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1.  Distinct modes of antigen presentation promote the formation, differentiation and activity of Foxp3+ regulatory T cells in vivo 
How the formation and activity of CD4+Foxp3+ regulatory T cells (Tregs) is shaped by TCR recognition of the diverse array of peptide:MHC complexes that can be generated from self- and/or foreign Ags in vivo remains poorly understood. We show that a self-peptide with low (but not high) stimulatory potency promotes thymic Treg formation, and can induce conventional CD4+ T cells in the periphery to become Tregs that express different levels of the transcription factor Helios according to anatomical location. When Tregs generated in response to this self-peptide subsequently encountered the same peptide derived instead from influenza virus in the lung-draining lymph nodes of infected mice, they proliferated, acquired a T-bet+CXCR3+ phenotype, and suppressed the antiviral effector T cell response in the lungs. However, these self-Ag-selected Tregs were unable to suppress the anti-viral immune response based on recognition of the peptide as a self-Ag rather than a viral-Ag. Notably, when expressed in a more immunostimulatory form, the self-peptide inhibited the formation of T-bet+CXCR3+ Tregs in response to viral-Ag, and Ag-expressing B cells from these mice induced Treg division without upregulation of CXCR3. These studies show that a weakly immunostimulatory self-peptide can induce thymic and peripheral Foxp3+ Treg formation but is unable to activate self-Ag-selected Tregs to modulate an anti-viral immune response. Moreover, a strongly immunostimulatory self-peptide expressed by B cells induced Tregs to proliferate without acquiring an effector phenotype that allows trafficking from the draining lymph node to the lungs, and thereby prevented the Tregs from suppressing the anti-viral immune response.
PMCID: PMC4390510  PMID: 25780041
2.  Regulatory cells in health and disease 
Immunological reviews  2014;259(1):5-10.
PMCID: PMC4017240  PMID: 24712455
3.  The role of T-cell receptor recognition of peptide:MHC complexes in the formation and activity of Foxp3+ regulatory T cells 
Immunological reviews  2014;259(1):11-22.
Foxp3+ regulatory T (Treg) cells are required to prevent the immune system from spontaneously mounting a severe autoaggressive lymphoproliferative disease and can modulate immune responses in a variety of settings, including infections. In this review, we describe studies that use transgenic mice to determine how signals through the T-cell receptor (TCR) contribute to the development, differentiation and activity of Treg cells in in vivo settings. By varying the amount and quality of the self-peptide recognized by an autoreactive TCR, we have shown that the interplay between autoreactive thymocyte deletion and Treg cell formation leads to a Treg cell repertoire that is biased toward low abundance agonist self-peptides. In an autoimmune disease setting, we have demonstrated that diverse TCR specificities can be required in order for Treg cells to prevent disease in a mouse model of autoimmune inflammatory arthritis. Lastly, we have shown that Treg cells initially selected based on specificity for a self-peptide can be activated by TCR recognition of a viral peptide, and that they can acquire a specialized phenotype and suppress anti-viral effector cell activity at the site of infection. These studies provide insights into the pivotal role that TCR specificity plays in the formation and activity of Treg cells.
PMCID: PMC4034456  PMID: 24712456
autoimmunity; tolerance; transgenic mice; thymus; viral
4.  The Degree of CD4+ T Cell Autoreactivity Determines Cellular Pathways Underlying Inflammatory Arthritis 
Although therapies targeting distinct cellular pathways (e.g. anti-cytokine versus anti-B cell therapy) have been found to be an effective strategy for at least some patients with inflammatory arthritis, the mechanisms that determine which pathways promote arthritis development are poorly understood. We have used a transgenic mouse model to examine how variations in the CD4+ T cell response to a surrogate self-peptide can affect the cellular pathways that are required for arthritis development. CD4+ T cells that are highly reactive with the self-peptide induce inflammatory arthritis that affects male and female mice equally. Arthritis develops by a B cell-independent mechanism, although it can be suppressed by an anti-TNF treatment, which prevented the accumulation of effector CD4+ Th17 cells in the joints of treated mice. By contrast, arthritis develops with a significant female bias in the context of a more weakly autoreactive CD4+ T cell response, and B cells play a prominent role in disease pathogenesis. In this setting of lower CD4+ T cell autoreactivity, B cells promote the formation of autoreactive CD4+ effector T cells (including Th17 cells), and IL-17 is required for arthritis development. These studies show that the degree of CD4+ T cell reactivity for a self-peptide can play a prominent role in determining whether distinct cellular pathways can be targeted to prevent the development of inflammatory arthritis.
PMCID: PMC3974175  PMID: 24591372
5.  Strength of TCR signal from self-peptide modulates autoreactive thymocyte deletion and Foxp3+ regulatory T cell formation 
European journal of immunology  2013;44(3):785-793.
We have analyzed the relationship between CD4+CD8− (CD4SP) thymocyte deletion and CD4SPFoxp3+ Treg formation using mice in which signals transmitted by the TCR have been attenuated by mutation of one of three tyrosine residues (Y145) of the adapter protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76). In mice containing polyclonal TCR repertoires, the Y145 mutation caused increased frequencies of CD4SPFoxp3+ thymocytes. CD4SP thymocytes expressing TCR Vβ-chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies in the context of the Y145 mutation, particularly amongst CD4SPFoxp3+ thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg formation in response to a defined self-peptide, the Y145 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3+ Treg formation still occurred, albeit with a reduced efficiency, and the Y145 mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg formation.
PMCID: PMC3959276  PMID: 24307208
Immune regulation; Tolerance; Thymic selection
6.  Pneumolysin Expression by Streptococcus pneumoniae Protects Colonized Mice from Influenza Virus-induced Disease 
Virology  2014;0:254-265.
The response to influenza virus (IAV) infection and severity of disease is highly variable in humans. We hypothesized that one factor contributing to this variability is the presence of specific respiratory tract (RT) microbes. One such microbe is Streptococcus pneumoniae (Sp) that is carried asymptomatically in the RT of many humans. In a mouse co-infection model we found that in contrast to secondary bacterial infection that exacerbates disease, Sp colonization 10 days prior to IAV protects from virus-induced morbidity and lung pathology. Using mutant Sp strains, we identified a critical role for the bacterial virulence factor pneumolysin (PLY) in mediating this protection. Colonization with the PLY-sufficient Sp strain induces expression of the immune-suppressive enzyme arginase 1 in alveolar macrophages (aMø) and correlates with attenuated recruitment and function of pulmonary inflammatory cells. Our study demonstrates a novel role for PLY in Sp-mediated protection by maintaining aMø as "gatekeepers" against virus-induced immunopathology.
PMCID: PMC4157663  PMID: 24999050
influenza virus; Streptococcus pneumoniae; pneumolysin; respiratory tract co-infection; immunopathology; alveolar macrophages; inflammatory monocytes; arginase I; iNOS; protection
7.  Autoreactive Th1 cells activate monocytes to support regional Th17 responses in inflammatory arthritis 
We have examined mechanisms underlying the formation of pathologic Th17 cells using a transgenic mouse model in which autoreactive CD4+ T cells recognize influenza virus hemagglutinin (HA) as a ubiquitously expressed self-antigen, and induce inflammatory arthritis. The lymph nodes of arthritic mice contain elevated numbers of inflammatory monocytes (iMO) with an enhanced capacity to promote CD4+ Th17 cell differentiation, and a regional inflammatory response develops in the paw-draining lymph nodes by an IL-17-dependent mechanism. The activation of these Th17-trophic iMO precedes arthritis development, and occurs in the context of an autoreactive CD4+ Th1 cell response. Adoptive transfer of HA-specific CD4+ T cells into non-arthritic mice expressing HA as a self-antigen similarly led to the formation of Th1 cells and of iMO that could support Th17 cell formation, and notably, the accumulation of these iMO in the lymph nodes was blocked by IFN-γ neutralization. These studies show that autoreactive CD4+ Th1 cells directed to a systemically distributed self-antigen can promote the development of a regional Th17 cell inflammatory response by driving the recruitment of Th17-trophic iMO to the lymph nodes.
PMCID: PMC3608750  PMID: 23420889
8.  Coinfection with Streptococcus pneumoniae Modulates the B Cell Response to Influenza Virus 
Journal of Virology  2014;88(20):11995-12005.
Pathogen-specific antibodies (Abs) protect against respiratory infection with influenza A virus (IAV) and Streptococcus pneumoniae and are the basis of effective vaccines. Sequential or overlapping coinfections with both pathogens are common, yet the impact of coinfection on the generation and maintenance of Ab responses is largely unknown. We report here that the B cell response to IAV is altered in mice coinfected with IAV and S. pneumoniae and that this response differs, depending on the order of pathogen exposure. In mice exposed to S. pneumoniae prior to IAV, the initial virus-specific germinal center (GC) B cell response is significantly enhanced in the lung-draining mediastinal lymph node and spleen, and there is an increase in CD4+ T follicular helper (TFH) cell numbers. In contrast, secondary S. pneumoniae infection exaggerates early antiviral antibody-secreting cell formation, and at later times, levels of GCs, TFH cells, and antiviral serum IgG are elevated. Mice exposed to S. pneumoniae prior to IAV do not maintain the initially robust GC response in secondary lymphoid organs and exhibit reduced antiviral serum IgG with diminished virus neutralization activity a month after infection. Our data suggest that the history of pathogen exposures can critically affect the generation of protective antiviral Abs and may partially explain the differential susceptibility to and disease outcomes from IAV infection in humans.
IMPORTANCE Respiratory tract coinfections, specifically those involving influenza A viruses and Streptococcus pneumoniae, remain a top global health burden. We sought to determine how S. pneumoniae coinfection modulates the B cell immune response to influenza virus since antibodies are key mediators of protection.
PMCID: PMC4178749  PMID: 25100838
9.  Requirement for diverse TCR specificities determines regulatory T cell activity in a mouse model of autoimmune arthritis1 
CD4+CD25+Foxp3+ regulatory T cells (Tregs) are required to restrain the immune system from mounting an autoaggressive systemic inflammatory response, but why their activity can prevent (or allow) organ-specific autoimmunity remains poorly understood. We have examined how TCR specificity contributes to Treg activity using a mouse model of spontaneous autoimmune arthritis, in which CD4+ T cells expressing a clonotypic TCR induce disease by an IL-17-dependent mechanism. Administration of polyclonal Tregs suppressed Th17 cell formation and prevented arthritis development; notably, Tregs expressing the clonotypic TCR did not. These clonotypic Tregs exerted antigen-specific suppression of effector CD4+ T cells using the clonotypic TCR in vivo, but failed to mediate bystander suppression and did not prevent Th17 cells using nonclonotypic TCRs from accumulating in joint-draining lymph nodes of arthritic mice. These studies indicate that the availability of Tregs with diverse TCR specificities can be crucial to their activity in autoimmune arthritis.
PMCID: PMC3331886  PMID: 22450809
10.  LIMD2 Is a Small LIM-Only Protein Overexpressed in Metastatic Lesions That Regulates Cell Motility and Tumor Progression by Directly Binding to and Activating the Integrin-Linked Kinase 
Cancer research  2014;74(5):1390-1403.
Proteins that communicate signals from the cytoskeleton to the nucleus are prime targets for effectors of metastasis as they often transduce signals regulating adhesion, motility, and invasiveness. LIM domain proteins shuttle between the cytoplasm and the nucleus, and bind to partners in both compartments, often coupling changes in gene expression to extracellular cues. In this work, we characterize LIMD2, a mechanistically undefined LIM-only protein originally found to be overexpressed in metastatic lesions but absent in the matched primary tumor. LIMD2 levels in fresh and archival tumors positively correlate with cell motility, metastatic potential, and grade, including bladder, melanoma, breast, and thyroid tumors. LIMD2 directly contributes to these cellular phenotypes as shown by overexpression, knockdown, and reconstitution experiments in cell culture models. The solution structure of LIMD2 that was determined using nuclear magnetic resonance revealed a classic LIM-domain structure that was highly related to LIM1 of PINCH1, a core component of the integrin-linked kinase–parvin–pinch complex. Structural and biochemical analyses revealed that LIMD2 bound directly to the kinase domain of integrin-linked kinase (ILK) near the active site and strongly activated ILK kinase activity. Cells that were null for ILK failed to respond to the induction of invasion by LIMD2. This strongly suggests that LIMD2 potentiates its biologic effects through direct interactions with ILK, a signal transduction pathway firmly linked to cell motility and invasion. In summary, LIMD2 is a new component of the signal transduction cascade that links integrin-mediated signaling to cell motility/metastatic behavior and may be a promising target for controlling tumor spread.
PMCID: PMC4183205  PMID: 24590809
11.  CD4+CD25+ regulatory T cells in autoimmune arthritis 
Immunological reviews  2010;233(1):97-111.
CD4+CD25+ regulatory T (Treg) cells can play a critical role in the prevention of autoimmunity, as evidenced by the cataclysmic autoimmune disease that develops in mice and humans lacking the key transcription factor forkhead box protein 3 (Foxp3). At present, however, how and whether Treg cells participate in the development of rheumatoid arthritis (RA), which has both systemic manifestations and a joint-targeted pathology that characterizes the disease, remains unclear. In this review, we describe work that has been carried out aimed at determining the role of Treg cells in disease development in RA patients and in mouse models of inflammatory arthritis. We also describe studies in a new model of spontaneous autoimmune arthritis (TS1×HACII mice), in which disease is caused by CD4+ T cells recognizing a neo-self-antigen expressed by systemically distributed antigen-presenting cells. We show that TS1×HACII mice develop arthritis despite the presence of CD4+CD25+Foxp3+ Treg cells that recognize this target autoantigen, and we outline steps in the development of arthritis at which Treg cells might potentially act, or fail to act, in the development of inflammatory arthritis.
PMCID: PMC2908310  PMID: 20192995
autoimmunity; tolerance/suppression/anergy; transgenic/knockout mice
12.  Thymocyte deletion can bias regulatory T cell formation toward low abundance self-peptide 
European journal of immunology  2009;39(12):3301-3306.
Autoreactive CD4+ T cells can undergo deletion and/or become CD25+Foxp3+ Treg cells as they develop intrathymically, but how these alternative developmental fates are specified based on interactions with self-peptide(s) is not understood. We show here that thymocytes expressing an autoreactive TCR can be subjected to varying degrees of deletion that correlate with the amount of self-peptide. Strikingly, among thymocytes that evade deletion, similar proportions acquire Foxp3 expression. These findings provide evidence that Foxp3+ Treg cells can develop among members of a cohort of autoreactive thymocytes that have evaded deletion by a self-peptide, and that deletion and Treg cell formation can act together to bias the Treg cell repertoire toward low abundance self-peptide(s).
PMCID: PMC2829603  PMID: 19768697
Tolerance; Thymic selection; Transgenic models
13.  Generation of Adaptive Regulatory T Cells by Alloantigen Is Required for Some But Not All Transplant Tolerance Protocols 
Transplantation  2011;91(7):707-713.
Because CD4+CD25+Foxp3+ regulatory T cells (Tregs) are essential for the maintenance of self-tolerance, significant interest surrounds the developmental cues for thymic-derived natural Tregs (nTregs) and periphery-generated adaptive Tregs (aTregs). In the transplant setting, the allograft may play a role in the generation of alloantigen-specific Tregs, but this role remains undefined. We examined whether the immune response to a transplant allograft results in the peripheral generation of aTregs.
To identify generation of aTregs, purified graft-reactive CD4+CD25− T cells were adoptively transferred to mice-bearing skin allograft. To demonstrate that aTregs are necessary for tolerance, DBA/2 skin was transplanted onto C57BL/6-RAG-1-deficient recipients adoptively transferred with purified sorted CD4+CD25− T cells; half of the recipients undergo tolerance induction treatment.
By tracking adoptively transferred cells, we show that purified graft-reactive CD4+CD25− T lymphocytes up-regulate Foxp3 in mice receiving skin allografts in the absence of any treatment. Interestingly, cotransfer of antigen-specific nTregs suppresses the up-regulation of Foxp3 by inhibiting the proliferation of allograft-responsive T cells. In vitro data are consistent with our in vivo data—Foxp3+ cells are generated on antigen activation, and this generation is suppressed on coculture with antigen-specific nTregs. Finally, blocking aTreg generation in grafted, rapamycin-treated mice disrupts alloantigen-specific tolerance induction. In contrast, blocking aTreg generation in grafted mice treated with nondepleting anti-CD4 plus anti-CD40L antibodies does not disrupt graft tolerance.
We conclude that graft alloantigen stimulates the de novo generation of aTregs, and this generation may represent a necessary step in some but not all protocols of tolerance induction.
PMCID: PMC3727173  PMID: 21386770
Treg; Adaptive Tregs; Tolerance
14.  Flow cytometric profiling of mature and developing regulatory T cells in the thymus 
Natural Regulatory T (Treg) cells are a subset of CD4+ T cells characterized by expression of the transcription factor Foxp3 and the ability to suppress immune responses. Treg cells develop in the thymus in response to highly specific interactions between the T cell receptor (TCR) and self-antigens. These processes can be recapitulated in antigen-specific systems using transgenic mice that co-express a TCR with its cognate peptide as a neo-self antigen. Here we describe a method for using such a system to establish a flow cytometric profile of phenotype markers expressed by developing and mature Treg cells in the thymus. Our approach is to compare antigen-specific thymocytes developing in the presence or absence of Treg cell-selecting ligands to identify phenotypic changes that characterize thymocytes undergoing selection into the Treg cell lineage.
PMCID: PMC3576838  PMID: 21287329
Thymocyte; Foxp3; Immune regulation; Treg progenitor cell; immunophenotyping
15.  Pharmacologic Activation of the Innate Immune System to Prevent Respiratory Viral Infections 
Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-β and subsequent high-level induction of IFN-β–dependent proteins, such as myxovirus resistance 1 (Mx1) and 2′,5′-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-β system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and “first responders” in the early stages of viral pandemics or bioterror attacks.
PMCID: PMC3265219  PMID: 21148741
innate immunity; interferon; influenza; pneumonia; bronchial epithelium
16.  Vaccination with M2e-Based Multiple Antigenic Peptides: Characterization of the B Cell Response and Protection Efficacy in Inbred and Outbred Mice 
PLoS ONE  2011;6(12):e28445.
The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains.
Methodology/Principal Findings
Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs.
Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population.
PMCID: PMC3236751  PMID: 22180783
17.  Protective antiviral antibody responses in a mouse model of influenza virus infection require TACI 
The Journal of Clinical Investigation  2011;121(10):3954-3964.
Antiviral Abs, for example those produced in response to influenza virus infection, are critical for virus neutralization and defense against secondary infection. While the half-life of Abs is short, Ab titers can last a lifetime due to a subset of the Ab-secreting cells (ASCs) that is long lived. However, the mechanisms governing ASC longevity are poorly understood. Here, we have identified a critical role for extrinsic cytokine signals in the survival of respiratory tract ASCs in a mouse model of influenza infection. Irradiation of mice at various time points after influenza virus infection markedly diminished numbers of lung ASCs, suggesting that they are short-lived and require extrinsic factors in order to persist. Neutralization of the TNF superfamily cytokines B lymphocyte stimulator (BLyS; also known as BAFF) and a proliferation-inducing ligand (APRIL) reduced numbers of antiviral ASCs in the lungs and bone marrow, whereas ASCs in the spleen and lung-draining lymph node were surprisingly unaffected. Mice deficient in transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a receptor for BLyS and APRIL, mounted an initial antiviral B cell response similar to that generated in WT mice but failed to sustain protective Ab titers in the airways and serum, leading to increased susceptibility to secondary viral challenge. These studies highlight the importance of TACI signaling for the maintenance of ASCs and protection against influenza virus infection.
PMCID: PMC3195469  PMID: 21881204
18.  ICOS expression by effector T cells influences the ability of regulatory T cells to inhibit anti-chromatin B cell responses in recipient mice 
Journal of autoimmunity  2009;34(4):460-468.
T regulatory cells are critical for the prevention of autoimmunity. Specifically, Treg cells can control anti-chromatin antibody production in vivo, and this correlates with decreased ICOS expression on CD4+ T helper cells. Here we test the significance of high ICOS expression by T effector cells, firstly in terms of the anti-chromatin B cell response, and secondly on the ability of Treg cells to suppress T cell help. We bred CD4+ T cell receptor transgenic mice with mice that carry the Roquinsan/san mutation. The Roquin gene functions to limit ICOS mRNA such that CD4 T cells from mutant mice express elevated ICOS. Using an in vivo model, TS1.Roquinsan/san Th cells were compared with wild-type TS1 Th cells with regard to their ability to help anti-chromatin B cells in the presence or absence of Treg cells. Both TS1 and TS1.Roquinsan/san Th cells induced anti-chromatin IgMa antibodies, but the TS1.Roquinsan/san Th cells resulted in the recovery of more class-switched and germinal center B cells. Neither source of Th cells were capable of inducing long-lived autoantibodies. Treg cells completely suppressed anti-chromatin IgMa antibody production and reduced anti-chromatin B cell recovery induced by TS1 Th cells. Importantly, this suppression was less effective when TS1.Roquinsan/san Th cells were used. Thus, high ICOS levels on effector T cells results in autoimmunity by augmenting the autoreactive B cell response and by dampening the effect of Treg cell suppression.
PMCID: PMC2859982  PMID: 20022728
regulatory T cells; autoreactive B cells; ICOS expression
19.  Efficient help for autoreactive B cell activation requires CD4+ T cell recognition of an agonist peptide at the effector stage 
European journal of immunology  2009;39(9):2377-2382.
T cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4+ T cell priming, and subsequent T-B cell cognate interactions, on antibody production by anti-chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4+ T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self-peptide during CD4+ T-B cell interaction can be a critical determinant in restraining anti-chromatin autoantibody production.
PMCID: PMC3015147  PMID: 19662636
T cells; B cells; antibodies; autoimmunity
20.  GITR blockade facilitates T-reg mediated allograft survival 
Transplantation  2009;88(10):1169-1177.
Many models of transplant tolerance have been found to depend upon the induction of regulatory T cells (T-regs). Innate immune signals are known to down-regulate T-regs thereby augmenting immunity by abrogating regulatory T cell function. Such signals may also provide a barrier to transplantation tolerance mediated by T-regs. A number of cell surface molecules expressed by T-regs have been found to inhibit T-reg activity, the best characterized of which is the glucocorticoid-induced TNF receptor-related protein (GITR).
Using an adoptive transfer model of allograft rejection we can study the effects of inflammation and antigen-specific T-regs on graft survival. Inflammation resulting from the transplant procedure counter-regulates the suppressor activity of T-regs. To assess whether T-reg activity could be enhanced by blocking GITR signaling we compared the capacity of T-regs to prolong the survival of grafts in the presence or absence of AITRL-Fc, a novel construct that binds GITR.
We report that interruption of GITR-GITRL binding by AITRL-Fc resulted in long-term T-reg-dependent acceptance of skin grafts in the setting of innate immune signals that otherwise interfere with T-reg activity.
Inflammation and other innate immune signals may activate antigen presenting cells (APC) to upregulate GITRL. GITR-GITRL interaction is one pathway by which APCs may enhance the adaptive response to foreign antigen by counter-regulating T-regs and by costimulating effector T cells. By blocking this interaction with AITRL-Fc, one can sustain the benefit conferred by graft-protective T-regs.
PMCID: PMC2803064  PMID: 19935370
21.  Regulatory T cell counter-regulation by innate immunity is a barrier to transplantation tolerance 
Innate immune signals foster adaptive immunity through activation of antigen presenting cells. Recent in vitro evidence suggests that innate signaling may also contribute to immunity by countering the effects of regulatory T cells (T-regs), counter-regulation. We present in vivo evidence using a transgenic skin allograft model that the function of T-regs is lost in the setting of acute skin transplantation but remains intact when grafts were transplanted one month prior to allow surgery-induced inflammation to abate. Our findings identify T-reg counter-regulation as a naturally occurring process that accompanies transplantation and an important barrier to T-reg mediated tolerance. Our finding further highlights the central role of regulatory cell deactivation in the initiation of the immune response.
PMCID: PMC2796697  PMID: 19845585
T-reg; inflammation; transplant; tolerance; allograft
22.  Blockade of GITR–GITRL interaction maintains Treg function to prolong allograft survival 
European journal of immunology  2010;40(5):1369-1374.
Involvement of Treg in transplant tolerance has been demonstrated in multiple models. During the active process of graft rejection, these regulatory cells are themselves regulated and inactivated, a process termed counter-regulation. We hypothesize that ligation of the costimulatory molecule glucocorticoid-induced TNF receptor-related protein (GITR) on Treg inhibits their ability to promote graft survival, and by blocking GITR ligation graft survival can be prolonged. To this aim, we have designed a soluble GITR fusion protein (GITR-Fc), which binds GITR ligand and inhibits activation of GITR. Here, we show that GITR-Fc prolonged mouse skin graft survival, and this prolongation is dependent on Treg. In a full MHC-mismatched skin graft setting, GITR-Fc significantly improved graft survival when used in combination with MR1, anti-CD40L, while GITR-Fc alone did not demonstrate graft prolongation. These results demonstrate that disruption of binding of GITR with GITR ligand may be an important strategy in prolonging allograft survival.
PMCID: PMC2935584  PMID: 20148423
GITR; Inflammation; Treg; Tolerance; Transplant
23.  Inducible MHC class II expression by mast cells supports effector and regulatory T cell activation1 
In addition to their well-established role as regulators of allergic response, recent evidence supports a role for mast cells in influencing the outcome of physiologic and pathologic T cell responses. One mechanism by which mast cells influence T cell function is indirectly through secretion of various cytokines. It remains unclear, however, whether mast cells can directly activate T cells through antigen presentation, as the expression of MHC class II by mast cells has been controversial. In this report, we demonstrate that in vitro stimulation of mouse mast cells with LPS and IFNγ induces the expression of MHC class II and co-stimulatory molecules. Although freshly isolated peritoneal mast cells do not express MHC class II, an in vivo inflammatory stimulus increases numbers of MHC class II-positive mast cells in situ. Expression of MHC class II granted mast cells the ability to process and present antigens directly to T cells with preferential expansion of antigen-specific regulatory T cells over naive T cells. These data support the notion that, in the appropriate setting, mast cells may regulate T cell responses through the direct presentation of antigen.
PMCID: PMC2922395  PMID: 19342644
Mast cells; T cells; antigen presentation/processing; MHC
24.  A direct comparison of rejection by CD8 and CD4 T cells in a transgenic model of allotransplantation 
The relative contributions of CD4+ and CD8+ T cells to transplant rejection remain unknown. The authors integrated a previous model of CD4-mediated graft rejection with a complementary model of CD8-mediated rejection to directly compare the function of graft-reactive CD4+ and CD8+ lymphocytes in vivo in a model where rejection requires transgenic T cells. These studies allow direct comparison of CD4 and CD8 T cell responses to the same antigen without the confounding effects of T cell depletion or homeostatic proliferation.
Materials and Methods:
Clone 4 and TS1 mice possess MHC class I- and II-restricted CD8+ and CD4+ T cells, respectively, which express transgenic T cell receptors that recognize the influenza hemagglutinin antigen (HA). We compared the in vivo response of CFSE-labeled, HA-specific transgenic CD8+ and CD4+ T cells after adoptive transfer into syngeneic BALB/c mice grafted with HA-expressing skin.
As in the authors’ CD4+ model, HA104 skin was consistently rejected by both Clone 4 mice (n=9, MST: 14.2) and by 5×105 Clone 4 lymphocytes transferred to naive BALB/c hosts that do not otherwise reject HA+ grafts. Rejection correlated with extensive proliferation of either graft-reactive T cell subset in the draining lymph nodes, and antigen-specific CD4+ and CD8+ cells acquired effector function and proliferated with similar kinetics.
These data extend the authors’ unique transgenic transplantation model to the investigation of CD8 T cell function. The initial results confirm fundamental functional similarity between the CD4 and CD8 T cell subsets and provide insight into the considerable redundancy underlying T cell mechanisms mediating allograft rejection.
PMCID: PMC2766493  PMID: 18512028
CD4; CD8; proliferation; differentiation; transplantation; rejection
25.  Autoantibody production in lpr/lpr gld/gld mice reflects accumulation of CD4+ effector cells that are resistant to regulatory T cell activity1 
Journal of autoimmunity  2008;31(2):98-109.
In Fas/FasL deficient mice anti-chromatin Ab production is T cell dependent and is not apparent until after 10 weeks of age. Early control of anti-chromatin antibodies may be due to the counterbalancing influence of Treg cells. Here we show that Treg cells block lpr/lpr gld/gld Th cells from providing help to anti-chromatin B cells in an in vivo transfer system. Interestingly, the percentage and absolute numbers of Foxp3+ Treg cells is elevated in BALB/c-lpr/lpr gld/gld mice and increases with age compared to BALB/c mice. The majority of Foxp3 expression is found in the B220− CD4+ T cell population, and Foxp3-expressing cells are localized in the splenic PALS (periarteriolar lymphocyte sheath). Strikingly, although the lack of functional Fas/FasL does not affect the ability of Treg cells to block Th cell proliferation, Treg cells can block the IFN-γ differentiation of Th cells from BALB/c or young BALB-lpr/lpr gld/gld mice but not of pre-existing Th1 cells from older BALB/c-lpr/lpr gld/gld mice. Thus, we suggest autoantibody production is not caused by the lack of Treg cells but by a defect in activation-induced cell death that leads to the accumulation of T effector cells that are resistant to regulatory T cell activity.
PMCID: PMC2585757  PMID: 18539433
autoantibody; Fas/FasL; IFN-γ; T regulatory

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