Matthew A. Burchill

IL-2 Receptor β-Dependent STAT5 Activation Is Required for the Development of Foxp3+ Regulatory T Cells

IL-2−/− mice develop autoimmunity despite having relatively normal numbers of regulatory T cells (Tregs). In contrast, we demonstrate that IL-2−/− × IL-15−/− and IL-2Rβ−/− mice have a significant decrease in Treg numbers. Ectopic expression of foxp3 in a subset of CD4+ T cells rescued Treg development and prevented autoimmunity in IL-2Rβ−/− mice, suggesting that IL-2Rβ-dependent signals regulate foxp3 expression in Tregs. Subsequent analysis of IL-2Rβ-dependent signal transduction pathways established that the transcription factor STAT5 is necessary and sufficient for Treg development. Specifically, T cell-specific deletion of STAT5 prevented Treg development; conversely, reconstitution of IL-2Rβ−/− mice with bone marrow cells expressing an IL-2Rβ mutant that exclusively activates STAT5 restored Treg development. Finally, STAT5 binds to the promoter of the foxp3 gene suggesting that IL-2Rβ-dependent STAT5 activation promotes Treg differentiation by regulating expression of foxp3.

Linked T Cell Receptor and Cytokine Signaling Govern the Development of the Regulatory T cell Repertoire

Appropriate development of regulatory T (Treg) cells is necessary to prevent autoimmunity. Neonatal mice, unlike adults, lack factors required for Treg cell development. It is unclear what these missing factors are. However, signals emanating from the T cell receptor (TCR), the costimulatory receptor CD28, and the family of gammac-dependent cytokine receptors are required for Treg cell development. Herein we demonstrate that expression of a constitutively active Stat5b transgene (Stat5b-CA) allowed for Treg cell development in neonatal mice and restored Treg cell numbers in Cd28(-/-) mice. Sequence analysis of TCR genes in Stat5b-CA Treg cells indicated that ectopic STAT5 activation resulted in a TCR repertoire that more closely resembled that of naive T cells. Using MHCII tetramers to identify antigen-specific T cells, we showed that STAT5 signals diverted thymocytes normally destined to become naive T cells into the Treg cell lineage. Our data support a two-step model of Treg cell differentiation in which TCR and CD28 signals induce cytokine responsiveness and STAT5-inducing cytokines then complete the program of Treg cell differentiation.

Distinct IL-2 Receptor Signaling Pattern in CD4+CD25 + Regulatory T Cells

Despite expression of the high-affinity IL-2R, CD4+CD25+ regulatory T cells (Tregs) are hypoproliferative upon IL-2R stimulation in vitro. However the mechanisms by which CD4+CD25+ T cells respond to IL-2 signals are undefined. In this report, we examine the cellular and molecular responses of CD4+CD25+ Tregs to IL-2. IL-2R stimulation results in a G1 cell cycle arrest, cellular enlargement and increased cellular survival of CD4+CD25+ T cells. We find a distinct pattern of IL-2R signaling in which the Janus kinase/STAT pathway remains intact, whereas IL-2 does not activate downstream targets of phosphatidylinositol 3-kinase. Negative regulation of phosphatidylinositol 3-kinase signaling and IL-2-mediated proliferation of CD4+CD25+ T cells is inversely associated with expression of the phosphatase and tensin homologue deleted on chromosome 10, PTEN.

IL-2, -7, and -15, but Not Thymic Stromal Lymphopoeitin, Redundantly Govern CD4+Foxp3+ Regulatory T Cell Development

Common γ chain (γc)-receptor dependent cytokines are required for regulatory T cell (Treg) development as γc−/− mice lack Tregs. However, it is unclear which γc-dependent cytokines are involved in this process. Furthermore, thymic stromal lymphopoietin (TSLP) has also been suggested to play a role in Treg development. In this study, we demonstrate that developing CD4+Foxp3+ Tregs in the thymus express the IL-2Rβ, IL-4Rα, IL-7Rα, IL-15Rα, and IL-21Rα chains, but not the IL9Rα or TSLPRα chains. Moreover, only IL-2, and to a much lesser degree IL-7 and IL-15, were capable of transducing signals in CD4+Foxp3+ Tregs as determined by monitoring STAT5 phosphorylation. Likewise, IL-2, IL-7, and IL-15, but not TSLP, were capable of inducing the conversion of CD4+CD25+Foxp3− thymic Treg progenitors into CD4+Foxp3+ mature Tregs in vitro. To examine this issue in more detail, we generated IL-2Rβ−/− × IL-7Rα−/− and IL-2Rβ−/− × IL-4Rα−/− mice. We found that IL-2Rβ−/− × IL-7Rα−/− mice were devoid of Tregs thereby recapitulating the phenotype observed in γc−/− mice; in contrast, the phenotype observed in IL-2Rβ−/− × IL-4Rα−/− mice was comparable to that seen in IL-2Rβ−/− mice. Finally, we observed that Tregs from both IL-2−/− and IL-2Rβ−/− mice show elevated expression of IL-7Rα and IL-15Rα chains. Addition of IL-2 to Tregs from IL-2−/− mice led to rapid down-regulation of these receptors. Taken together, our results demonstrate that IL-2 plays the predominant role in Treg development, but that in its absence the IL-7Rα and IL-15Rα chains are up-regulated and allow for IL-7 and IL-15 to partially compensate for loss of IL-2.

Distinct Effects of STAT5 Activation on CD4+ and CD8+ T Cell Homeostasis: Development of CD4+CD25+ Regulatory T Cells versus CD8+ Memory T Cells

Using transgenic mice that express a constitutively active version of STAT5b, we demonstrate that STAT5 plays a key role in governing B cell development and T cell homeostasis. STAT5 activation leads to a 10-fold increase in pro-B, but not pro-T, cells. Conversely, STAT5 signaling promotes the expansion of mature αβ T cells (6-fold increase) and γδ and NK T cells (3- to 4-fold increase), but not of mature B cells. In addition, STAT5 activation has dramatically divergent effects on CD8+ vs CD4+ T cells, leading to the selective expansion of CD8+ memory-like T cells and CD4+CD25+ regulatory T cells. These results establish that activation of STAT5 is the primary mechanism underlying both IL-7/IL-15-dependent homeostatic proliferation of naive and memory CD8+ T cells and IL-2-dependent development of CD4+CD25+ regulatory T cells.

STAT5 Activation Underlies IL7 Receptor-Dependent B Cell Development

Signals initiated by the IL7R are required for B cell development. However, the roles that distinct IL7R-induced signaling pathways play in this process remains unclear. To identify the function of the Raf and STAT5 pathways in IL7R-dependent B cell development, we used transgenic mice that express constitutively active forms of Raf (Raf-CAAX) or STAT5 (STAT5b-CA) throughout lymphocyte development. Both Raf-CAAX and STAT5b-CA mice exhibit large increases in pro-B cells. However, crossing the Raf-CAAX transgene onto the IL7R−/− background fails to rescue B cell development. In contrast, STAT5 activation selectively restores B cell expansion in IL7R−/− mice. Notably, the expansion of pro-B cells in STAT5b-CA mice correlated with an increase in cyclin D2, pim-1, and bcl-xL expression, suggesting that STAT5 directly affects pro-B cell proliferation and survival. In addition, STAT5 activation also restored B cell differentiation in IL7R−/− mice as determined by 1) the restoration of VH Ig gene rearrangement and 2) the appearance of immature and mature B cell subsets. These findings establish STAT5 as the key player entraining B cell development downstream of the IL7R.

TLR7 enables cross-presentation by multiple dendritic cell subsets through a type I IFN-dependent pathway

Conjugation of TLR agonists to protein or peptide antigens has been demonstrated in many studies to be an effective vaccine formula in inducing cellular immunity. However, the molecular and cellular mediators involved in TLR-induced immune responses have not been carefully examined. In this study, we identify Type I IFN and IL-12 as critical mediators of cross-priming induced by a TLR7 agonist-antigen conjugate. We demonstrate that TLR7-driven cross-priming requires both Type I IFN and IL-12. Signaling through the IFN-αβR was required for the timely recruitment and accumulation of activated dendritic cells in the draining lymph nodes. Although IL-12 was indispensable during cross-priming, it did not regulate DC function. Therefore, the codependency for these 2 cytokines during TLR7-induced cross-priming is the result of their divergent effects on different cell-types. Furthermore, although dermal and CD8α(+) DCs were able to cross-prime CD8(+) T cells, Langerhans cells were unexpectedly found to potently cross-present antigen and support CD8(+) T-cell expansion, both in vitro and in vivo. Collectively, the data show that a TLR7 agonist-antigen conjugate elicits CD8(+) T-cell responses by the coordinated recruitment and activation of both tissue-derived and lymphoid organ-resident DC subsets through a Type I IFN and IL-12 codependent mechanism.

Antigen capture and archiving by lymphatic endothelial cells following vaccination or viral infection

Antigen derived from viral infections with influenza and Vesicular Stomatitis Virus (VSV) can persist after resolution of infection. Here we show that antigen can similarly persist for weeks following viral challenge and vaccination. Antigen is captured by Lymphatic Endothelial Cells (LECs), under conditions that induce LEC proliferation. Consistent with published data showing that viral antigen persistence impacts on the function of circulating memory T cells, we find that vaccine elicited antigen persistence, found on LECs, positively influences the degree of protective immunity elicited by circulating memory CD8+ T cells. The coupling of LEC proliferation and antigen capture identifies a mechanism by which the LECs store, or “archive”, antigens for extended periods of time after antigen challenge, thereby increasing IFNγ/IL-2 production and enhancing protection against infection. These findings therefore have the potential to impact future vaccination strategies and our understanding of the role for persisting antigen in both vaccine and infectious settings.

Restricted STAT5 activation dictates appropriate thymic B versus T cell lineage commitment.

The molecular mechanisms regulating lymphocyte lineage commitment remain poorly characterized. To explore the role of the IL7R in this process, we generated transgenic mice that express a constitutively active form of STAT5 (STAT5b-CA), a key downstream IL7R effector, throughout lymphocyte development. STAT5b-CA mice exhibit a 40-fold increase in pro-B cells in the thymus. As documented by BrdU labeling studies, this increase is not due to enhanced B cell proliferation. Thymic pro-B cells in STAT5b-CA mice show a modest increase in cell survival (∼4-fold), which correlates with bcl-xL expression. However, bcl-xL transgenic mice do not show increases in thymic B cell numbers. Thus, STAT5-dependent bcl-xL up-regulation and enhanced B cell survival are not sufficient to drive the thymic B cell development observed in STAT5b-CA mice. Importantly, thymic pro-B cells in STAT5b-CA mice are derived from early T cell progenitors (ETPs), suggesting that STAT5 acts by altering ETP lineage commitment. Supporting this hypothesis, STAT5 binds to the pax5 promoter in ETPs from STAT5b-CA mice and induces pax5, a master regulator of B cell development. Conversely, STAT5b-CA mice exhibit a decrease in the DN1b subset of ETPs, demonstrating that STAT5 activation inhibits early T cell differentiation or lineage commitment. On the basis of these findings, we propose that the observed expression of the IL-7R on common lymphoid progenitors, but not ETPs, results in differential STAT5 signaling within these distinct progenitor populations and thus helps ensure appropriate development of B cells and T cells in the bone marrow and thymic environments, respectively.

Comparison of OX40 Ligand and CD70 in the Promotion of CD4+ T Cell Responses

The TNF superfamily members CD70 and OX40 ligand (OX40L) were reported to be important for CD4+ T cell expansion and differentiation. However, the relative contribution of these costimulatory signals in driving CD4+ T cell responses has not been addressed. In this study, we found that OX40L is a more important determinant than CD70 of the primary CD4+ T cell response to multiple immunization regimens. Despite the ability of a combined TLR and CD40 agonist (TLR/CD40) stimulus to provoke appreciable expression of CD70 and OX40L on CD8+ dendritic cells, resulting CD4+ T cell responses were substantially reduced by Ab blockade of OX40L and, to a lesser degree, CD70. In contrast, the CD8+ T cell responses to combined TLR/CD40 immunization were exclusively dependent on CD70. These requirements for CD4+ and CD8+ T cell activation were not limited to the use of combined TLR/CD40 immunization, because vaccinia virus challenge elicited primarily OX40L-dependent CD4 responses and exclusively CD70-dependent CD8+ T cell responses. Attenuation of CD4+ T cell priming induced by OX40L blockade was independent of signaling through the IL-12R, but it was reduced further by coblockade of CD70. Thus, costimulation by CD70 or OX40L seems to be necessary for primary CD4+ T cell responses to multiple forms of immunization, and each may make independent contributions to CD4+ T cell priming.