Cutting edge: IL-2 signals determine the degree of TCR signaling necessary to support regulatory T cell proliferation in vivo

To ensure immune tolerance, regulatory T cell (Treg) numbers must be maintained by cell division. This process has been thought to be strictly dependent on the Treg TCR interacting with MHC class II. In this study, we report that Treg division does not absolutely require cell-autonomous TCR signaling in vivo, depending on the degree of IL-2-mediated stimulation provided. At steady state IL-2 levels, Tregs require cell-autonomous TCR signaling to divide. However, when given exogenous IL-2 or when STAT5 is selectively activated in Tregs, Treg division can occur independently of MHC class II and TCR signaling. Thus, depending on the amount of IL-2R stimulation, a wide range of TCR signals supports Treg division, which may contribute to preservation of a diverse repertoire of Treg TCR specificities. These findings also have therapeutic implications, as TCR signaling by Tregs may not be required when using IL-2 to increase Treg numbers for treatment of inflammatory disorders.     

Cutting edge: contributions of apoptosis and anergy to systemic T cell tolerance

Multiple pathways can induce and maintain peripheral T cell tolerance. The goal of this study was to define the contributions of apoptosis and anergy to the maintenance of self-tolerance to a systemic Ag. Upon transfer into mice expressing OVA systemically, OVA-specific DO11 CD4+ T cells are activated transiently, cease responding, and die. Bim is the essential apoptosis-inducing trigger and apoptosis proceeds despite increased expression of Bcl-2 and Bcl-x. However, preventing apoptosis by eliminating Bim does not restore proliferation or cytokine production by DO11 cells. While Foxp3 is transiently induced, anergy is not associated with the stable development of regulatory T cells. Thus, apoptosis is dispensable for tolerance to a systemic self-Ag and cell-intrinsic anergy is sufficient to tolerize T cells.     

Cutting edge: Modulation of intestinal autoimmunity and IL-2 signaling by sphingosine kinase 2 independent of sphingosine 1-phosphate

Sphingosine kinase (Sphk) phosphorylates sphingosine into sphingosine-1-phosphate (S1P), but its recently identified isoform Sphk2 has been suggested to have distinct subcellular localization and substrate specificity. We demonstrate here that, surprisingly, Sphk2(-/-) CD4(+) T cells exhibit a hyperactivated phenotype with significantly enhanced proliferation and cytokine secretion in response to IL-2 as well as reduced sensitivity to regulatory T cell-mediated suppression in vitro, apparently independent of effects upon S1P. Such findings appear to reflect a requirement for Sphk2 to suppress IL-2 signaling because, in Sphk2(-/-) CD4(+) T cells, IL-2 induced abnormally accentuated STAT5 phosphorylation and small interfering RNA knockdown of STAT5 abrogated their hyperactive phenotype. This pathway physiologically modulates autoinflammatory responses, because Sphk2(-/-) T cells induced more rapid and robust inflammatory bowel disease in scid recipients. Thus, Sphk2 regulates IL-2 pathways in T cells, and the modulation of Sphk2 activity may be of therapeutic utility in inflammatory and/or infectious diseases.     

Cutting edge: Evidence for a dynamically driven T cell signaling mechanism

T cells use the αβ TCR to bind peptides presented by MHC proteins (pMHC) on APCs. Formation of a TCR-pMHC complex initiates T cell signaling via a poorly understood process, potentially involving changes in oligomeric state, altered interactions with CD3 subunits, and mechanical stress. These mechanisms could be facilitated by binding-induced changes in the TCR, but the nature and extent of any such alterations are unclear. Using hydrogen/deuterium exchange, we demonstrate that ligation globally rigidifies the TCR, which via entropic and packing effects will promote associations with neighboring proteins and enhance the stability of existing complexes. TCR regions implicated in lateral associations and signaling are particularly affected. Computational modeling demonstrated a high degree of dynamic coupling between the TCR constant and variable domains that is dampened upon ligation. These results raise the possibility that TCR triggering could involve a dynamically driven, allosteric mechanism.     

Cutting edge: murine cytomegalovirus induces a polyfunctional CD4 T cell response

CD4 T lymphocytes regulate the adaptive immune response to most viruses, both by providing help to CD8 T cells and B cells as well as through direct antiviral activity. Currently, no mouse cytomegalovirus (MCMV)-specific CD4 T cell responses are known. In this study, we identify and characterize 15 I-A(b)-restricted CD4 T cell responses specific for MCMV epitopes. CD4 T cells accumulate to high levels in the spleen and lungs during acute infection and produce multiple cytokines (IFN-gamma, TNF, IL-2, IL-10, and IL-17). Interestingly, IL-17 and IFN-gamma production within epitope-specific cells was found to be mutually exclusive. CD4 T cells recognizing a peptide derived from m09 were only detectable at later times of infection and displayed a unique cytokine production profile. In total, this study reveals that the MCMV-specific CD4 T cell response is complex and functionally diverse, highlighting its important role in controlling this persistent pathogen.     

Cutting edge: mechanisms of IL-2-dependent maintenance of functional regulatory T cells

IL-2 controls the survival of regulatory T cells (Tregs), but it is unclear whether IL-2 also directly affects Treg suppressive capacity in vivo. We have found that eliminating Bim-dependent apoptosis in IL-2- and CD25-deficient mice restored Treg numbers but failed to cure their lethal autoimmune disease, demonstrating that IL-2-dependent survival and suppressive activity can be uncoupled in Tregs. Treatment with IL-2-anti-IL-2-Ab complexes enhanced the numbers and suppressive capacity of IL-2-deprived Tregs with striking increases in CD25, CTLA-4, and CD39/CD73 expression. Although cytokine treatment induced these suppressive mechanisms in both IL-2(-/-) and IL-2(-/-)Bim(-/-) mice, it only reversed autoimmune disease in the latter. Our results suggest that successful IL-2 therapy of established autoimmune diseases will require a threshold quantity of Tregs present at the start of treatment and show that the suppressive capacity of Tregs critically depends on IL-2 even when Treg survival is independent of this cytokine.     

Cutting edge: T cells respond to lipopolysaccharide innately via TLR4 signaling

LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1alpha (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.     

Cutting edge: CD4 T cell-mast cell interactions alter IgE receptor expression and signaling

Mast cell activation is associated with atopic and inflammatory diseases, but the natural controls of mast cell homeostasis are poorly understood. We hypothesized that CD4(+)CD25(+) regulatory T cells (Treg) could function in mast cell homeostasis. In this study, we demonstrate that mast cells can recruit both Treg and conventional CD4(+) T cells (Tconv). Furthermore, Treg, but not Tconv, suppress mast cell FcepsilonRI expression. Despite the known inhibitory functions of IL-10 and TGFbeta1, FcepsilonRI suppression was independent of IL-10 and TGF-beta1 and required cell contact. Surprisingly, coculture with either Treg or Tconv cells suppressed IgE-mediated leukotriene C(4) production but enhanced cytokine production by mast cells. This was accompanied by a selective increase in FcepsilonRI-mediated Stat5 phosphorylation, which is a critical mediator of IgE-mediated cytokine secretion. These data are the first direct demonstration that mast cells can recruit Treg and illustrate that T cell interactions can alter the mast cell response.     

Cutting edge: Lipopolysaccharide induces IL-10-producing regulatory CD4+ T cells that suppress the CD8+ T cell response

TLR ligands are potent activators of dendritic cells and therefore function as adjuvants for the induction of immune responses. We analyzed the capacity of TLR ligands to enhance CD8+ T cell responses toward soluble protein Ag. Immunization with OVA together with LPS or poly(I:C) elicited weak CD8+ T cell responses in wild-type C57BL/6 mice. Surprisingly, these responses were greatly increased in mice lacking CD4+ T cells indicating the induction of regulatory CD4+ T cells. In vivo, neutralization of IL-10 completely restored CD8+ T cell responses in wild-type mice and OVA-specific IL-10 producing CD4+ T cells were detected after immunization with OVA plus LPS. Our study shows that TLR ligands not only activate the immune system but simultaneously induce Ag specific, IL-10-producing regulatory Tr1 cells that strongly suppress CD8+ T cell responses. In this way, excessive activation of the immune system may be prevented.     

Cutting edge: Thymic NK cells develop independently from T cell precursors

Although NK cells in the mouse are thought to develop in the bone marrow, a small population of NK cells in the thymus has been shown to derive from a GATA3-dependent pathway. Characteristically, thymic NK cells express CD127 and few Ly49 molecules and lack CD11b. Because these NK cells develop in the thymus, the question of their relationship to the T cell lineage has been raised. Using several different mouse models, we find that unlike T cells, thymic NK cells are not the progeny of Rorc-expressing progenitors and do not express Rag2 or rearrange the TCRγ locus. We further demonstrate that thymic NK cells develop independently of the Notch signaling pathway, supporting the idea that thymic NK cells represent bona fide NK cells that can develop independently of all T cell precursors.     

Cutting edge: antigen-independent CD8 T cell proliferation

Recent analyses of CD8 T cell responses to Listeria monocytogenes infection demonstrate that the duration of in vivo T cell proliferation is not determined by the amount or duration of Ag presentation. However, the extent to which T lymphocytes are capable of proliferating in the absence of Ag is unknown. Herein we demonstrate that CD8 T lymphocytes undergo up to eight rounds of proliferation in the absence of Ag following transient, 2.5-h in vitro antigenic stimulation. Ag-independent expansion of CD8 T cells is driven by IL-2 and is further augmented by IL-7 or IL-15. These experiments clearly demonstrate that CD8 T cells undergo prolonged proliferation following transient Ag exposure and support the notion that in vivo CD8 T cell expansion following infection can be uncoupled from Ag presentation.     

Cutting edge: early IFN-gamma signaling directly enhances primary antiviral CD4+ T cell responses

IFN-gamma drives CD4+ T cell differentiation toward the Th1 phenotype (Th1) and suppresses Th2 development. Current evidence indicates that IFN-gamma inhibits T cell proliferation and decreases T cell survival. In contrast to the above, we show here that antiviral CD4+ T cell generation after infection is reduced in the absence of IFN-gamma signals. The deficient expansion of cells was not due to perturbations in T cell sensitivity to peptide or to altered migratory patterns through nonlymphoid tissues. Instead, IFN-gamma enhanced early antiviral CD4 responses largely through direct signals into these cells. Our data challenge prevailing dogma and have implications for how the sizes of the CD8+ and CD4+ T cell responses are established.     

Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes

Gamma delta intraepithelial lymphocytes are thought to coordinate responses to pathogens that penetrate the epithelial barrier. To directly test this, mice were inoculated with Nocardia asteroides. At doses that were nonlethal for control mice, gamma delta-deficient mice became severely ill and died within 14 days. Histologic examination of these lungs demonstrated the presence of severe tissue damage and unimpeded bacterial growth in the gamma delta-deficient mice compared with neutrophilic lesions and clearance of the organism in control mice. Interestingly, ozone exposure that targets a comparable lung region also resulted in diffuse epithelial necrosis associated with a similar lack of neutrophil recruitment in gamma delta-deficient mice. These data demonstrate that gamma delta intraepithelial lymphocytes can protect the host from pathogenic and nonpathogenic insults by targeting the inflammatory response to epithelial necrosis.     

Cutting edge: Th17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment

Th17 cells play an active role in inflammation and autoimmune diseases. However, the nature and regulation of Th17 in the context of tumor immunity remain unknown. In this study, we show that parallel to regulatory T (Treg) cells, IL-17(+) CD4(+) and CD8(+) T cells are kinetically induced in multiple tumor microenvironments in mice and humans. Treg cells play a crucial role in tumor immune pathogenesis and temper immune therapeutic efficacy. IL-2 is crucial for the production and function of Treg cells. We now show that IL-2 reduces IL-17(+) T cell differentiation in the tumor microenvironment accompanied with an enhanced Treg cell compartment in vitro and in vivo. Altogether, our work demonstrates a dynamic differentiation of IL-17(+) T cells in the tumor microenvironment, reveals a novel role for IL-2 in controlling the balance between IL-17(+) and Treg cells, and provides new insight of IL-17(+) T cells in tumor immune pathology and therapy.     

Cutting edge: IL-7-independent regulation of IL-7 receptor alpha expression and memory CD8 T cell development

Expression of IL-7Ralpha on a subset of Ag-specific effector CD8 T cells is believed to identify memory cell precursors. However, whether IL-7 regulates IL-7Ralpha expression in vivo and is responsible for selective survival of IL-7Ralpha(+) effector cells is unknown. Our results show that in the absence of IL-7, IL-7Ralpha expression was extinguished on the majority of CD8 T cells responding to virus infection, sustained on a subset of effector cells transitioning to memory, and expressed at high levels by memory cells. Additionally, an IL-7-deficient environment was capable of supporting bcl-2 up-regulation and memory cell development in response to virus infection. Thus, IL-7Ralpha regulation occurs independently of IL-7 in responding CD8 T cells, indicating that CD8 memory T cell precursors are not selected by IL-7/IL-7Ralpha interactions.