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.

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.

Summary

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.

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).

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.

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.

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.

BACKGROUND

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).

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.

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.

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.

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.

Introduction:

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.

Results:

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.

Conclusions:

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.

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.

CD4+25+ T cells are a unique population of immunoregulatory T cells which are critical for the prevention of autoimmunity. To address the thymic selection of these cells we have used two models of attenuated thymic deletion. In K14-Aβb mice, major histocompatibility complex (MHC) class II I-Ab expression is limited to thymic cortical epithelium and deletion by hematopoietic antigen-presenting cells does not occur. In H2-DMα–deficient mice, MHC class II molecules contain a limited array of self-peptides resulting in inefficient clonal deletion. We find that CD4+25+ T cells are present in the thymus and periphery of K14-Aβb and H2-DMα–deficient mice and, like their wild-type counterparts, suppress the proliferation of cocultured CD4+25− effector T cells. In contrast, CD4+25+ T cells from MHC class II–deficient mice do not suppress responder CD4+ T cells in vitro or in vivo. Thus, development of regulatory CD4+25+ T cells is dependent on MHC class II-positive thymic cortical epithelium. Furthermore, analysis of the specificities of CD4+25+ T cells in K14-Aβb and H2-DMα–deficient mice suggests that a subset of CD4+25+ T cells is subject to negative selection on hematopoietic antigen-presenting cells.

We have examined B cell populations that participate in distinct phases of the immune response to the influenza virus A/PR/8/34 hemagglutinin (HA) for their susceptibility to negative selection in mice that express the HA as a neo–self-antigen (HA104 mice). We demonstrated previously that specificity for the neo–self-HA causes a population of immunoglobulin G antibody-secreting cells, which dominate the primary response to virus immunization in BALB/c mice, to be negatively selected in HA104 mice. We find here that in contrast to these primary response B cells, HA-specific memory response B cells developed equivalently in HA104 and nontransgenic (BALB/c) mice. Indeed, there was no indication that HA-specific B cells were negatively selected during memory formation in influenza virus–immunized HA104 mice, even though the neo–self-HA can be recognized by memory B cells. Furthermore, HA-specific autoantibodies were induced in the absence of virus immunization by mating HA104 mice with mice transgenic for a CD4+ HA-specific T cell receptor. These findings indicate that specificity for a self-antigen does not prevent the maturation of autoreactive B cells in the germinal center pathway. Rather, the availability of CD4+ T cell help may play a crucial role in regulating autoantibody responses to the HA in HA104 mice.

Background

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.

Conclusion/Significance

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.