We compared how CD4 vs CD8 cells attain the capacity to express the effector cytokine IFN-γ under both immunogenic and tolerogenic conditions. Although the Ifng gene locus was epigenetically repressed in naive Ag-inexperienced CD4 cells, it had already undergone partial remodeling toward a transcriptionally competent configuration in naive CD8 cells. After TCR stimulation, CD8 cells fully remodeled the Ifng locus and gained the capacity to express high levels of IFN-γ more rapidly than CD4 cells. Enforced dual costimulation through OX40 and 4-1BB redirected CD8 cells encountering soluble exogenous peptide to expand and differentiate into IFN-γ and TNF-α double-producing effectors rather than becoming tolerant. Despite this and the stronger tendency of CD8 compared with CD4 cells to differentiate into IFN-γ-expressing effectors, when parenchymal self-Ag was the source of tolerizing Ag, enforced dual costimulation selectively boosted expansion but did not push effector differentiation in CD8 cells while both expansion and effector differentiation were dramatically boosted in CD4 cells. Notably, enforced dual costimulation was able to push effector differentiation in CD8 cells encountering cognate parenchymal self-Ag when CD4 cells were simultaneously engaged. Thus, the ability of enforced OX40 plus 4-1BB dual costimulation to redirect CD8 cells to undergo effector differentiation was unexpectedly influenced by the source of tolerizing Ag and help was selectively required to facilitate CD8 cell effector differentiation when the tolerizing Ag derived from self.

When Th1 effector CD4 cells encounter tolerizing Ag in vivo, their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than noneffector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with Ag vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and -distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ-expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggested that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure.

BACKGROUND-Prostate cancer promotes the development of T cell tolerance towards prostatic antigens, potentially limiting the efficacy of prostate cancer vaccines targeting these antigens. Here, we sought to determine the stage of disease progression when T cell tolerance develops, as well as the role of steady state dendritic cells (DC) and CD4 + CD25 + T regulatory cells (Tregs) in programming tolerance.

BackgroundNeuropathic pain is one of the most devastating kinds of chronic pain. Neuroinflammation has been shown to contribute to the development of neuropathic pain. We have previously demonstrated that lumbar spinal cord-infiltrating CD4+ T lymphocytes contribute to the maintenance of mechanical hypersensitivity in spinal nerve L5 transection (L5Tx), a murine model of neuropathic pain. Here, we further examined the phenotype of the CD4+ T lymphocytes involved in the maintenance of neuropathic pain-like behavior via intracellular flow cytometric analysis and explored potential interactions between infiltrating CD4+ T lymphocytes and spinal cord glial cells.ResultsWe consistently observed significantly higher numbers of T-Bet+, IFN-γ+, TNF-α+, and GM-CSF+, but not GATA3+ or IL-4+, lumbar spinal cord-infiltrating CD4+ T lymphocytes in the L5Tx group compared to the sham group at day 7 post-L5Tx. This suggests that the infiltrating CD4+ T lymphocytes expressed a pro-inflammatory type 1 phenotype (Th1). Despite the observation of CD4+ CD40 ligand (CD154)+ T lymphocytes in the lumbar spinal cord post-L5Tx, CD154 knockout (KO) mice did not display significant changes in L5Tx-induced mechanical hypersensitivity, indicating that T lymphocyte-microglial interaction through the CD154-CD40 pathway is not necessary for L5Tx-induced hypersensitivity. In addition, spinal cord astrocytic activation, represented by glial fibillary acidic protein (GFAP) expression, was significantly lower in CD4 KO mice compared to wild type (WT) mice at day 14 post-L5Tx, suggesting the involvement of astrocytes in the pronociceptive effects mediated by infiltrating CD4+ T lymphocytes.ConclusionsIn all, these data indicate that the maintenance of L5Tx-induced neuropathic pain is mostly mediated by Th1 cells in a CD154-independent manner via a mechanism that could involve multiple Th1 cytokines and astrocytic activation.

Bone marrow-derived APC are critical for both priming effector/memory T cell responses to pathogens and inducing peripheral tolerance in self-reactive T cells. In particular, dendritic cells (DC) can acquire peripheral self-Ags under steady state conditions and are thought to present them to cognate T cells in a default tolerogenic manner, whereas exposure to pathogen-associated inflammatory mediators during the acquisition of pathogen-derived Ags appears to reprogram DCs to prime effector and memory T cell function. Recent studies have confirmed the critical role of DCs in priming CD8 cell effector responses to certain pathogens, although the necessity of steady state DCs in programming T cell tolerance to peripheral self-Ags has not been directly tested. In the current study, the role of steady state DCs in programming self-reactive CD4 cell peripheral tolerance was assessed by combining the CD11c-diphtheria toxin receptor transgenic system, in which DC can be depleted via treatment with diphtheria toxin, with a TCR-transgenic adoptive transfer system in which either naive or Th1 effector CD4 cells are induced to undergo tolerization after exposure to cognate parenchymally derived self-Ag. Although steady state DCs present parenchymal self-Ag and contribute to the tolerization of cognate naive and Th1 effector CD4 cells, they are not essential, indicating the involvement of a non-DC tolerogenic APC population(s). Tolerogenic APCs, however, do not require the cooperation of CD4+CD25+ regulatory T cells. Similarly, DC were required for maximal priming of naive CD4 cells to vaccinia viral-Ag, but priming could still occur in the absence of DC.

When naive CD4+ Th cells encounter cognate pathogen-derived Ags they expand and develop the capacity to express the appropriate effector cytokines for neutralizing the pathogen. Central to this differentiation process are epigenetic modifications within the effector cytokine genes that allow accessibility to the transcriptional machinery. In contrast, when mature self-reactive CD4 cells encounter their cognate epitopes in the periphery they generally undergo a process of tolerization in which they become hyporesponsive/anergic to antigenic stimulation. In the current study, we used a TCR transgenic adoptive transfer system to demonstrate that in a dose-dependent manner parenchymal self-Ag programs cognate naive CD4 cells to acetylate histones bound to the promoter region of the Ifng gene (which encodes the signature Th1 effector cytokine) during peripheral tolerization. Although the Ifng gene gains transcriptional competence, these tolerized CD4 cells fail to express substantial amounts of IFN-γ in response to antigenic stimulation apparently because a blockage in TCR-mediated signaling also develops. Nevertheless, responsiveness to antigenic stimulation is partially restored when self-Ag-tolerized CD4 cells are retransferred into mice infected with a virus expressing the same Ag. Additionally, there is preferential boosting in the ability of these CD4 cells to express IFN-γ relative to other cytokines with expression that also becomes impaired. Taken together, these results suggest that epigenetic modification of the Ifng locus during peripheral CD4 cell tolerization might allow for preferential expression of IFN-γ during recovery from tolerance.

When Th1 effector CD4 cells encounter tolerizing antigen in vivo their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than non-effector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with antigen vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ-expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggests that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN γ expression potential through alterations in chromatin structure.

We previously demonstrated that CD4+ T cells contribute to the maintenance of L5 spinal nerve transection (L5Tx) induced neuropathic pain in BALB/c mice. It is proposed that a particular subtype(s) of the L5Tx-induced spinal cord infiltrating CD4+ T cells mediates this maintenance of neuropathic pain via T cell - glial interactions. Here, we determined the subtype of L5Tx-induced spinal cord infiltrating CD4+ T cells. There were significantly higher numbers of T-Bet+ lumbar spinal cord infiltrating CD4+ T cells in the L5Tx group compared to the sham group at day 7 post-L5Tx, the peak time for detecting lumbar infiltrating CD4+ T cells, while no significant GATA3+ CD4+ T cells were observed. There were no significant differences in the levels of T-Bet+ or GATA3+ CD4+ T cells in the spleen or lumbar lymph nodes detected at this time. Interestingly, although no significantly higher levels of IFNγ were found in L5Tx mice in the lumbar spinal cord post-surgery, there was a notable increase in splenic levels of IFNγ in the L5Tx group at day 3 post-L5Tx compared to both sham and naïve mice. As we also have data indicating no differences in Th17 responses between groups, altogether our data suggests the involvement of Th1 cells in L5Tx-induced neuropathic pain and that the maintenance of neuropathic pain is likely a Th1-mediated process. Specific downstream responses mediated by these infiltrating Th1 cells require further investigation.