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1.  The transforming growth factor beta signaling pathway is critical for the formation of CD4 T follicular helper cells and isotype-switched antibody responses in the lung mucosa 
eLife  null;4:e04851.
T follicular helper cells (Tfh) are crucial for the initiation and maintenance of germinal center (GC) reactions and high affinity, isotype-switched antibody responses. In this study, we demonstrate that direct TGF-β signaling to CD4 T cells is important for the formation of influenza-specific Tfh cells, GC reactions, and development of isotype-switched, flu-specific antibody responses. Early during infection, TGF-β signaling suppressed the expression of the high affinity IL-2 receptor α chain (CD25) on virus-specific CD4 T cells, which tempered IL-2 signaling and STAT5 and mammalian target of rapamycin (mTOR) activation in Tfh precursor CD4 T cells. Inhibition of mTOR allowed for the differentiation of Tfh cells in the absence of TGF-βR signaling, suggesting that TGF-β insulates Tfh progenitor cells from IL-2-delivered mTOR signals, thereby promoting Tfh differentiation during acute viral infection. These findings identify a new pathway critical for the generation of Tfh cells and humoral responses during respiratory viral infections.
eLife digest
The influenza virus is thought to cause illness in up to 10% of adults and 30% of children each year worldwide. Most of these cases resolve on their own and don’t require treatment, but three to five million people are hospitalized and up to half a million people die each year.
Unfortunately, the vaccines currently available to protect against influenza only target particular varieties or “strains” of the virus. The strains that circulate vary from year-to-year so it is necessary to develop new influenza vaccines every year. However, it is difficult to correctly predict which strains will circulate, so a more effective solution would be to develop a new vaccine that can help the body defend itself against many, or ideally any influenza strain.
During a viral infection, a type of immune cell in the host can specialize into two different types of cells to help fight the virus: T helper 1 cells and CD4 T follicular helper cells. T helper 1 cells help to kill host cells that have become infected. CD4 T follicular helper cells promote the production of proteins called antibodies, which identify and neutralize the virus.
Here, Marshall et al. studied how T helper 1 cells and CD4 T follicular helper cells form in mice suffering from a lung infection similar to influenza. It was already known that a protein called transforming growth factor beta (TGF-β) helps the immune response to mount an effective defense against an infection without causing too much harm to the host. Marshall et al. show that TGF-β increases the number of CD4 T follicular helper cells in the mice by suppressing the production of another protein—called IL-2—on the surface of CD4 T cells. Treating mice lacking the ability to detect TGF-β with a drug that blocks a protein controlled by IL-2 also allows more CD4 T follicular helper cells to be produced.
Marshall et al.’s findings reveal that TGF-β is involved in controlling the balance of T helper 1 cells and CD4 T follicular helper cells produced during viral infections of the respiratory tract. Since TGF-β also has other roles in immune responses against viruses, it is now an attractive target for the development of a vaccine that may protect us against all strains of the influenza virus.
PMCID: PMC4337607  PMID: 25569154
T follicular helper cells; viral infection; antibody; TGF-beta; cytokines; mouse
2.  CD28 expression is required after T cell priming for helper T cell responses and protective immunity to infection 
eLife  2014;3:e03180.
The co-stimulatory molecule CD28 is essential for activation of helper T cells. Despite this critical role, it is not known whether CD28 has functions in maintaining T cell responses following activation. To determine the role for CD28 after T cell priming, we generated a strain of mice where CD28 is removed from CD4+ T cells after priming. We show that continued CD28 expression is important for effector CD4+ T cells following infection; maintained CD28 is required for the expansion of T helper type 1 cells, and for the differentiation and maintenance of T follicular helper cells during viral infection. Persistent CD28 is also required for clearance of the bacterium Citrobacter rodentium from the gastrointestinal tract. Together, this study demonstrates that CD28 persistence is required for helper T cell polarization in response to infection, describing a novel function for CD28 that is distinct from its role in T cell priming.
eLife digest
Invasion by a bacterium or virus typically activates a mammalian host's immune system to eliminate the pathogen. The cells of the so-called ‘innate immune system’ are the body's first line of defense against infection, and these cells patrol the organs and tissues in an effort to locate and eliminate pathogens quickly. The innate immune response is rapid and non-specific, but often cannot completely clear an infection. When necessary, innate immune cells will escalate the immune response by activating the second branch of the immune system, called the ‘adaptive immune system’. This specifically targets and eradicates an invading pathogen.
T cells are essential components of the adaptive immune system, and these cells can be readily distinguished from other types of cell by proteins called T cell receptors (or TCRs) found on their surface. There are also different types of T cell, each with a specific function. T helper cells, for example, help other adaptive immune cells to mature and activate, which involves these immune cells proliferating and developing into more specialized cells.
For a T cell to activate, two events must occur at the same time. First, the TCR must recognize and bind to a fragment of the pathogen that is presented to it by an innate immune cell. And second, ‘co-stimulatory molecules’ present on the surfaces of both the T cell and the same innate immune cell must interact. Using these two signals to activate a T cell helps to ensure the adaptive immune response is not ‘unleashed‘ unnecessarily.
Co-stimulatory molecules have become popular targets for therapies aimed at treating autoimmune disorders—where the immune system attacks and destroys the body's own tissues. One of the most well studied co-stimulatory molecules expressed by T cells is called CD28; however, it remained unknown whether CD28 is involved in any processes after T cell activation.
Now, Linterman et al. reveal that the CD28 co-stimulatory molecule plays a number of roles in addition to T cell activation. For example, a newly developed mouse model showed that CD28 must remain on the surface of T helper cells after they have been activated for these cells to effectively specialize. Linterman et al. also discovered that CD28 helps different T helper cell subtypes to develop.
Linterman et al. demonstrate that CD28 is critical throughout a host's response to infection, and suggest that if CD28 is lost on activated T cells (which happens during aging, HIV infection and autoimmune diseases) the responses of T helper cells become limited. Furthermore, these findings reveal that treatments that target the CD28 co-stimulatory molecule will also affect on-going immune responses.
PMCID: PMC4241536  PMID: 25347065
CD28; helper T cells; infection; mouse
3.  Loss of Circulating CD4 T Cells with B Cell Helper Function during Chronic HIV Infection 
PLoS Pathogens  2014;10(1):e1003853.
The interaction between follicular T helper cells (TFH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7highCXCR5highCCR6highPD-1high CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory TFH cells.
Author Summary
Follicular T helper cells (TFH) interact with B cells within germinal centers of lymphoid tissue to promote the survival, isotype switching and generation of high affinity memory B cells and plasma cells. Recently, a population of circulating CD4 T cells that shares phenotypic and functional characteristics with TFH cells, named peripheral TFH cells, has been identified. The relationship between peripheral TFH cells in the blood and TFH cells within the lymphoid tissue remains unclear, and whether or not peripheral TFH cells can provide insight into T cell and B cell dynamics within lymphoid tissue during infection or vaccination is not understood. Here we characterize peripheral TFH cells and show that unlike TFH cells, peripheral TFH cells secrete a diverse array of cytokines and decrease, rather than increase, during chronic HIV infection. Furthermore, we did not observe a relationship between peripheral TFH cells and memory B cells, or with the production of neutralizing antibodies to HIV. Overall, our data indicate that while peripheral TFH cells share some characteristics with TFH cells, they may not represent a good surrogate to study T cell and B cell dynamics within lymphoid tissue.
PMCID: PMC3911819  PMID: 24497824
4.  Phenotype, effector function, and tissue localization of PD-1-expressing human follicular helper T cell subsets 
BMC Immunology  2011;12:53.
It is well established that PD-1 is expressed by follicular T cells but its function in regulation of human T helper cells has been unclear. We investigated the expression modality and function of PD-1 expressed by human T cells specialized in helping B cells.
We found that PD-1-expressing T cells are heterogeneous in PD-1 expression. We identified three different PD-1-expressing memory T cell subsets (i.e. PD-1low (+), PD-1medium (++), and PD-1high (+++) cells). PD-1+++ T cells expressed CXCR5 and CXCR4 and were localized in the rim of germinal centers. PD-1+ or PD-1++ cells expressed CCR7 and were present mainly in the T cell area or other parts of the B cell follicles. Utilizing a novel antigen density-dependent magnetic sorting (ADD-MS) method, we isolated the three T cell subsets for functional characterization. The germinal center-located PD-1+++ T cells were most efficient in helping B cells and in producing IL-21 and CXCL13. Other PD-1-expressing T cells, enriched with Th1 and Th17 cells, were less efficient than PD-1+++ T cells in these capacities. PD-1+++ T cells highly expressed Ki-67 and therefore appear active in cell activation and proliferation in vivo. IL-2 is a cytokine important for proliferation and survival of the PD-1+++ T cells. In contrast, IL-21, while a major effector cytokine produced by the PD-1-expressing T helper cells, had no function in generation, survival, or proliferation of the PD-1-expressing helper T cells at least in vitro. PD-1 triggering has a suppressive effect on the proliferation and B cell-helping function of PD-1+++ germinal center T cells.
Our results revealed the phenotype and effector function of PD-1-expressing T helper cell subsets and indicate that PD-1 restrains the B cell-helping function of germinal center-localized T cells to prevent excessive antibody response.
PMCID: PMC3184275  PMID: 21914188
5.  Follicular Helper NKT Cells Induce Limited B Cell Responses and Germinal Center Formation in the Absence of CD4+ T Cell Help 
B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4+ T follicular helper (TFH) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4+ T cells (MHC II−/−) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II−/− mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II−/− mice display a typical CXCR5hiprogrammed death-1hiICOShiBcl-6hi TFH phenotype and induce germinal centers. The specific IgG response induced in MHC II−/− mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKTFH cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKTFH and CD4+ TFH cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.
PMCID: PMC3559029  PMID: 22379027
6.  Natural killer T cells and innate immune B cells from lupus-prone NZB/W mice interact to generate IgM and IgG autoantibodies 
European journal of immunology  2008;38(1):156-165.
Lupus-prone NZB/W F1 mice develop glomerulonephritis after T helper cell dependent isotype switching of autoantibody secretion from IgM to IgG at about six months of age. We compared innate immune natural killer (NK) T cells and conventional T cells for their capacity to help spontaneous in vitro immunoglobulin and autoantibody secretion of innate immune (B-1 and marginal zone) and conventional (follicular) B cell subsets from NZB/W F1 mice. We found that purified NKT cells not only increased spontaneous secretion of IgM and IgM anti-dsDNA antibodies by B-1 and marginal zone B cells, but also facilitated secretion of IgG anti-dsDNA antibodies predominantly by B-1 B cells. Little IgM or IgG anti-dsDNA antibodies was secreted by follicular B cells, and conventional T cells failed to provide potent helper activity to any B cell subset. All combinations of T and B cell subsets from normal C57BL/6 mice failed to generate vigorous IgM and IgG secretion. NZB/W NKT cell helper activity was blocked by anti-CD1 and anti-CD40L mAbs. In conclusion, direct interactions between innate immune T and B cells form a pathway for the development of IgM and IgG lupus autoantibody secretion in NZB/W mice.
PMCID: PMC2915938  PMID: 18050273
B cells; T cells; Systemic lupus erythematosus; Autoantibodies
7.  CD4 T-Cell Subsets in Malaria: TH1/TH2 Revisited 
CD4+ T-cells have been shown to play a central role in immune control of infection with Plasmodium parasites. At the erythrocytic stage of infection, IFN-γ production by CD4+ T-cells and CD4+ T-cell help for the B-cell response are required for control and elimination of infected red blood cells. CD4+ T-cells are also important for controlling Plasmodium pre-erythrocytic stages through the activation of parasite-specific CD8+ T-cells. However, excessive inflammatory responses triggered by the infection have been shown to drive pathology. Early classical experiments demonstrated a biphasic CD4+ T-cell response against erythrocytic stages in mice, in which T helper (Th)1 and antibody-helper CD4+ T-cells appear sequentially during a primary infection. While IFN-γ-producing Th1 cells do play a role in controlling acute infections, and they contribute to acute erythrocytic-stage pathology, it became apparent that a classical Th2 response producing IL-4 is not a critical feature of the CD4+ T-cell response during the chronic phase of infection. Rather, effective CD4+ T-cell help for B-cells, which can occur in the absence of IL-4, is required to control chronic parasitemia. IL-10, important to counterbalance inflammation and associated with protection from inflammatory-mediated severe malaria in both humans and experimental models, was originally considered be produced by CD4+ Th2 cells during infection. We review the interpretations of CD4+ T-cell responses during Plasmodium infection, proposed under the original Th1/Th2 paradigm, in light of more recent advances, including the identification of multifunctional T-cells such as Th1 cells co-expressing IFN-γ and IL-10, the identification of follicular helper T-cells (Tfh) as the predominant CD4+ T helper subset for B-cells, and the recognition of inherent plasticity in the fates of different CD4+ T-cells.
PMCID: PMC4290673  PMID: 25628621
malaria; Plasmodium; multifunctional CD4 T-cells; CD4 T-cell subsets; Tfh; Th1; Th2; Th22
8.  Germinal Center T Follicular Helper Cell IL-4 Production Is Dependent on Signaling Lymphocytic Activation Molecule Receptor (CD150) 
CD4 T cell help is critical for the generation and maintenance of germinal centers (GCs), and T follicular helper (TFH) cells are the CD4 T cell subset required for this process. Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP [SH2D1A]) expression in CD4 T cells is essential for GC development. However, SAP-deficient mice have only a moderate defect in TFH differentiation, as defined by common TFH surface markers. CXCR5+ TFH cells are found within the GC, as well as along the boundary regions of T/B cell zones. In this study, we show that GC-associated T follicular helper (GC TFH) cells can be identified by their coexpression of CXCR5 and the GL7 epitope, allowing for phenotypic and functional analysis of TFH and GC TFH populations. GC TFH cells are a functionally discrete subset of further polarized TFH cells, with enhanced B cell help capacity and a specialized ability to produce IL-4 in a TH2-independent manner. Strikingly, SAP-deficient mice have an absence of the GC TFH cell subset and SAP− TFH cells are defective in IL-4 and IL-21 production. We further demonstrate that SLAM (Slamf1, CD150), a surface receptor that uses SAP signaling, is specifically required for IL-4 production by GC TFH cells. GC TFH cells require IL-4 and -21 production for optimal help to B cells. These data illustrate complexities of SAP-dependent SLAM family receptor signaling, revealing a prominent role for SLAM receptor ligation in IL-4 production by GC CD4 T cells but not in TFH cell and GC TFH cell differentiation.
PMCID: PMC2913439  PMID: 20525889
9.  Memory T Follicular Helper CD4 T Cells 
T follicular helper (Tfh) cells are the subset of CD4 T helper cells that are required for generation and maintenance of germinal center reactions and the generation of long-lived humoral immunity. This specialized T helper subset provides help to cognate B cells via their expression of CD40 ligand, IL-21, IL-4, and other molecules. Tfh cells are characterized by their expression of the chemokine receptor CXCR5, expression of the transcriptional repressor Bcl6, and their capacity to migrate to the follicle and promote germinal center B cell responses. Until recently, it remained unclear whether Tfh cells differentiated into memory cells and whether they maintain Tfh commitment at the memory phase. This review will highlight several recent studies that support the idea of Tfh-committed CD4 T cells at the memory stage of the immune response. The implication of these findings is that memory Tfh cells retain their capacity to recall their Tfh-specific effector functions upon reactivation to provide help for B cell responses and play an important role in prime and boost vaccination or during recall responses to infection. The markers that are useful for distinguishing Tfh effector and memory cells, as well as the limitations of using these markers will be discussed. Tfh effector and memory generation, lineage maintenance, and plasticity relative to other T helper lineages (Th1, Th2, Th17, etc.) will also be discussed. Ongoing discoveries regarding the maintenance and lineage stability versus plasticity of memory Tfh cells will improve strategies that utilize CD4 T cell memory to modulate antibody responses during prime and boost vaccination.
PMCID: PMC4313784  PMID: 25699040
T follicular helper cells; memory T cells; Bcl6; CXCR5; helper T cells
10.  STAT5 is a potent negative regulator of TFH cell differentiation 
Interleukin 2, STAT5, and Blimp-1 work together to suppress differentiation of follicular helper T cells in mice.
Follicular helper T cells (TFH cells) constitute the CD4+ T cell subset that is specialized to provide help to germinal center (GC) B cells and, consequently, mediate the development of long-lived humoral immunity. TFH cell differentiation is driven by the transcription factor Bcl6, and recent studies have identified cytokine and cell–cell signals that drive Bcl6 expression. However, although TFH dysregulation is associated with several major autoimmune diseases, the mechanisms underlying the negative regulation of TFH cell differentiation are poorly understood. In this study, we show that STAT5 inhibits TFH cell differentiation and function. Constitutive STAT5 signaling in activated CD4+ T cells selectively blocked TFH cell differentiation and GCs, and IL-2 signaling was a primary inducer of this pathway. Conversely, STAT5-deficient CD4+ T cells (mature STAT5fl/fl CD4+ T cells transduced with a Cre-expressing vector) rapidly up-regulated Bcl6 expression and preferentially differentiated into TFH cells during T cell priming in vivo. STAT5 signaling failed to inhibit TFH cell differentiation in the absence of the transcription factor Blimp-1, a direct repressor of Bcl6 expression and TFH cell differentiation. These results demonstrate that IL-2, STAT5, and Blimp-1 collaborate to negatively regulate TFH cell differentiation.
PMCID: PMC3281266  PMID: 22271576
11.  Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production 
Follicular T helper cells are the major reservoir for HIV infection and accumulate during chronic HIV infection.
In the present study, we have investigated the distribution of HIV-specific and HIV-infected CD4 T cells within different populations of memory CD4 T cells isolated from lymph nodes of viremic HIV-infected subjects. Four memory CD4 T cell populations were identified on the basis of the expression of CXCR5, PD-1, and Bcl-6: CXCR5−PD-1−Bcl-6−, CXCR5+PD-1−Bcl-6−, CXCR5−PD-1+Bcl-6−, and CXCR5+PD-1+Bcl-6+. On the basis of Bcl-6 expression and functional properties (IL-21 production and B cell help), the CXCR5+PD-1+Bcl-6+ cell population was considered to correspond to the T follicular helper (Tfh) cell population. We show that Tfh and CXCR5−PD-1+ cell populations are enriched in HIV-specific CD4 T cells, and these populations are significantly increased in viremic HIV-infected subjects as compared with healthy subjects. The Tfh cell population contained the highest percentage of CD4 T cells harboring HIV DNA and was the most efficient in supporting productive infection in vitro. Replication competent HIV was also readily isolated from Tfh cells in subjects with nonprogressive infection and low viremia (<1,000 HIV RNA copies). However, only the percentage of Tfh cells correlated with the levels of plasma viremia. These results demonstrate that Tfh cells serve as the major CD4 T cell compartment for HIV infection, replication, and production.
PMCID: PMC3549706  PMID: 23254284
12.  Follicular helper T cells: implications in neoplastic hematopathology 
Seminars in Diagnostic Pathology  2011;28(3):202-213.
A distinct subset of T helper cells, named follicular T helper cells (TFH), has been recently described. TFH cells are characterized by their homing capacities in the germinal centres of B-cell follicles where they interact with B cells, supporting B-cell survival and antibody responses. TFH cells can be identified by the expression of several markers including the chemokine CXCL13, the co-stimulatory molecules PD1 and ICOS, and the transcription factor BCL6. They appear to be relevant markers for the diagnosis of angioimmunoblastic T-cell lymphoma (AITL) and have helped to recognize subsets of peripheral T-cell lymphoma, NOS with nodal or cutaneous presentation expressing TFH antigens that might be related to AITL. In B-cell neoplasms, TFH cells are present within the microenvironment of nodular lymphocyte-predominant Hodgkin lymphoma and follicular lymphoma where they likely support the growth of neoplastic germinal centre-derived B cells. Interestingly, the amount of PD1+ cells in the neoplastic follicles might have a favourable impact on the outcome of FL patients.
Altogether, the availability of antibodies directed to TFH -associated molecules has important diagnostic and prognostic implications in haematopathology. In addition, TFH cells could represent interesting targets in TFH-derived lymphomas such as AITL, or in some B-cell neoplasms where they act as part of the tumour microenvironment.
PMCID: PMC4019516  PMID: 21850986
Germinal Center; immunology; pathology; Humans; Lymphoma; immunology; metabolism; pathology; T-Lymphocyte Subsets; immunology; metabolism; pathology; T-Lymphocytes, Helper-Inducer; immunology; metabolism; pathology; follicular T helper cells (TFH), angioimmunoblastic T-cell lymphoma (AITL), TFH-derived lymphomas, nodular lymphocyte predominant Hodgkin lymphoma, follicular lymphoma
13.  Subspecialization of Cxcr5+ T Cells 
The Journal of Experimental Medicine  2001;193(12):1373-1382.
The T helper (Th) cell pool is composed of specialized cells with heterogeneous effector functions. Apart from Th1 and 2 cells, CXCR5+ T cells have been suggested to be another type of effector T cell specialized for B cell help. We show here that CXCR5+ T cells are heterogeneous, and we identify subsets of CXCR5+ CD4 T cells that differ in function and microenvironmental localization in secondary lymphoid tissues. CD57+CXCR5 T cells, hereafter termed germinal center Th (GC-Th) cells, are localized only in GCs, lack CCR7, and are highly responsive to the follicular chemokine B lymphocyte chemoattractant but not to the T cell zone EBI1-ligand chemokine. Importantly, GC-Th cells are much more efficient than CD57−CXCR5+ T cells or CXCR5− T cells in inducing antibody production from B cells. Consistent with their function, GC-Th cells produce elevated levels of interleukin 10 upon stimulation which, with other cytokines and costimulatory molecules, may help confer their B cell helper activity. Our results demonstrate that CXCR5+ T cells are functionally heterogeneous and that the GC-Th cells, a small subset of CXCR5+ T cells, are the key helpers for B cell differentiation and antibody production in lymphoid tissues.
PMCID: PMC2193300  PMID: 11413192
germinal center; chemokine receptor; CXCR5; B cell help; T helper cells
14.  GITR Intrinsically Sustains Early Type 1 and Late Follicular Helper CD4 T Cell Accumulation to Control a Chronic Viral Infection 
PLoS Pathogens  2015;11(1):e1004517.
CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during chronic infection remain incompletely defined. While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection. Here we show that during a persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13, mice lacking the glucocorticoid-induced tumor necrosis factor receptor related protein (GITR) exhibit defective CD8 T cell accumulation, increased T cell exhaustion and impaired viral control. Differences in CD8 T cells and viral control between GITR+/+ and GITR-/- mice were lost when CD4 T cells were depleted. Moreover, mixed bone marrow chimeric mice, as well as transfer of LCMV epitope-specific CD4 or CD8 T cells, demonstrated that these effects of GITR are largely CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IFNγ+IL-2+ Th1 cells, facilitating CD8 T cell expansion and early viral control. GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CD8 T cells to between days four and eight p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.
Author Summary
The natural rodent pathogen LCMV clone 13 causes a persistent viral infection in mice and has successfully predicted several immunological factors that are relevant to human chronic viral infection such as HIV. LCMV clone 13 infection is ultimately controlled by cell-mediated and humoral immune responses by day 60–90 post-infection in CD4 T cell-sufficient mice. While it has been known for several years that CD4 T cell help is critical for control of LCMV clone 13, research to date has been largely limited to the regulatory factors that contribute to late CD4 T cell dysfunction, with little knowledge of the role of T cell co-stimulatory factors in sustaining CD4 T cells to help cell-mediated and humoral immune responses. Using GITR-deficient mice, we show that the co-stimulatory molecule GITR plays a critical cell-intrinsic role in early CD4 T cell accumulation to support cytotoxic T cell responses and late LCMV-specific IgG production. The early effects of Th1 on CTL responses are IL-2-dependent. Mice lacking GITR have up to 35-fold higher viral burden relative to GITR-sufficient controls. Taken together, we demonstrate a critical cell-intrinsic role for GITR is sustaining CD4 T cell responses to control chronic LCMV infection. Thus, GITR on CD4 T cells may critically contribute to the initial viral set-point in infections such as HIV.
PMCID: PMC4295864  PMID: 25590581
15.  Viral persistence redirects CD4 T cell differentiation toward T follicular helper cells 
Persistent virus infection drives follicular T helper cell differentiation.
CD4 T cell responses are crucial to prevent and control viral infection; however, virus-specific CD4 T cell activity is considered to be rapidly lost during many persistent viral infections. This is largely caused by the fact that during viral persistence CD4 T cells do not produce the classical Th1 cytokines associated with control of acute viral infections. Considering that CD4 T cell help is critical for both CD8 T cell and B cell functions, it is unclear how CD4 T cells can lose responsiveness but continue to sustain long-term control of persistent viral replication. We now demonstrate that CD4 T cell function is not extinguished as a result of viral persistence. Instead, viral persistence and prolonged T cell receptor stimulation progressively redirects CD4 T cell development away from the Th1 response induced during an acute infection toward T follicular helper cells. Importantly, this sustained CD4 T cell functionality is critical to maintain immunity and ultimately aid in the control of persistent viral infection.
PMCID: PMC3092345  PMID: 21536743
16.  Molecular programming of B cell memory 
Nature reviews. Immunology  2011;12(1):24-34.
The development of high-affinity B cell memory is regulated through three separable phases, each involving antigen recognition by specific B cells and cognate T helper cells. Initially, antigen-primed B cells require cognate T cell help to gain entry into the germinal centre pathway to memory. Once in the germinal centre, B cells with variant B cell receptors must access antigens and present them to germinal centre T helper cells to enter long-lived memory B cell compartments. Following antigen recall, memory B cells require T cell help to proliferate and differentiate into plasma cells. A recent surge of information — resulting from dynamic B cell imaging in vivo and the elucidation of T follicular helper cell programmes — has reshaped the conceptual landscape surrounding the generation of memory B cells. In this Review, we integrate this new information about each phase of antigen-specific B cell development to describe the newly unravelled molecular dynamics of memory B cell programming.
PMCID: PMC3947622  PMID: 22158414
17.  Nasal Anti-CD3 Antibody Ameliorates Lupus by Inducing an IL-10-Secreting CD4+CD25-LAP+ Regulatory T Cell and Is Associated with Down-Regulation of IL-17+CD4+ICOS+CXCR5+ Follicular Helper T Cells1 
Lupus is an Ab-mediated autoimmune disease. One of the potential contributors to the development of systemic lupus erythematosus is a defect in naturally occurring CD4+CD25+ regulatory T cells. Thus, the generation of inducible regulatory T cells that can control autoantibody responses is a potential avenue for the treatment of systemic lupus erythematosus. We have found that nasal administration of anti-CD3 mAb attenuated lupus development as well as arrested ongoing lupus in two strains of lupus-prone mice. Nasal anti-CD3 induced a CD4+CD25-latency-associated peptide (LAP)+ regulatory T cell that secreted high levels of IL-10 and suppressed disease in vivo via IL-10- and TFG-β-dependent mechanisms. Disease suppression also occurred following adoptive transfer of CD4+CD25-LAP+ regulatory T cells from nasal anti-CD3-treated animals to lupus-prone mice. Animals treated with nasal anti-CD3 had less glomerulonephritis and diminished levels of autoantibodies as measured by both ELISA and autoantigen microarrays. Nasal anti-CD3 affected the function of CD4+ICOS+CXCR5+ follicular helper T cells that are required for autoantibody production. CD4+ICOS+CXCR5+ follicular helper T cells express high levels of IL-17 and IL-21 and these cytokines were down-regulated by nasal anti-CD3. Our results demonstrate that nasal anti-CD3 induces CD4+CD25-LAP+ regulatory T cells that suppress lupus in mice and that it is associated with down-regulation of T cell help for autoantibody production.
PMCID: PMC2753458  PMID: 18941193
18.  The effect of aging on the frequency, phenotype and cytokine production of human blood CD4 + CXCR5 + T follicular helper cells: comparison of aged and young subjects 
T cell-dependent B-cell responses decline with age, indicating declined cognate helper activity of aged CD4 + T cells for B cells. However, the mechanisms remain unclear. T follicular helper (Tfh) cells, a novel T helper subset, play an essential role in helping B cells differentiation into long-lived plasma cells in germinal center (GC) or short-lived plasma cells. In the present study, we proposed that there might existe changes of proportion, phenotype or cytokine production of blood Tfh cells in healthy elderly individuals compared with healthy young individuals.
The results showed that frequencies of aged blood CXCR5 + CD4 + Tfh cells increased compared with young subjects. Both aged and young blood CXCR5 + CD4 + Tfh cells constitutively expressed CD45RO, CCR7 and CD28, and few of these cells expressed CD69 or HLA-DR, which indicated that they were resting memory cells. There was no significant difference of IL-21 frequency production by aged blood CXCR5 + CD4 + Tfh determined by FACS compared with young individuals, however, aged PBMCs produced significantly higher levels of IL-21 evaluated by ELISA. Furthermore, there were no significant differences of percentages of IFN-γ, IL-4, IL-17 or IL-22 production by aged Tfh cells compared with their counterparts of young individuals respectively. However, frequencies of IL-17+ cells within aged CD4 + CXCR5-T cells were markedly lower than in the young individuals. Furthermore we observed different frequencies of IFN-γ, IL-17, IL-4 or IL-22 production by Tfh or by CD4 + CXCR5- cells in aged and young subjects respectively.
Our data demonstrated that the frequencies of blood memory CXCR5 + CD4 + Tfh cells increased in the elderly population. There were similar frequencies of Th characterized cytokine production such as IL-21, IFN-γ, IL-4, IL-17 or IL-22 in aged and young Tfh cells. However, aged PBMCs produced a significantly higher amount of IL-21 compare to young subjects.
PMCID: PMC4148677  PMID: 25177353
Tfh cells; Aging; IL-21
19.  Decreased Frequencies of Circulating Follicular Helper T Cell Counterparts and Plasmablasts in Ankylosing Spondylitis Patients Naïve for TNF Blockers 
PLoS ONE  2014;9(9):e107086.
Follicular helper T cells (Tfh), localized in lymphoid organs, promote B cell differentiation and function. Circulating CD4 T cells expressing CXCR5, ICOS and/or PD-1 are counterparts of Tfh. Three subpopulations of circulating CD4+CXCR5+ cells have been described: CXCR3+CCR6- (Tfh-Th1), CXCR3-CCR6+ (Tfh-Th17), and CXCR3-CCR6- (Tfh-Th2). Only Tfh-Th17 and Tfh-Th2 function as B cell helpers. Our objective was to study the frequencies of circulating Tfh (cTfh), cTfh subsets and plasmablasts (CD19+CD20-CD27+CD38high cells), and the function of cTfh cells, in patients with Ankylosing Spondylitis (AS). To this end, peripheral blood was drawn from healthy controls (HC) (n = 50), AS patients naïve for TNF blockers (AS/nb) (n = 25) and AS patients treated with TNF blockers (AS/b) (n = 25). The frequencies of cTfh and plasmablasts were determined by flow cytometry. Cocultures of magnetically sorted CD4+CXCR5+ T cells with autologous CD19+CD27- naïve B cells were established from 3 AS/nb patients and 3 HC, and concentrations of IgG, A and M were measured in supernatants. We obseved that AS/nb but not AS/b patients, demonstrated decreased frequencies of circulating CD4+CXCR5+ICOS+PD-1+ cells and plasmablasts, together with a decreased (Tfh-Th17+Tfh-Th2)/Tfh-Th1 ratio. The amounts of IgG and IgA produced in cocultures of CD4+CXCR5+ T cells with CD19+CD27- B cells of AS/nb patients were significantly lower than observed in cocultures established from HC. In summary, AS/nb but not AS/b patients, demonstrate a decreased frequency of cTfh and plasmablasts, and an underrepresentation of cTfh subsets bearing a B helper phenotype. In addition, peripheral blood CD4+CXCR5+ T cells of AS/nb patients showed a decreased capacity to help B cells ex vivo.
PMCID: PMC4159293  PMID: 25203742
20.  Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period 
Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation.
Follicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry.
The concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml).
Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively.
Our results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.
PMCID: PMC2990747  PMID: 21040564
21.  Multiple checkpoints keep follicular helper T cells under control to prevent autoimmunity 
Follicular helper T (Tfh) cells select mutated B cells in germinal centres, which can then differentiate into long-lived high affinity memory B cells and plasma cells. Tfh cells are regulated by a unique molecular programme orchestrated by the transcriptional repressor Bcl6. This transcription factor turns down expression of multiple genes, including transcriptional regulators of other T helper lineages and a vast amount of microRNAs. This enables Tfh cells to express a suite of chemokine receptors, stimulatory ligands and cytokines that enable migration into B-cell follicles, and provision of effective help to B cells. Not surprisingly, dysregulation of this powerful helper subset can lead to a range of autoantibody-mediated diseases; indeed, aberrant accumulation of Tfh cells has been linked with systemic lupus erythematosus, Sjogren's disease and autoimmune arthritis. Here we dissect multiple checkpoints that operate throughout Tfh cell development and maturation to maintain immunological tolerance while mounting robust and long-lasting antibody responses.
PMCID: PMC4002912  PMID: 20364160
autoimmunity; germinal centre; Tfh
22.  Modulation of SAP dependent T:B cell interactions as a strategy to improve vaccination 
Current opinion in virology  2013;3(3):363-370.
Generating long-term humoral immunity is a crucial component of successful vaccines and requires interactions between T cells and B cells in germinal centers (GC). In GCs, a specialized subset of CD4+ helper T cells, called T follicular helper cells (Tfh), provide help to B cells; this help directs the magnitude and quality of the antibody response. Tfh cell help influences B cell survival, proliferation, somatic hypermutation, class switch recombination, and differentiation. Sustained contact between Tfh cells and B cells is necessary for the provision of help to B cells. SAP (Signaling lymphocytic activation molecule (SLAM)-associated protein, encoded by Sh2d1a) regulates the duration of T:B cell interactions and is required for long-term humoral immunity in animal models and in humans. SAP binds to SLAM family receptors and mediates signaling that affects cell adhesion, cytokine secretion, and TCR signaling strength. Therefore, the modulation of SAP and SLAM family receptor expression represents a major axis by which the quality and duration of an antibody response is controlled after vaccination.
PMCID: PMC3763741  PMID: 23743125
23.  Pathogenesis of Renal Disease in Systemic Lupus Erythematosus—The Role of Autoantibodies and Lymphocytes Subset Abnormalities 
Lupus nephritis (LN) is a common and severe organ manifestation of systemic lupus erythematosus (SLE), and is associated with significant patient morbidity and mortality. Autoantibodies and aberrations in lymphocyte subsets have putative roles in the pathogenesis of SLE and LN, and might reflect disease activity and are amenable to immunosuppressive treatments. Anti-DNA is one of the well-studied autoantibodies, which correlates with disease activity and has direct nephritogenic effects on resident renal cells and various glomerular components. Other important autoantibodies in the pathogenesis of LN include anti-C1q, anti-α-actinin and anti-nucleosome antibodies. Changes in naive and memory B cells and plasma cells have been observed in SLE and LN patients. These B cell subsets exert diverse effects during pathogenesis of LN such as production of autoantibodies, secretion of proinflammatory and anti-inflammatory cytokines and presentation of auto-antigens to effector cells. Aberration of T lymphocytes, especially the T-helper subsets, is also highly pertinent in the development of LN. In this context, important T helper subsets include Th1, Th2, Th9, Th17, TReg and follicular T-helper cells. The growing knowledge on these autoantibodies and lymphocyte subset abnormalities will enhance our understanding of SLE and LN, and hence help devise better strategies for disease monitoring and treatment.
PMCID: PMC4425058  PMID: 25860947
autoantibodies; lupus nephritis; lymphocytes; pathogenesis; subsets
24.  Germinal centre alloantibody responses are mediated exclusively by indirect-pathway CD4 T follicular helper cells 
The durable alloantibody responses that develop in organ transplant patients indicate long-lived plasma cell output from T-dependent germinal centres (GCs), but which of the two pathways of CD4 T cell allorecognition are responsible for generating allospecific T follicular helper (TFH) cells remains unclear. This was addressed by reconstituting T-cell deficient mice with monoclonal populations of TCR-transgenic CD4 T cells that recognised alloantigen only as conformationally-intact protein (direct pathway) or only as self-restricted allopeptide (indirect pathway), and then assessing the alloantibody response to a heart graft.
Recipients reconstituted with indirect-pathway CD4 T cells developed long-lasting IgG alloantibody responses, with splenic GCs and allospecific bone marrow plasma cells readily detectable 50 days after heart transplantation. Differentiation of the transferred CD4 T cells into TFH cells was confirmed by follicular localisation and by acquisition of signature phenotype. In contrast, IgG alloantibody was not detectable in recipient mice reconstituted with direct-pathway CD4 T cells. Neither prolongation of the response by preventing NK cell killing of donor dendritic cells, nor prior immunisation to develop CD4 T cell memory altered the inability of the direct-pathway to provide allospecific B cell help.
CD4 T cell help for GC alloantibody responses is provided exclusively via the indirect-allorecognition pathway.
PMCID: PMC3378630  PMID: 22323543
25.  Suppression of murine SLE by oral anti-CD3 
Lupus  2009;18(7):586-596.
Lupus is an antibody-mediated autoimmune disease. The production of pathogenic, class switched and affinity maturated autoantibodies in lupus is dependent on T cell help. A potential mechanism of disease pathogenesis is a lack of control of pathogenic T helper cells by regulatory T cells in lupus. It has been repeatedly shown that the naturally occurring CD4+CD25+ regulatory T cells in lupus prone mice and patients with SLE are defective both in frequency and function. Thus, the generation of inducible regulatory T cells that can control T cell help for autoantibody production is a potential avenue for the treatment of SLE. We have found that oral administration of anti-CD3 monoclonal antibody attenuated lupus development and arrested on-going disease in lupus prone SNF1 mice. Oral anti-CD3 induces a CD4+CD25-LAP+ regulatory T cell that secrets high levels of TGF-β and suppresses in vitro in TFG-β-dependent fashion. Animals treated with oral anti-CD3 had less glomerulonephritis and diminished levels of anti-dsDNA autoantibodies. Oral anti-CD3 led to a downregulation of IL-17+CD4+ICOS-CXCR5+ follicular helper T cells, CD138+ plasma cells and CD73+ mature memory B cells. Our results show that oral anti-CD3 induces CD4+CD25-LAP+ regulatory T cells that suppress lupus in mice and is associated with down regulation of T cell help for autoantibody production.
PMCID: PMC2753460  PMID: 19433458
CD4+CD25-LAP+ regulatory T cells; IL-17+ follicular helper T cells; oral anti-CD3; spontaneous lupus

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