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) are shaped by TCR recognition of the diverse array of peptide:MHC complexes that can be generated from self-antigens 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-antigen-selected Tregs were unable to suppress the antiviral immune response based on recognition of the peptide as a self-antigen 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-antigen-selected Tregs to modulate an antiviral 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 antiviral immune response.

The degree of CD4+ T cell autoreactivity determines cellular pathways underlying inflammatory arthritis.

Although therapies targeting distinct cellular pathways (e.g., anticytokine 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.

Strength of TCR signal from self-peptide modulates autoreactive thymocyte deletion and Foxp3(+) Treg-cell formation.

Autoreactive CD4(+) CD8(-) (CD4SP) thymocytes can be subjected to deletion when they encounter self-peptide during their development, but they can also undergo selection to become CD4SPFoxp3(+) Treg cells. We have analyzed the relationship between these distinct developmental fates using mice in which signals transmitted by the TCR have been attenuated by mutation of a critical tyrosine residue of the adapter protein SLP-76. In mice containing polyclonal TCR repertoires, the mutation caused increased frequencies of CD4SPFoxp3(+) thymocytes. CD4SP thymocytes expressing TCR Vß-chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies, particularly among Foxp3(+) thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg-cell formation in response to a defined self-peptide, SLP-76 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3(+) Treg-cell formation still occurred, albeit with a reduced efficiency, and the mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of the TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg-cell differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg-cell formation.

Viral antigen induces differentiation of Foxp3+ natural regulatory T cells in influenza virus-infected mice.

We examined the formation, participation, and functional specialization of virus-reactive Foxp3(+) regulatory T cells (Tregs) in a mouse model of influenza virus infection. "Natural" Tregs generated intrathymically, based on interactions with a self-peptide, proliferated in response to a homologous viral Ag in the lungs and, to a lesser extent, in the lung-draining mediastinal lymph nodes (medLNs) of virus-infected mice. In contrast, conventional CD4(+) T cells with identical TCR specificity underwent little or no conversion to become "adaptive" Tregs. The virus-reactive Tregs in the medLNs and the lungs of infected mice upregulated a variety of molecules associated with Treg activation, as well as acquired expression of molecules (T-bet, Blimp-1, and IL-10) that confer functional specialization to Tregs. Notably, however, the phenotypes of the T-bet(+) Tregs obtained from these sites were distinct, because Tregs isolated from the lungs expressed significantly higher levels of T-bet, Blimp-1, and IL-10 than did Tregs from the medLNs. Adoptive transfer of Ag-reactive Tregs led to decreased proliferation of antiviral CD4(+) and CD8(+) effector T cells in the lungs of infected hosts, whereas depletion of Tregs had a reciprocal effect. These studies demonstrate that thymically generated Tregs can become activated by a pathogen-derived peptide and acquire discrete T-bet(+) Treg phenotypes while participating in and modulating an antiviral immune response.

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-Ag 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 nonarthritic mice expressing HA as a self-Ag 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-Ag can promote the development of a regional Th17 cell inflammatory response by driving the recruitment of Th17-trophic iMO to the lymph nodes.

Requirement for diverse TCR specificities determines regulatory T cell activity in a mouse model of autoimmune arthritis.

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 Ag-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.

CD4⁺CD25⁺Foxp3⁺ regulatory T cell formation requires more specific recognition of a self-peptide than thymocyte deletion.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are generated during thymocyte development and play a crucial role in preventing the immune system from attacking the body's cells and tissues. However, how the formation of these cells is directed by T-cell receptor (TCR) recognition of self-peptide:major histocompatibility complex (MHC) ligands remains poorly understood. We show that an agonist self-peptide with which a TCR is strongly reactive can induce a combination of thymocyte deletion and CD4(+)CD25(+)Foxp3(+) Treg cell formation in vivo. A weakly cross-reactive partial agonist self-peptide could similarly induce thymocyte deletion, but failed to induce Treg cell formation. These studies indicate that CD4(+)CD25(+)Foxp3(+) Treg cell formation can require highly stringent recognition of an agonist self-peptide by developing thymocytes. They also refine the "avidity" model of thymocyte selection by demonstrating that the quality of the signal mediated by agonist self-peptides, rather than the overall intensity of TCR signaling, can be a critical factor in directing autoreactive thymocytes to undergo CD4(+)CD25(+)Foxp3(+) Treg cell formation and/or deletion during their development.

How specificity for self-peptides shapes the development and function of regulatory T cells.

The cataclysmic disease that develops in mice and humans lacking CD4+ T cells expressing the transcription factor Foxp3 has provided abundant evidence that Foxp3+CD4+ Tregs are required to suppress a latent autoreactivity of the immune system. There is also evidence for the existence of tissue-specific Tregs that can act to suppress regional autoimmune responses, suggesting that Tregs exert their effects, in part, through responding to self-peptides. However, how the immune system generates a repertoire of Tregs that is designed to recognize and direct regulatory function to self-peptides is incompletely understood. This review describes studies aimed at determining how T cell recognition of self-peptide(s) directs Treg formation in the thymus, including discussion of a modified "avidity" model of thymocyte development. Studies aimed at determining how TCR specificity contributes to the ability of Tregs to suppress autoimmune diseases are also discussed.

Thymocyte deletion can bias Treg formation toward low-abundance self-peptide.

Autoreactive CD4+ T cells can undergo deletion and/or become CD25+Foxp3+ Treg 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 can develop among members of a cohort of autoreactive thymocytes that have evaded deletion by a self-peptide, and that deletion and Treg formation can act together to bias the Treg repertoire toward low-abundance self-peptide(s).

CD4+ T cells recognizing a single self-peptide expressed by APCs induce spontaneous autoimmune arthritis.

We have examined processes leading to the spontaneous development of autoimmune inflammatory arthritis in transgenic mice containing CD4+ T cells targeted to a nominal Ag (hemagglutinin (HA)) and coexpressing HA driven by a MHC class II promoter. Despite being subjected to multiple tolerance mechanisms, autoreactive CD4+ T cells accumulate in the periphery of these mice and promote systemic proinflammatory cytokine production. The majority of mice spontaneously develop inflammatory arthritis, which is accompanied by an enhanced regional immune response in lymph nodes draining major joints. Arthritis development is accompanied by systemic B cell activation; however, neither B cells nor Ab is required for arthritis development, since disease develops in a B cell-deficient background. Moreover, arthritis also develops in a recombinase activating gene-deficient background, indicating that the disease process is driven by CD4+ T cells recognizing the neo-self HA Ag. These findings show that autoreactive CD4+ T cells recognizing a single self-Ag, expressed by systemically distributed APCs, can induce arthritis via a mechanism that is independent of their ability to provide help for autoantibody production.