expression 198. More recently, it was proposed that after Yap is phosphorylated by LATS1/2 (large tumor suppressor 1/2) kinases, it is retained in the cytoplasm where it interacts indirectly with disheveled (Dvl) and inhibits nuclear translocation of Dvl, blocking TCF transcription of Wnt target genes 199.

Another potential promoter of stemness in HIV-infected CD4+ T cells comes from cytokines secreted at the site of the immune synapse. One example is signal transducer and activator of transcription-3 (STAT-3), a transcription factor that links cytokine signals to pluripotent stem cell potential. Activation of STAT-3 is required for self-renewal of ES cells 200, 201, and its critical role in T cells is supported by the identification of patients with a mutation in STAT-3 that have dysfunction in the maintenance of T-cell memory 202. Furthermore, recent observations in CD4+ T cells point to STAT-3 activation downstream of IL-21 as a mechanism for maintaining stem cell–like qualities in Th17 cells 202. In addition to the importance of STAT-3 signaling in stem cell potential, these experiments also point to a role for β-catenin Wnt signaling and Notch signaling.

Wnt signaling pathways and gene regulation

Wnt signaling is a fundamental component in embryonic stem cell proliferation, differentiation, and cell fate decisions 203206, homeostasis in adult stem cells and tissues, and self-renewal in cancer stem cells (reviewed in 207). The diversity of receptors, ligands, and regulators 208 that comprise the Wnt signaling pathway provide the foundation to influence cell development and homeostasis in a variety of organs/tissues.

Wnt signaling is triggered through either a β-catenin–dependent (canonical) or -independent (non-canonical) pathway by interactions between one of 19 Wnt ligands with one of 10 seven-transmembrane receptors termed Frizzled (Fzd). Non-canonical signaling does not rely on gene expression associated with β-catenin. Signaling occurs when Wnt ligands specific to non-canonical signaling binding to Fzd receptors and coreceptors other than Lrp4 [Ldl (low density lipoprotein) receptor-related protein 4], Lrp5, Lrp6, or no coreceptor 209. The most well-characterized non-canonical pathways are the planar cell polarity pathway and the Ca2+-dependent pathway (reviewed in 210, 211).

β-catenin is the key component to canonical Wnt signaling and mediates both structural and signaling functions. In the absence of Wnt signaling, a destruction complex comprised of glycogen synthase kinase 3β (GSK3β), casein kinase 1 (CK1), axin and adenomatous polyposis coli (APC) phosphorylate β-catenin, and target the phosphorylated form for unbiquitin-dependent degradation by the E3 ligase β-transducin repeat-containing protein (β-TrCP) (Fig. 2).2). In the presence of Wnt ligands, Wnt binds to its receptor, Fzd, and coreceptor, Lrp, to induce Lrp phosphorylation mediated by GSK3β and CK1. Through an unclear disheveled (Dvl)-dependent mechanism, unphosphorylated β-catenin accumulates and translocates into the nucleus where β-catenin replaces Groucho and corepressors from DNA-bound T-cell factor (TCF)/lymphoid enhancer factor (LEF) and recruits transcriptional coactivators and histone modifiers to drive expression of genes that promote cell cycle and survival (cyclinD1, c-Jun, fra-1, c-myc). CTLA-4, a negative regulator of T-cell activation and correlate of HIV latency, is upregulated by Wnt3a in human melanoma tumors 212 and differentially expressed along with the negative regulator PD-1 in activated CD4+ T cells (J.H. Brehm and R.-P. Sekaly, unpublished data). A comprehensive list of Wnt target genes can be found at 213, 214.

Fig. 2
Canonical Wnt signaling and its potential role in T-cell differentiation, self-renewal, and HIV latency

The structural function of β-catenin is defined by interaction with cadherins at the plasma membrane, mediating an indirect role in cell-to-cell adhesion and reduced proliferation. Increased expression of cadherins in human SW480 human colon carcinoma cells recruits β-catenin to adherin junctions at the cell surface, decreasing nuclear pools of β-catenin, and thereby antagonizing β-catenin–LEF/TCF transcription of genes associated with proliferation 215, 216.

Wnt signaling transcription factors TCF1 and TCF3 have activator and repressor functions, respectively, in embryonic stem cell self-renewal and determination of stem cell fate. In the absence of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) and GSK3β inhibition, TCF3 bound to DNA represses genes specific to self-renewal (Oct4, Sox2, Nanog, and Klf2/4) and drives stem cell differentiation. Conversely, in the presence of MAPK (ERK) kinase and GSK3β (Wnt signaling) inhibition, genes that maintain stem cell self-renewal are expressed after β-catenin removal of TCF3 repression followed by transcriptional activation of TCF1 and stimulation of biosynthetic and metabolic processes 204, 205. Genes regulating Wnt signaling activity are often differentially regulated in T-cell populations, as seen from microarray data analyzed at VGTI-FL. It will be interesting to interpret these data and what their expression means related to Wnt signaling activity specific to self-renewal and survival of memory T cells.

Role of Wnt/β-catenin signaling in stem cell memory T cells and HIV latency

Components of both the canonical and non-canonical Wnt signaling pathways are active in T lymphocytes. For instance, CD8+ naive T cells (Tn) and Tscm highly express Wnt signaling transducers, TCF7 and LEF1, that are lost during differentiation from Tn to Tscm to Tcm to effector memory T cells (Tem) 194. In fact, Wnt3a ligand and inhibitors of GSK3β inhibit the differentiation of Tn to short-lived effector T cells (Teff) while enhancing self-renewal of CD4+ and CD8+ Tcm cells 50, 51 and CD8+ Tscm 50 (Fig. 2).2). Furthermore, gain-of-function and loss-of-function studies of β-catenin and TCF1 in CD8+ T cells from mice confirm that canonical Wnt signaling is sufficient for the establishment of long-term Tcm cells 217219.

Consistent with these findings, preliminary observations in our laboratory indicate that memory CD4+ T cells express Wnt receptors (FZD1-10 and LRP6). After stimulation of memory CD4+ cells with anti-CD3/CD28 in the presence of Dickkopf-related protein 1 (Dkk1), an inhibitor for canonical Wnt/β-catenin signaling, the number of proliferating cells increased and phosphorylation of β-catenin (ser33/37/Thr41) decreased (C. Benne and R.-P. Sekaly, unpublished data), implying that Wnt/β-catenin signaling may maintain self-renewal and survival of memory CD4+ T cells. In fact, diminished ki67 and CD38 expression on memory CD4+ T cells and preservation of the Tcm subset were observed after a 7-day culture in the presence of Wnt3a and anti-CD3/CD28 (J.H. Brehm and R.-P. Sekaly, unpublished data). These findings in CD4+ T cells in combination with published results strongly suggest the canonical Wnt/β-catenin pathway as a critical component in self-renewal of CD4+ Tscm and Tcm populations and may be a mechanism for maintaining cells containing the HIV latent reservoir.

Notch signaling and gene regulation

Similar to Wnt, Notch signaling is a key factor in mediating stem cell proliferation, self-renewal, differentiation, and quiescence in various tissues associated with embryonic development and adult tissue homeostasis. Notch signaling is context dependent; thus, the microenvironment of cells in which Notch is signaling will strongly influence their fate. Notch signaling in stem cells and other tissues has been elegantly reviewed by Koch et al. 220. In context of the immunological synapse, combined Notch signaling and TLR stimulation of DCs can modulate TLR-induced cytokine expression compared with TLR or Notch signaling alone, including increased IL-2 and IL-10 expression and decreased IL-12 expression. This mechanism is dependent on PI3K activity after signaling through an alternate Notch pathway than the canonical Notch pathway described below 221. DC–T-cell interactions and the resulting immune response may be affected in an environment influenced by Notch/TLR ligand signaling.

Canonical Notch signaling occurs after a cell presenting ligand [Jagged1, Jagged2, Delta-like 1 (Dll1), Dll3, and Dll4] binds to a Notch receptor (Notch 1, 2, 3, and 4) on an adjacent cell (Fig. 3).3). The triggered Notch receptor undergoes two sequential proteolytic cleavages to form a free Notch intracellular domain (NICD). The NICD translocates into the nucleus followed by interaction with a transcriptional repressor complex comprised of CSL [C-promoter–binding factor-1 (CDF-1) suppressor of Hairless/Lag-1, also called recombining binding protein suppressor of hairless (RBP-J)] and other corepressors. The NICD–CSL interaction dissociates the repressor complex and recruits MAML (mastermind-like proteins) and coactivators such as p300 to release gene silencing and upregulate Notch target genes 222, 223.

Fig. 3
Role of the canonical Notch signaling pathway in asymmetric cell division and HIV latency

Notch signaling in T-cell differentiation and quiescence

Preservation of long-lived quiescent T cells is dependent on inhibition of apoptosis and maintenance of cells in a non-dividing (G0) state. Notch signaling regulates both cell cycle and apoptosis. For example, target gene Hes1 (hairy and enhancer of split-1) downregulates Notch ligands and expression of G1 cyclins in mouse neural progenitors 224, and Mathieu et al. 225 identify that Pdcd1 transcription of a negative regulator for T-cell activation, PD-1, is controlled through Notch signaling in activated CD8+ T cells. Several studies report that high levels of Notch signaling induce quiescence, whereas low levels promote proliferation and differentiation 226228. Furthermore, a recent study reported that Notch protects expanded CD4+ T-cell clones from apoptosis and stimulates genes found in the metabolic pathway (i.e. carbohydrate, lipid, glucose metabolism, glucose transport, biosynthesis, and energy generation) 229.

Although Wnt and Notch are separate pathways, numerous reports suggest that these two pathways are interlinked in cell self-renewal, quiescence, and cell fate 220, 222. For instance, Wnt signaling upregulates Notch ligands Jag1 230, Dll4 231, and expression of the Notch2 receptor 232. In contrast, Wnt pathway component Dvl binds NICD to block Notch signaling 233. Additional interactions between NICD and Wnt proteins have also been shown to disrupt Notch activity. These include axin-NICD 234, axin and adenomatous polyposis coli-NICD 235, and GSK3β phosphorylation of NICD 236, 237. Preselin1 (PS1) is another protein with Wnt and Notch activity. PS1 is the γ-secretase involved in the intramembranous proteolysis of Notch within the signal-receiving cell to form the NICD; however, PS1 is also a negative regulator of β-catenin degradation in the canonical Wnt signaling pathway 238, 239.

The role of Notch in memory T-cell differentiation (Tn, Tcm, Tem, and Teff) has not yet been investigated. However, the following characteristics support the notion that Notch signaling may be linked to memory T-cell homeostasis: (i) Notch increases cell survival in T cells; (ii) high levels of Notch signaling induce cell quiescence; (iii) Wnt and Notch pathways regulate one another 220, 222; and (iv) regulation of Notch signaling is associated with asymmetric division, as discussed below 240242. Combining these factors with studies in stem cells suggest that a versatile pathway such as Notch may also facilitate memory T-cell homeostasis and differentiation.

Notch–Wnt signaling and inhibition of HIV replication

Not only are Notch and Wnt important in CD4+ T-memory cell development, proliferation, and survival, recent studies have shown that Notch and canonical Wnt signaling modulate HIV replication in various target cells. For instance, Tyagi and Karn 243 demonstrate that CBF-1 (C-promoter–binding factor-1, the mammalian representative of the CSL family) of the Notch signaling pathway reduces RNA polymerase II on the HIV promoter and recruits HDACs to the LTR in Jurkat cells, inhibiting HIV transcription. In other studies, β-catenin and TCF4 of the Wnt/β-catenin pathway are strong inhibitors of HIV replication in astrocytes and peripheral blood mononuclear cells 244247. Of interest, IFN-γ, a type II interferon with antiviral activity that promotes proapoptotic response in infected cells, is found to be elevated in plasma, lymph nodes, and cerebrospinal fluid of HIV-infected individuals. IFN-γ upregulates expression of the Wnt/β-catenin pathway inhibitor Dkk1 through STAT3 signaling and enhances HIV replication 248. These data suggest that components of both the Notch and Wnt signaling pathways inhibit HIV replication, contributing to induction of HIV latency in CD4+ memory T cells, whereas components of immune response to infection (IFN-γ) antagonize Wnt signaling to increase HIV replication. It will be important to explore the mechanisms associated with Wnt/Notch signaling in HIV infection of CD4+ memory T cells and their relationship in HIV-infected populations (i.e. acute, chronic, elite controllers, and antiretroviral-treated immune responders or immune non-responders).

Asymmetric cell division as an inducer of latency

Dividing stem cells must have capacity to produce progeny with differentiation potential and progeny that retain the parental, stem cell qualities. One mechanism through which this is possible is called asymmetric cell division. This is typically observed in stem cells that are capable of polarization such that the composition of one daughter cell is distinct from the other. In certain types of stem cells, this mechanism will generate one daughter cell that retains the original stem cell phenotype and another that has distinct effector cell function 249, 250.

Asymmetric division of this kind is perhaps most apparent in T cells because prolonged engagement of the TCR in an immune synapse creates a highly polarized cell for division. This polarization results in unequal distribution of proteins between the two daughter cells and generates one cell with effector function and one with memory function 240. Many molecules important to effector function and differentiation such as IFN-γR, PKC-ζ, T-bet, and CD25 segregate preferentially toward the immunological synapse and segregation of the proteasome is responsible, at least partially, for this effect 251, 252. The process of polarization of dividing cells persists even after the dissemination of the immunological synapse and is retained by the networks of the partitioning-defective protein 3 (Par3) and Scribble complexes 241.

Although the segregation of HIV-specific proteins toward the immunological synapse has not been characterized, it seems plausible given the polarization of viral particles toward the virological synapse. The possibility that HIV particles and proteins could accumulate at the interface with an APC suggests a very appealing mechanism by which a cell division would result a daughter cell with active HIV and one with latent HIV. Mathematical predictions have suggested that this concept of asymmetric division within HIV-infected cells may account for the kinetics of detectible virus in patients undergoing ART 253.

The basic model of asymmetric cell division in the maintenance of HIV latency is as follows. An HIV-infected CD4+ T cell engages its cognate antigen on an APC forming a stable immunological synapse. Along with cellular components that polarize toward the APC, HIV proteins and particles would also move toward the active TCR interface. After division, the daughter cell engaged with the APC would have the effector cell phenotype and would contain the active HIV. The daughter cell that is distal to the immunological synapse would maintain a memory phenotype, or possibly a stem cell memory phenotype, and would contain only the integrated HIV proviral DNA. In this case, the memory cell would lack the essential transcription machinery, such as Tat protein, to drive HIV virus production. If this model were accurate, it would predict that the latent HIV reservoir would be smaller in the effector cell population than in memory population, as the effector cells would contain mostly active virus and would be purged during ART. In fact, when the latent reservoir is quantified in antigen-experienced CD4+ T-cell subsets, the latent reservoir mostly persists in the less differentiated Tcm subset and is significantly decreased in Tem cells 17.

Notch–Wnt signaling and asymmetric division in HIV latency

Unequal distribution of Numb, an inhibitor of notch signaling, is a classical indicator for asymmetric division in antigen-stimulated cells and essential in determining cell fate 240, 241. Numb is polarized to the distal side of a cell undergoing division, along with Par3 and atypical protein kinase C (aPKC), whereas Scribble and disks large family (DlgF) polarize to the proximal cell 241. In the proposed model for asymmetric cell division for HIV latency, Numb would be allocated to the cell distal to the APC, restricting Notch signaling in cells that maintain a memory or Tscm phenotype, and replicating HIV would be polarized to effector cells harboring ongoing Notch signaling (Fig. 3).3). Initial studies indicate that Notch signaling is required for CD4+ and CD8+ T-cell proliferation, NFκB activity and IFN-γ production 254, and will increase IL-2 receptor expression followed by production of IL-2 on CD4+ T cells 255. Inhibition of Notch signaling by Numb during asymmetric division would prevent activation and differentiation of distal daughter cells, contributing to the Tcm and Tscm phenotype.

Habib et al. 256 recently designed a single-cell embryonic stem cell in vitro model for Wnt-induced asymmetric division. Localized Wnt signal stimulates Wnt signaling ‘Wnt-on’ in the dividing cell proximal to Wnt3a ligand and maintains embryonic stem cell pluripotency, whereas the dividing cell distal to the Wnt3a ligand is in a ‘Wnt-off’ state and differentiates toward an epiblast stem cell (EpiSC) fate 256. To our knowledge, the role of Wnt3a in asymmetric cell division and its association with Numb and Notch signaling in vivo has not yet been investigated. Although these observations indicate a role for Notch and Wnt signaling to promote asymmetric division of memory CD4+ T cells and HIV latency, directed studies will help elucidate these novel hypotheses.


The immunological synapse provides strong potential mechanisms for the establishment and maintenance of a latent HIV reservoir. The immunological synapse can dictate functionality of T cells through expression of a multitude of cytokines and cofactors. In the context of an HIV-infected T cell, this means that components of the immune synapse may be able to facilitate latency as well as active infection. In addition, the observation of asymmetric cell division in response to T-cell stimulation by the immune synapse provides a potential mechanism by which HIV infection can be maintained in a latent state, while at the same time producing progeny that actively express HIV virus. As the precise mechanisms of HIV latency establishment and maintenance become clearer, interventions that disrupt specific components of the immunological synapse may represent the final step in the process of HIV eradication.


The authors are grateful for the funding from DARE (U19 AI096109), Ahmed (P01 AI080192-01), and Lederman (AI076174-01A1) (R. P. S.), amfAR (107175-44RGRL and fellowship 108264) (N. C. and R. F.), the NIH (1U19AI096109) (N. C.), and the Merck Cure Project (105171) (J. H. B., A. C., C. C., D. A. K.). The authors have no conflicts of interest to declare.


1. Pantaleo G, et al. Lymphoid organs function as major reservoirs for human immunodeficiency virus. Proc Natl Acad Sci USA. 1991;88:9838–9842. [PubMed]
2. Pantaleo G, et al. HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature. 1993;362:355–358. [PubMed]
3. Couedel-Courteille A, Butor C, Juillard V, Guillet JG, Venet A. Dissemination of SIV after rectal infection preferentially involves paracolic germinal centers. Virology. 1999;260:277–294. [PubMed]
4. Veazey RS, et al. Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science. 1998;280:427–431. [PubMed]
5. Fox CH, Kotler D, Tierney A, Wilson CS, Fauci AS. Detection of HIV-1 RNA in the lamina propria of patients with AIDS and gastrointestinal disease. J Infect Dis. 1989;159:467–471. [PubMed]
6. Zuckerman RA, et al. Higher concentration of HIV RNA in rectal mucosa secretions than in blood and seminal plasma, among men who have sex with men, independent of antiretroviral therapy. J Infect Dis. 2004;190:156–161. [PubMed]
7. Chun TW, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature. 1997;387:183–188. [PubMed]
8. Kiviat NB, et al. Determinants of human immunodeficiency virus DNA and RNA shedding in the anal-rectal canal of homosexual men. J Infect Dis. 1998;177:571–578. [PubMed]
9. Lampinen TM, et al. Association of antiretroviral therapy with detection of HIV-1 RNA and DNA in the anorectal mucosa of homosexual men. AIDS. 2000;14:F69–F75. [PubMed]
10. Chun TW, et al. Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy. J Infect Dis. 2008;197:714–720. [PubMed]
11. Guadalupe M, et al. Viral suppression and immune restoration in the gastrointestinal mucosa of human immunodeficiency virus type 1-infected patients initiating therapy during primary or chronic infection. J Virol. 2006;80:8236–8247. [PMC free article] [PubMed]
12. Yukl SA, et al. Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. J Infect Dis. 2010;202:1553–1561. [PMC free article] [PubMed]
13. Sigal A, et al. Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy. Nature. 2011;477:95–98. [PubMed]
14. Deeks SG, et al. Towards an HIV cure: a global scientific strategy. Nat Rev Immunol. 2012;12:607–614. [PMC free article] [PubMed]
15. Eisele E, Siliciano RF. Redefining the viral reservoirs that prevent HIV-1 eradication. Immunity. 2012;37:377–388. [PubMed]
16. Chun TW, Finzi D, Margolick J, Chadwick K, Schwartz D, Siliciano RF. In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency. Nat Med. 1995;1:1284–1290. [PubMed]
17. Chomont N, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med. 2009;15:893–900. [PMC free article] [PubMed]
18. Finzi D, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science. 1997;278:1295–1300. [PubMed]
19. Wong JK, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science. 1997;278:1291–1295. [PubMed]
20. Finzi D, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med. 1999;5:512–517. [PubMed]
21. Siliciano JD, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med. 2003;9:727–728. [PubMed]
22. Strain MC, et al. Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: intrinsic stability predicts lifelong persistence. Proc Natl Acad Sci USA. 2003;100:4819–4824. [PubMed]
23. Ananworanich J, et al. Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection. PLoS ONE. 2012;7:e33948. [PMC free article] [PubMed]
24. Strain MC, et al. Effect of treatment, during primary infection, on establishment and clearance of cellular reservoirs of HIV-1. J Infect Dis. 2005;191:1410–1418. [PubMed]
25. Colin L, Van Lint C. Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies. Retrovirology. 2009;6:111. [PMC free article] [PubMed]
26. Zack JA, Arrigo SJ, Weitsman SR, Go AS, Haislip A, Chen IS. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990;61:213–222. [PubMed]
27. Stevenson M, Stanwick TL, Dempsey MP, Lamonica CA. HIV-1 replication is controlled at the level of T cell activation and proviral integration. EMBO J. 1990;9:1551–1560. [PubMed]
28. Zhou Y, Zhang H, Siliciano JD, Siliciano RF. Kinetics of human immunodeficiency virus type 1 decay following entry into resting CD4+ T cells. J Virol. 2005;79:2199–2210. [PMC free article] [PubMed]
29. Han Y, et al. Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes. J Virol. 2004;78:6122–6133. [PMC free article] [PubMed]
30. Shan L, et al. Influence of host gene transcription level and orientation on HIV-1 latency in a primary-cell model. J Virol. 2011;85:5384–5393. [PMC free article] [PubMed]
31. Van Lint C, Emiliani S, Ott M, Verdin E. Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation. EMBO J. 1996;15:1112–1120. [PubMed]
32. Kauder SE, Bosque A, Lindqvist A, Planelles V, Verdin E. Epigenetic regulation of HIV-1 latency by cytosine methylation. PLoS Pathog. 2009;5:e1000495. [PMC free article] [PubMed]
33. Agbottah E, Deng L, Dannenberg LO, Pumfery A, Kashanchi F. Effect of SWI/SNF chromatin remodeling complex on HIV-1 Tat activated transcription. Retrovirology. 2006;3:48. [PMC free article] [PubMed]
34. Blazkova J, et al. CpG methylation controls reactivation of HIV from latency. PLoS Pathog. 2009;5:e1000554. [PMC free article] [PubMed]
35. Nabel G, Baltimore D. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature. 1987;326:711–713. [PubMed]
36. Bohnlein E, Lowenthal JW, Siekevitz M, Ballard DW, Franza BR, Greene WC. The same inducible nuclear proteins regulates mitogen activation of both the interleukin-2 receptor-alpha gene and type 1 HIV. Cell. 1988;53:827–836. [PubMed]
37. Adams M, et al. Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts. Proc Natl Acad Sci USA. 1994;91:3862–3866. [PubMed]
38. Tyagi M, Pearson RJ, Karn J. Establishment of HIV latency in primary CD4+ cells is due to epigenetic transcriptional silencing and P-TEFb restriction. J Virol. 2010;84:6425–6437. [PMC free article] [PubMed]
39. Archin NM, et al. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature. 2012;487:482–485. [PMC free article] [PubMed]
40. Lassen KG, Ramyar KX, Bailey JR, Zhou Y, Siliciano RF. Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells. PLoS Pathog. 2006;2:e68. [PMC free article] [PubMed]
41. Chomont N, DaFonseca S, Vandergeeten C, Ancuta P, Sekaly RP. Maintenance of CD4+ T-cell memory and HIV persistence: keeping memory, keeping HIV. Curr Opin HIV AIDS. 2011;6:30–36. [PubMed]
42. Ellis JS, et al. APCs expressing high levels of programmed death ligand 2 sustain the development of CD4 T cell memory. J Immunol. 2010;185:3149–3157. [PMC free article] [PubMed]
43. Seddon B, Tomlinson P, Zamoyska R. Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells. Nat Immunol. 2003;4:680–686. [PubMed]
44. van Leeuwen EM, Sprent J, Surh CD. Generation and maintenance of memory CD4(+) T Cells. Curr Opin Immunol. 2009;21:167–172. [PMC free article] [PubMed]
45. Kondrack RM, Harbertson J, Tan JT, McBreen ME, Surh CD, Bradley LM. Interleukin 7 regulates the survival and generation of memory CD4 cells. J Exp Med. 2003;198:1797–1806. [PMC free article] [PubMed]
46. Wagner TA, McKernan JL, Tobin NH, Tapia KA, Mullins JI, Frenkel LM. An increasing proportion of monotypic HIV-1 DNA sequences during antiretroviral treatment suggests proliferation of HIV-infected cells. J Virol. 2013;87:1770–1778. [PMC free article] [PubMed]
47. Kim H, Perelson AS. Viral and latent reservoir persistence in HIV-1-infected patients on therapy. PLoS Comp Biol. 2006;2:e135. [PMC free article] [PubMed]
48. Bosque A, Famiglietti M, Weyrich AS, Goulston C, Planelles V. Homeostatic proliferation fails to efficiently reactivate HIV-1 latently infected central memory CD4+ T cells. PLoS Pathog. 2011;7:e1002288. [PMC free article] [PubMed]
49. Gianella S, et al. Cytomegalovirus DNA in semen and blood is associated with higher levels of proviral HIV DNA. J Infect Dis. 2013;207:898–902. [PMC free article] [PubMed]
50. Gattinoni L, et al. Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat Med. 2009;15:808–813. [PMC free article] [PubMed]
51. Muralidharan S, et al. Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes. J Immunol. 2011;187:5221–5232. [PMC free article] [PubMed]
52. Scripture-Adams DD, Brooks DG, Korin YD, Zack JA. Interleukin-7 induces expression of latent human immunodeficiency virus type 1 with minimal effects on T-cell phenotype. J Virol. 2002;76:13077–13082. [PMC free article] [PubMed]
53. Wang FX, et al. IL-7 is a potent and proviral strain-specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART. J Clin Invest. 2005;115:128–137. [PMC free article] [PubMed]
54. Chun TW, Engel D, Mizell SB, Ehler LA, Fauci AS. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J Exp Med. 1998;188:83–91. [PMC free article] [PubMed]
55. Chun TW, et al. Gene expression and viral production in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals. Proc Natl Acad Sci USA. 2003;100:1908–1913. [PubMed]
56. Chun TW, et al. HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir. J Clin Invest. 2005;115:3250–3255. [PMC free article] [PubMed]
57. Iannello A, et al. HIV-1 causes an imbalance in the production of interleukin-18 and its natural antagonist in HIV-infected individuals: implications for enhanced viral replication. J Infect Dis. 2010;201:608–617. [PubMed]
58. Cameron P, et al. Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton. Proc Natl Acad Sci USA. 2010;107:16934–16939. [PubMed]
59. Saleh S, et al. Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells. Retrovirology. 2011;8:80. [PMC free article] [PubMed]
60. Zhang L, et al. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med. 1999;340:1605–1613. [PubMed]
61. Palmer S, et al. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy. Proc Natl Acad Sci USA. 2008;105:3879–3884. [PubMed]
62. Bailey JR, et al. Residual human immunodeficiency virus type 1 viremia in some patients on antiretroviral therapy is dominated by a small number of invariant clones rarely found in circulating CD4+ T cells. J Virol. 2006;80:6441–6457. [PMC free article] [PubMed]
63. Kieffer TL, et al. Genotypic analysis of HIV-1 drug resistance at the limit of detection: virus production without evolution in treated adults with undetectable HIV loads. The J Infect Dis. 2004;189:1452–1465. [PubMed]
64. Dinoso JB, et al. Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy. Proc Natl Acad Sci USA. 2009;106:9403–9408. [PubMed]
65. Gandhi RT, et al. The effect of raltegravir intensification on low-level residual viremia in HIV-infected patients on antiretroviral therapy: a randomized controlled trial. PLoS Med. 2010;7:e1000321. [PMC free article] [PubMed]
66. Yukl SA, et al. Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. AIDS. 2010;24:2451–2460. [PMC free article] [PubMed]
67. Borrow P, et al. Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nat Med. 1997;3:205–211. [PubMed]
68. Kastenmuller W, Torabi-Parizi P, Subramanian N, Lammermann T, Germain RN. A spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread. Cell. 2012;150:1235–1248. [PMC free article] [PubMed]
69. Gonzalez PA, Carreno LJ, Figueroa CA, Kalergis AM. Modulation of immunological synapse by membrane-bound and soluble ligands. Cytokine Growth Factor Rev. 2007;18:19–31. [PubMed]
70. Miller MJ, Wei SH, Parker I, Cahalan MD. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science. 2002;296:1869–1873. [PubMed]
71. Miller MJ, Wei SH, Cahalan MD, Parker I. Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy. Proc Natl Acad Sci USA. 2003;100:2604–2609. [PubMed]
72. Mempel TR, Henrickson SE, Von Andrian UH. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature. 2004;427:154–159. [PubMed]
73. Dustin ML, Bromley SK, Kan Z, Peterson DA, Unanue ER. Antigen receptor engagement delivers a stop signal to migrating T lymphocytes. Proc Natl Acad Sci USA. 1997;94:3909–3913. [PubMed]
74. Dustin ML. Multiscale analysis of T cell activation: correlating in vitro and in vivo analysis of the immunogical synpase. Curr Top Microbiol Immunol. 2009;334:47–70. [PubMed]
75. Irvine DJ, Purbhoo MA, Krogsgaard M, Davis MM. Direct observation of ligand recognition by T cells. Nature. 2002;419:845–849. [PubMed]
76. Maggi E, Cosmi L, Liotta F, Romagnani P, Romagnani S, Annunziato F. Thymic regulatory T cells. Autoimmun Rev. 2005;4:579–586. [PubMed]
77. Sumoza-Toledo A, Eaton AD, Sarukhan A. Regulatory T cells inhibit protein kinase C theta recruitment to the immune synapse of naive T cells with the same antigen specificity. J Immunol. 2006;176:5779–5787. [PubMed]
78. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature. 1998;395:82–86. [PubMed]
79. Piguet V, Sattentau QJ. Dangerous liaisons at the virological synapse. J Clin Invest. 2004;114:605–610. [PMC free article] [PubMed]
80. Bachmann MF, et al. Distinct roles for LFA-1 and CD28 during activation of naive T cells: adhesion versus costimulation. Immunity. 1997;7:549–557. [PubMed]
81. Freiberg BA, et al. Staging and resetting T cell activation in SMACs. Nat Immunol. 2002;3:911–917. [PubMed]
82. Yokosuka T, et al. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat Immunol. 2005;6:1253–1262. [PubMed]
83. Varma R, Campi G, Yokosuka T, Saito T, Dustin ML. T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity. 2006;25:117–127. [PMC free article] [PubMed]
84. Martinez-Martin N, et al. T cell receptor internalization from the immunological synapse is mediated by TC21 and RhoG GTPase-dependent phagocytosis. Immunity. 2011;35:208–222. [PubMed]
85. Shaw AS, Dustin ML. Making the T cell receptor go the distance: a topological view of T cell activation. Immunity. 1997;6:361–369. [PubMed]
86. Allespach EJ, et al. ERM-dependent movement of CD43 defines a novel protein complex distal to the immunological synpase. Immunity. 2001;15:739–750. [PubMed]
87. Delon J, Kaibuchi K, Germain RN. Exclusion of CD43 from the immunological synpase is mediated by phosphorylation-regulated relocation of the cytoskeletal adaptor moesin. Immunity. 2001;15:691–701. [PubMed]
88. Donnadieu E, Bismuth G, Trautmann A. Antigen recognition by helper T cells elicits a sequence of distinct changes of their shape and intracellular calcium. Curr Biol. 1994;4:584–595. [PubMed]
89. Negulescu PA, Krasieva TB, Khan A, Kerschbaum HH, Cahalan MD. Polarity of T cell shape, motility, and sensitivity to antigen. Immunity. 1996;4:421–430. [PubMed]
90. Sims TN, et al. Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse. Cell. 2007;129:773–785. [PubMed]
91. Kao H, Lin J, Littman DR, Shaw AS, Allen PM. Regulated movement of CD4 in and out of the immunological synpase. J Immunol. 2008;181:8248–8257. [PMC free article] [PubMed]
92. Molon B, et al. T cell costimulation by chemokine receptors. Nat Immunol. 2005;6:465–471. [PubMed]
93. Bousso P. T-cell activation by dendritic cells in the lymph node: lessons from the movies. Nat Rev Immunol. 2008;8:675–684. [PubMed]
94. Scholer A, Hugues S, Boissonnas A, Fetler L, Amigorena S. Intercellular adhesion molecule-1-dependent stable interactions between T cells and dendritic cells determine CD8+ T cell memory. Immunity. 2008;28:258–270. [PubMed]
95. Barber EK, Dasgupta JD, Schlossman SF, Trevillyan JM, Rudd CE. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex. Proc Natl Acad Sci USA. 1989;86:3277–3281. [PubMed]
96. Alon R, Dustin ML. Force as a facilitator of integrin conformational changes during leukocyte arrest on blood vessels and antigen-presenting cells. Immunity. 2007;26:17–27. [PubMed]
97. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell. 2002;110:673–687. [PubMed]
98. Hogg N, Patzak I, Willenbrock F. The insider's guide to leukocyte integrin signalling and function. Nat Rev Immunol. 2011;11:416–426. [PubMed]
99. Shamri R, et al. Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat Immunol. 2005;6:497–506. [PubMed]
100. Kaiser A, Donnadieu E, Abastado JP, Trautmann A, Nardin A. CC chemokine ligand 19 secreted by mature dendritic cells increases naive T cell scanning behavior and their response to rare cognate antigen. J Immunol. 2005;175:2349–2356. [PubMed]
101. Saleh S, Solomon A, Wightman F, Xhilaga M, Cameron P, Lewin S. CCR7 ligands CCL19 and CCL21 increase permissiveness of resting memory CD4+ T cells to HIV-1 infection: a novel model of HIV-1 latency. Blood. 2007;110:4161–4164. [PubMed]
102. Norton SD, Zuckerman L, Urdahl KB, Shefner R, Miller J, Jenkins MK. The CD28 ligand, B7, enhances IL-2 production by providing a costimulatory signal to T cells. J Immunol. 1992;149:1556–1561. [PubMed]
103. Yokosuka T, et al. Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation. Immunity. 2008;29:589–601. [PMC free article] [PubMed]
104. Boomer JS, Green JM. An enigmatic tail of CD28 signaling. Cold Spring Harbor Perp Biol. 2010;2:a002436. [PMC free article] [PubMed]
105. Green JM, Karpitskiy V, Kimzey SL, Shaw AS. Coordinate regulation of T cell activation by CD2 and CD28. J Immunol. 2000;164:3591–3595. [PubMed]
106. Espagnolle N, et al. CD2 and TCR synergize for the activation of phospholipase Cgamma1/calcium pathway at the immunological synapse. Int Immunol. 2007;19:239–248. [PubMed]
107. Douglass AD, Vale RD. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell. 2005;121:937–950. [PMC free article] [PubMed]
108. Altin JG, Sloan EK. The role of CD45 and CD45-associated molecules in T cell activation. Immunol Cell Biol. 1997;75:430–445. [PubMed]
109. Ng DH, Watts JD, Aebersold R, Johnson P. Demonstration of a direct interaction between p56lck and the cytoplasmic domain of CD45 in vitro. J Biol Chem. 1996;271:1295–1300. [PubMed]
110. Stone J, et al. Aberrant TCR-mediated signaling in CD45-null thymocytes involves dysfunctional regulation of Lck, Fyn, TCR-zeta, and ZAP-70. J Immunol. 1997;158:5773–5782. [PubMed]
111. Barbeau B, Robichaud G, Fortin J, Tremblay M. Negative regulation of the NFAT1 factor by CD45: implication in HIV-1 long terminal repeat activation. J Immunol. 2001;167:2700–2713. [PubMed]
112. Fos C, et al. ICOS ligation recruits the p50alpha PI3K regulatory subunit to the immunological synapse. J Immunol. 2008;181:1969–1977. [PubMed]
113. Boisvert J, Edmondson S, Krummel MF. Immunological synapse formation licenses CD40-CD40L accumulations at T-APC contact sites. J Immuol. 2004;173:3647–3652. [PubMed]
114. Gimferrer I, et al. Relevance of CD6-mediated interactions in T cell activation and proliferation. J Immunol. 2004;173:2262–2270. [PubMed]
115. Pacheco R, et al. CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse. Proc Natl Acad Sci USA. 2005;102:9583–9588. [PubMed]
116. Kaufmann DE, et al. Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction. Nat Immunol. 2007;8:1246–1254. [PubMed]
117. Cecchinato V, et al. Immune activation driven by CTLA-4 blockade augments viral replication at mucosal sites in simian immunodeficiency virus infection. J Immunol. 2008;180:5439–5447. [PMC free article] [PubMed]
118. El-Far M, et al. Down-regulation of CTLA-4 by HIV-1 Nef Protein. PLoS ONE. 2013;8:e54295. [PMC free article] [PubMed]
119. Linsley PS, Bradshaw J, Greene J, Peach R, Bennett KL, Mittler RS. Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement. Immunity. 1996;4:535–543. [PubMed]
120. Yokosuka T, et al. Spatiotemporal basis of CTLA-4 costimulatory molecule-mediated negative regulation of T cell activation. Immunity. 2010;33:326–339. [PubMed]
121. Saito T, Yokosuka T, Hashimoto-Tane A. Dynamic regulation of T cell activation and co-stimulation through TCR-microclusters. FEBS Lett. 2010;584:4865–4871. [PubMed]
122. Schneider H, et al. Reversal of the TCR stop signal by CTLA-4. Science. 2006;313:1972–1975. [PubMed]
123. Schneider H, Smith X, Liu H, Bismuth G, Rudd CE. CTLA-4 disrupts ZAP70 microcluster formation with reduced T cell/APC dwell times and calcium mobilization. Eur J Immunol. 2008;38:40–47. [PubMed]
124. Blanchard N, di Bartolo V, Hivroz C. In the immune synapse, ZAP-70 controls T cell polarization and recruitment of signaling proteins but not formation of the synaptic pattern. Immunity. 2002;17:389–399. [PubMed]
125. Gomez TS, et al. HS1 functions as an essential actin-regulatory adpator protein at the immune synapse. Immunity. 2006;24:741–752. [PMC free article] [PubMed]
126. Sasahara Y, et al. Mechanism of recruitment of WASP to the immunological synapse and its activation following TCR ligation. Mol Cell. 2002;10:1269–1281. [PubMed]
127. Das V, et al. Activation-induced polarized recycling targets T cell antigen receptors to the immunological synapse; involvement of SNARE complexes. Immunity. 2004;20:577–588. [PubMed]
128. Freeman GJ, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192:1027–1034. [PMC free article] [PubMed]
129. Kinter AL, et al. The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands. J Immunol. 2008;181:6738–6746. [PubMed]
130. Golden-Mason L, Palmer B, Klarquist J, Mengshol JA, Castelblanco N, Rosen HR. Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction. J Virol. 2007;81:9249–9258. [PMC free article] [PubMed]
131. Boni C, et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. J Virol. 2007;81:4215–4225. [PMC free article] [PubMed]
132. Trautmann L, et al. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med. 2006;12:1198–1202. [PubMed]
133. Day CL, et al. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature. 2006;443:350–354. [PubMed]
134. Petrovas C, et al. PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J Exp Med. 2006;203:2281–2292. [PMC free article] [PubMed]
135. Hatano H, et al. Cell-based measures of viral persistence are associated with immune activation and programmed cell death protein 1 (PD-1)-expressing CD4+ T cells. J Infect Dis. 2012 doi: 10.1093/infdis/jis630. [PMC free article] [PubMed]
136. Da Fonesca S. Purging the HIV-1 Reservoir Through the Disruption of the PD-1 Pathway. Suppl 3. Vol. 13. J Int AIDS Soc: 2010. p. O15.
137. Sheppard KA, et al. PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta. FEBS Lett. 2004;574:37–41. [PubMed]
138. Saunders PA, Hendrycks VR, Lidinsky WA, Woods ML. PD-L2:PD-1 involvement in T cell proliferation, cytokine production, and integrin-mediated adhesion. Eur J Immunol. 2005;35:3561–3569. [PubMed]
139. Parry RV, et al. CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol Cell Biol. 2005;25:9543–9553. [PMC free article] [PubMed]
140. Yokosuka T, Takamatsu M, Kobayashi-Imanishi W, Hashimoto-Tane A, Azuma M, Saito T. Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2. J Exp Med. 2012;209:1201–1217. [PMC free article] [PubMed]
141. Hannier S, Triebel F. The MHC class II ligand lymphocyte activation gene-3 is co-distributed with CD8 and CD3-TCR molecules after their engagement by mAb or peptide-MHC class I complexes. Int Immunol. 1999;11:1745–1752. [PubMed]
142. Wu TH, Zhen Y, Zeng C, Yi HF, Zhao Y. B and T lymphocyte attenuator interacts with CD3zeta and inhibits tyrosine phosphorylation of TCRzeta complex during T-cell activation. Immunol Cell Biol. 2007;85:590–595. [PubMed]
143. Youngblood B, et al. Chronic virus infection enforces demethylation of the locus that encodes PD-1 in antigen-specific CD8(+) T cells. Immunity. 2011;35:400–412. [PMC free article] [PubMed]
144. Martin N, Welsch S, Jolly C, Briggs JA, Vaux D, Sattentau QJ. Virological synapse-mediated spread of human immunodeficiency virus type 1 between T cells is sensitive to entry inhibition. J Virol. 2010;84:3516–3527. [PMC free article] [PubMed]
145. Chen P, Hubner W, Spinelli MA, Chen BK. Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Env-dependent neutralization-resistant virological synapses. J Virol. 2007;81:12582–12595. [PMC free article] [PubMed]
146. Jolly C, Kashefi K, Hollinshead M, Sattentau QJ. HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse. J Exp Med. 2004;199:283–293. [PMC free article] [PubMed]
147. Dustin ML. Cell adhesion molecules and actin cytoskeleton at immune synapses and kinapses. Curr Opin Cell Biol. 2007;19:529–533. [PMC free article] [PubMed]
148. Vasiliver-Shamis G, Cho MW, Hioe CE, Dustin ML. Human immunodeficiency virus type 1 envelope gp120-induced partial T-cell receptor signaling creates an F-actin-depleted zone in the virological synapse. J Virol. 2009;83:11341–11355. [PMC free article] [PubMed]
149. Vasiliver-Shamis G, et al. Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells. J Virol. 2008;82:9445–9457. [PMC free article] [PubMed]
150. Campi G, Varma R, Dustin ML. Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling. J Exp Med. 2005;202:1031–1036. [PMC free article] [PubMed]
151. Blanco J, Bosch B, Fernandez-Figueras MT, Barretina J, Clotet B, Este JA. High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells. J Biol Chem. 2004;279:51305–51314. [PubMed]
152. Vasiliver-Shamis G, Dustin ML, Hioe CE. HIV-1 virological synapse is not simply a copycat of the immunological synapse. Virus. 2010;2:1239–1260. [PMC free article] [PubMed]
153. Monel B, Beaumont E, Vendrame D, Schwartz O, Brand D, Mammano F. HIV cell-to-cell transmission requires the production of infectious virus particles and does not proceed through env-mediated fusion pores. J Virol. 2012;86:3924–3933. [PMC free article] [PubMed]
154. Hubner W, et al. Quantitative 3D video microscopy of HIV transfer across T cell virological synapses. Science. 2009;323:1743–1747. [PMC free article] [PubMed]
155. Rudnicka D, et al. Simultaneous cell-to-cell transmission of human immunodeficiency virus to multiple targets through polysynapses. J Virol. 2009;83:6234–6246. [PMC free article] [PubMed]
156. Jolly C, Sattentau QJ. Human immunodeficiency virus type 1 assembly, budding, and cell-cell spread in T cells take place in tetraspanin-enriched plasma membrane domains. J Virol. 2007;81:7873–7884. [PMC free article] [PubMed]
157. Jolly C, Sattentau QJ. Human immunodeficiency virus type 1 virological synapse formation in T cells requires lipid raft integrity. J Virol. 2005;79:12088–12094. [PMC free article] [PubMed]
158. Jolly C, Mitar I, Sattentau QJ. Adhesion molecule interactions facilitate human immunodeficiency virus type 1-induced virological synapse formation between T cells. J Virol. 2007;81:13916–13921. [PMC free article] [PubMed]
159. Arthos J, et al. HIV-1 envelope protein binds to and signals through integrin alpah4beta7, the gut mucosal homing receptor for peripheral T cells. Nat Immunol. 2008;9:301–309. [PubMed]
160. Y B, Tremblay MJ. Susceptibility of HIV type 1 to the fusion inhibitor T-20 is reduced on insertion of host intercellular adhesion molecule 1 in the virus membrane. J Infect Dis. 2004;190:894–902. [PubMed]
161. Korin YD, Zack JA. Progression to the G1b phase of the cell cycle is required for completion of human immunodeficiency virus type 1 reverse transcription in T cells. J Virol. 1998;72:3161–3168. [PMC free article] [PubMed]
162. Rousso I, Mixon MB, Chen BK, Kim PS. Palmitoylation of the HIV-1 envelope glycoprotein is critical for viral infectivity. Proc Natl Acad Sci USA. 2000;97:13523–13525. [PubMed]
163. Ono A, Freed EO. Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc Natl Acad Sci USA. 2001;98:13925–13930. [PubMed]
164. Nguyen DH, Hildreth JE. Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. J Virol. 2000;74:3264–3272. [PMC free article] [PubMed]
165. Lindwasser OW, Resh MD. Multimerization of human immunodeficiency virus type 1 Gag promotes its localization to barges, raft-like membrane microdomains. J Virol. 2001;75:7913–7924. [PMC free article] [PubMed]
166. Holm K, Weclewicz K, Hewson R, Suomalainen M. Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag) associates with membrane domains that are largely resistant to Brij98 but sensitive to Triton X-100. J Virol. 2003;77:4805–4817. [PMC free article] [PubMed]
167. Ding L, Derdowski A, Wang JJ, Spearman P. Independent segregation of human immunodeficiency virus type 1 Gag protein complexes and lipid rafts. J Virol. 2003;77:1916–1926. [PMC free article] [PubMed]
168. Campbell SM, Crowe SM, Mak J. Lipid rafts and HIV-1: from viral entry to assembly of progeny virions. J Clin Virol. 2001;22:217–227. [PubMed]
169. Bhattacharya J, Peters PJ, Clapham PR. Human immunodeficiency virus type 1 envelope glycoproteins that lack cytoplasmic domain cysteines: impact on association with membrane lipid rafts and incorporation onto budding virus particles. J Virol. 2004;78:5500–5506. [PMC free article] [PubMed]
170. Jolly C, Mitar I, Sattentau QJ. Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1. J Virol. 2007;81:5547–5560. [PMC free article] [PubMed]
171. Jimenez-Baranda S, et al. Filamin-A regulates actin-dependent clustering of HIV receptorts. Nat Cell Biol. 2007;9:838–846. [PubMed]
172. Iyengar S, Hildreth JE, Schwartz DH. Actin-dependent receptor colocalization required for human immunodeficiency virus entry into host cells. J Virol. 1998;72:5251–5255. [PMC free article] [PubMed]
173. Barrero-Villar M, et al. Moesin is required for HIV-1-induced CD4-CXCR4 interaction, F-actin redistribution, membrane fusion and viral infection in lymphocytes. J Cell Sci. 2009;122:103–113. [PubMed]
174. Yoder A, et al. HIV envelope-CXCR4 signaling activates cofilin to overcome cortical actin restriction in resting CD4 T cells. Cell. 2008;134:782–792. [PMC free article] [PubMed]
175. Campbell EM, Nunez R, Hope TJ. Disruption of the actin cytoskeleton can complement the ability of Nef to enhance human immunodeficiency virus type 1 infectivity. J Virol. 2004;78:5745–5755. [PMC free article] [PubMed]
176. Sourisseau M, Sol-Foulon N, Porrot F, Blanchet F, Schwartz O. Inefficient human immunodeficiency virus replication in mobile lymphocytes. J Virol. 2007;81:1000–1012. [PMC free article] [PubMed]
177. Dimitrov DS, Willey RL, Sato H, Chang LJ, Blumenthal R, Martin MA. Quantitation of human immunodeficiency virus type 1 infection kinetics. J Virol. 1993;67:2182–2190. [PMC free article] [PubMed]
178. Carr JM, Hocking H, Li P, Burrell CJ. Rapid and efficient cell-to-cell transmission of human immunodeficiency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes. Virology. 1999;265:319–329. [PubMed]
179. Jung A, et al. Recombination: multiply infected spleen cells in HIV patients. Nature. 2002;418:144. [PubMed]
180. Del Portillo A, Tripodi J, Najfeld V, Wodarz D, Levy DN, Chen BK. Multiploid inheritance of HIV-1 during cell-to-cell infection. J Virol. 2011;85:7169–7176. [PMC free article] [PubMed]
181. Sherer NM, Lehmann MJ, Jimenez-Soto LF, Horensavitz C, Pypaert M, Mothes W. Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission. Nat Cell Biol. 2007;9:310–315. [PMC free article] [PubMed]
182. Jin J, Sherer NM, Heidecker G, Derse D, Mothes W. Assembly of the murine leukemia virus is directed towards sites of cell-cell contact. PLoS Biol. 2009;7:e1000163. [PMC free article] [PubMed]
183. Josefsson L, et al. Majority of CD4+ T cells from peripheral blood of HIV-1-infected individuals contain only one HIV DNA molecule. Proc Natl Acad Sci USA. 2011;108:11199–11204. [PubMed]
184. Murooka TT, et al. HIV-infected T cells are migratory vehicles for viral dissemination. Nature. 2012;490:283–287. [PMC free article] [PubMed]
185. Mazurov D, Ilinskaya A, Heidecker G, Lloyd P, Derse D. Quantitative comparison of HTLV-1 and HIV-1 cell-to-cell infection with new replication dependent vectors. PLoS Pathog. 2010;6:e1000788. [PMC free article] [PubMed]
186. Gordon-Alonso M, et al. Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion. J Immunol. 2006;177:5129–5137. [PubMed]
187. Weng J, Krementsov DN, Khurana S, Roy NH, Thali M. Formation of syncytia is repressed by tetraspanins in human immunodeficiency virus type 1-producing cells. J Virol. 2009;83:7467–7474. [PMC free article] [PubMed]
188. Krementsov DN, Weng J, Lambele M, Roy NH, Thali M. Tetraspanins regulate cell-to-cell transmission of HIV-1. Retrovirology. 2009;6:64. [PMC free article] [PubMed]
189. Zhang Z, et al. Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science. 1999;286:1353–1357. [PubMed]
190. Bukrinsky MI, Stanwick TL, Dempsey MP, Stevenson M. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science. 1991;254:423–427. [PubMed]
191. Durham ND, et al. Neutralization resistance of virological synapse-mediated HIV-1 Infection is regulated by the gp41 cytoplasmic tail. J Virol. 2012;86:7484–7495. [PMC free article] [PubMed]
192. Abela IA, et al. Cell-cell transmission enables HIV-1 to evade inhibition by potent CD4bs directed antibodies. PLoS Pathog. 2012;8:e1002634. [PMC free article] [PubMed]
193. Gattinoni L, Klebanoff CA, Restifo NP. Paths to stemness: building the ultimate antitumour T cell. Nat Rev Cancer. 2012;12:671–684. [PubMed]
194. Gattinoni L, et al. A human memory T cell subset with stem cell-like properties. Nat Med. 2011;17:1290–1297. [PMC free article] [PubMed]
195. Lugli E, et al. Superior T memory stem cell persistence supports long-lived T cell memory. J Clin Invest. 2013;123:594–599. [PMC free article] [PubMed]
196. Lian I, et al. The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes Dev. 2010;24:1106–1118. [PubMed]
197. Thaventhiran JE, et al. Activation of the Hippo pathway by CTLA-4 regulates the expression of Blimp-1 in the CD8+ T cell. Proc Natl Acad Sci USA. 2012;109:E2223–E2229. [PubMed]
198. Konsavage WM, Jr, Kyler SL, Rennoll SA, Jin G, Yochum GS. Wnt/beta-catenin signaling regulates Yes-associated protein (YAP) gene expression in colorectal carcinoma cells. J Biol Chem. 2012;287:11730–11739. [PubMed]
199. Barry ER, et al. Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature. 2013;493:106–110. [PMC free article] [PubMed]
200. Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 1998;12:2048–2060. [PubMed]
201. Matsuda T, et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J. 1999;18:4261–4269. [PubMed]
202. Siegel AM, et al. A critical role for STAT3 transcription factor signaling in the development and maintenance of human T cell memory. Immunity. 2011;35:806–818. [PMC free article] [PubMed]
203. Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med. 2004;10:55–63. [PubMed]
204. Wray J, et al. Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation. Nat Cell Biol. 2011;13:838–845. [PMC free article] [PubMed]
205. Yi F, et al. Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal. Nat Cell Biol. 2011;13:762–770. [PMC free article] [PubMed]
206. ten Berge DKD, et al. Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells. Nat Cell Biol. 2011;13:1070–1075. [PubMed]
207. Holland JD, Klaus A, Garratt AN, Birchmeier W. Wnt signaling in stem and cancer stem cells. Curr Opin Cell Biol. 2013;25:254–264. [PubMed]
208. Kim W, Kim M, Jho E. Wnt/β-catenin signalling: from plasma membrane to nucleus. Biochem J. 2013;450:9–21. [PubMed]
209. Cain CJ, Manilay JO. Hematopoietic stem cell fate decisions are regulated by Wnt antagonists: comparisons and current controversies. Exp Hematol. 2013;41:3–16. [PubMed]
210. Seifert JR, Mlodzik M. Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility. Nat Rev Genet. 2007;8:126–138. [PubMed]
211. Veeman MTAJ, Moon RT. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell. 2003;5:367–377. [PubMed]
212. Shah KV, Chien AJ, Yee C, Moon RT. CTLA-4 is a direct target of Wnt/beta-catenin signaling and is expressed in human melanoma tumors. J Invest Dermatol. 2008;128:2870–2879. [PMC free article] [PubMed]
213. Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012;149:1192–1205. [PubMed]
214. Li Vivian SW, et al. Wnt signaling through inhibition of β-catenin degradation in an intact Axin1 complex. Cell. 2012;149:1245–1256. [PubMed]
215. Sadot E, Simcha I, Shtutman M, Ben-Ze'ev A, Geiger B. Inhibition of b-catenin-mediated transactivation by cadherin derivatives. Proc Natl Acad Sci USA. 1998;95:15339–15344. [PubMed]
216. Simcha I, et al. Differential nuclear translocation and transactivation potential of b-catenin and plakoglobin. J Cell Biol. 1998;141:1433–1448. [PMC free article] [PubMed]
217. Zhao DM, et al. Constitutive activation of Wnt signaling favors generation of memory CD8 T cells. J Immunol. 2010;184:1191–1199. [PMC free article] [PubMed]
218. Jeannet G, Boudousquie C, Gardiol N, Kang J, Huelsken J, Held W. Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8 T cell memory. Proc Natl Acad Sci USA. 2010;107:9777–9782. [PubMed]
219. Zhou X, Yu S, Zhao DM, Harty JT, Badovinac VP, Xue HH. Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1. Immunity. 2010;33:229–240. [PMC free article] [PubMed]
220. Koch U, Lehal R, Radtke F. Stem cells living with a Notch. Development. 2013;140:689–704. [PubMed]
221. Gentle ME, Rose A, Bugeon L, Dallman MJ. Noncanonical Notch signaling modulates cytokine responses of dendritic cells to inflammatory stimuli. J Immunol. 2012;189:1274–1284. [PMC free article] [PubMed]
222. Andersson ER, Sandberg R, Lendahl U. Notch signaling: simplicity in design, versatility in function. Development. 2011;138:3593–3612. [PubMed]
223. Radtke F, Fasnacht N, Macdonald HR. Notch signaling in the immune system. Immunity. 2010;32:14–27. [PubMed]
224. Shimojo H, Ohtsuka T, Kageyama R. Oscillations in notch signaling regulate maintenance of neural progenitors. Neuron. 2008;58:52–64. [PubMed]
225. Mathieu M, Cotta-Grand N, Daudelin J-F, Thébault P, Labrecque N. Notch signaling regulates PD-1 expression during CD8+ T-cell activation. Immunol Cell Biol. 2012;91:82–88. [PubMed]
226. Ninov N, Borius M, Stainier DYR. Different levels of Notch signaling regulate quiescence, renewal and differentiation in pancreatic endocrine progenitors. Development. 2012;139:1557–1567. [PubMed]
227. Perdigoto CN, Schweisguth F, Bardin AJ. Distinct levels of Notch activity for commitment and terminal differentiation of stem cells in the adult fly intestine. Development. 2011;138:4585–4595. [PubMed]
228. Mazzone M, et al. Dose-dependent induction of distinct phenotypic responses to Notch pathway activation in mammary epithelial cells. Proc Natl Acad Sci USA. 2010;107:5012–5017. [PubMed]
229. Helbig C, et al. Notch controls the magnitude of T helper cell responses by promoting cellular longevity. Proc Natl Acad Sci USA. 2012;109:9041–9046. [PubMed]
230. Estrach S, Ambler CA, Lo Celso C, Hozumi K, Watt FM. Jagged 1 is a beta-catenin target gene required for ectopic hair follicle formation in adult epidermis. Development. 2006;133:4427–4438. [PubMed]
231. Corada M, et al. The Wnt/beta-catenin pathway modulates vascular remodeling and specification by upregulating Dll4/Notch signaling. Dev Cell. 2010;18:938–949. [PubMed]
232. Ungerbäck JEN, Grünberg J, Sigvardsson M, Söderkvist P. The Notch-2 gene is regulated by Wnt signaling in cultured colorectal cancer cells. PLoS ONE. 2011;6:e17957. [PMC free article] [PubMed]
233. Axelrod JDMK, Artavanis-Tsakonas S, Perrimon N. Interaction between Wingless and Notch signaling pathways mediated by Dishevelled. Science. 1996;271:1826–1832. [PubMed]
234. Hayward P, Balayo T, Martinez Arias A. Notch synergizes with axin to regulate the activity of armadillo in Drosophila. Dev Dyn. 2006;235:2656–2666. [PubMed]
235. Munoz-Descalzo S, Tkocz K, Balayo T, Arias AM. Modulation of the ligand-independent traffic of Notch by Axin and Apc contributes to the activation of Armadillo in Drosophila. Development. 2011;138:1501–1506. [PubMed]
236. Espinosa L, Ingles-Esteve J, Aguilera C, Bigas A. Phosphorylation by glycogen synthase kinase-3 beta down-regulates Notch activity, a link for Notch and Wnt pathways. J Biol Chem. 2003;278:32227–32235. [PubMed]
237. Foltz DRSM, Berechid BE, Nye JS. Glycogen synthase kinase-3beta modulates notch signaling and stability. Curr Biol. 2002;12:1006–1011. [PubMed]
238. Soriano SKD, Fu M, Pestell R, Chevallier N, Zheng H, Koo EH. Presenilin 1 negatively regulates beta-catenin/T cell factor/lymphoid enhancer factor-1 signaling independently of beta-amyloid precursor protein and notch processing. J Cell Biol. 2001;152:785–794. [PMC free article] [PubMed]
239. Kopan R, Goate A. A common enzyme connects Notch signaling and Alzheimer's disease. Genes Dev. 2000;14:2799–2806. [PubMed]
240. Chang JT, et al. Asymmetric T lymphocyte division in the initiation of adaptive immune responses. Science. 2007;315:1687–1691. [PubMed]
241. Oliaro J, et al. Asymmetric cell division of T cells upon antigen presentation uses multiple conserved mechanisms. J Immunol. 2010;185:367–375. [PubMed]
242. Dong Z, Yang N, Yeo S-Y, Chitnis A, Guo S. Intralineage directional notch signaling regulates self-renewal and differentiation of asymmetrically dividing radial glia. Neuron. 2012;74:65–78. [PMC free article] [PubMed]
243. Tyagi M, Karn J. CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency. EMBO J. 2007;26:4985–4995. [PubMed]
244. Kumar A, Zloza A, Moon RT, Watts J, Tenorio AR, Al-Harthi L. Active beta-catenin signaling is an inhibitory pathway for human immunodeficiency virus replication in peripheral blood mononuclear cells. J Virol. 2008;82:2813–2820. [PMC free article] [PubMed]
245. Narasipura SD, Henderson LJ, Fu SW, Chen L, Kashanchi F, Al-Harthi L. Role of beta-catenin and TCF/LEF family members in transcriptional activity of HIV in astrocytes. J Virol. 2012;86:1911–1921. [PMC free article] [PubMed]
246. Henderson LJ, Narasipura SD, Adarichev V, Kashanchi F, Al-Harthi L. Identification of novel T cell factor 4 (TCF-4) binding sites on the HIV long terminal repeat which associate with TCF-4, beta-catenin, and SMAR1 to repress HIV transcription. J Virol. 2012;86:9495–9503. [PMC free article] [PubMed]
247. Wortman B, Darbinian N, Sawaya BE, Khalili K, Amini S. Evidence for regulation of long terminal repeat transcription by Wnt transcription factor TCF-4 in human astrocytic cells. J Virol. 2002;76:11159–11165. [PMC free article] [PubMed]
248. Li W, Henderson LJ, Major EO, Al-Harthi L. IFN-gamma mediates enhancement of HIV replication in astrocytes by inducing an antagonist of the beta-catenin pathway (DKK1) in a STAT 3-dependent manner. J Immunol. 2011;186:6771–6778. [PMC free article] [PubMed]
249. Goulas S, Conder R, Knoblich JA. The Par complex and integrins direct asymmetric cell division in adult intestinal stem cells. Cell Stem Cell. 2012;11:529–540. [PMC free article] [PubMed]
250. Knoblich JA. Mechanisms of asymmetric stem cell division. Cell. 2008;132:583–597. [PubMed]
251. Chang JT, et al. Asymmetric proteasome segregation as a mechanism for unequal partitioning of the transcription factor T-bet during T lymphocyte division. Immunity. 2011;34:492–504. [PMC free article] [PubMed]
252. Ciocca ML, Barnett BE, Burkhardt JK, Chang JT, Reiner SL. Cutting edge: asymmetric memory T cell division in response to rechallenge. J Immunol. 2012;188:4145–4148. [PMC free article] [PubMed]
253. Rong L, Perelson AS. Asymmetric division of activated latently infected cells may explain the decay kinetics of the HIV-1 latent reservoir and intermittent viral blips. Math Biosci. 2009;217:77–87. [PMC free article] [PubMed]
254. Palaga TML, Golde TE, Osborne BA. TCR-mediated Notch signaling regulates proliferation and IFN-gamma production in peripheral T cells. J Immunol. 2003;171:3019–3024. [PubMed]
255. Adler SHCE, et al. Notch signaling augments T cell responsiveness by enhancing CD25 expression. J Immunol. 2003;171:2896–2903. [PubMed]
256. Habib SJ, et al. A localized Wnt signal orients asymmetric stem cell division in vitro. Science. 2013;339:1445–1448. [PubMed]

Articles from Wiley-Blackwell Online Open are provided here courtesy of Wiley-Blackwell, John Wiley & Sons