SUMMARY Exhausted CD8+ T cells (TEX) in chronic infections and cancer have limited effector function, high inhibitory receptor co-expression and extensive transcriptional changes compared to effector (TEFF) or memory (TMEM) CD8+ T cells. TEX are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, TEX are a distinct immune subset, with a unique chromatin landscape compared to TEFF and TMEM. However, the mechanisms governing the transcriptional and epigenetic development of TEX remain unknown. Here, we identify the HMG-box transcription factor TOX as a central regulator of TEX. TOX is largely dispensable for TEFF and TMEM formation, but is critical for exhaustion and without TOX TEX do not form. TOX is induced by calcineurin and NFAT2 and operates in a feed-forward loop to become calcineurin independent and sustained in TEX. Thus, robust TOX expression results in commitment to TEX by translating persistent stimulation into a distinct TEX transcriptional and epigenetic developmental program.

No abstract

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.

Positive selection of developing thymocytes is associated with changes in cell function, at least in part caused by alterations in expression of cell surface proteins. Surprisingly, however, few such proteins have been identified. We have analyzed the pattern of gene expression during the early stages of murine thymocyte differentiation. These studies led to identification of a cell surface protein that is a useful marker of positive selection and is a likely regulator of mature lymphocyte and APC function. The protein is a member of the Ig superfamily and contains conserved tyrosine-based signaling motifs. The gene encoding this protein was independently isolated recently and termed B and T lymphocyte attenuator (Btla). We describe in this study anti-BTLA mAbs that demonstrate that the protein is expressed in the bone marrow and thymus on developing B and T cells, respectively. BTLA is also expressed by all mature lymphocytes, splenic macrophages, and mature, but not immature bone marrow-derived dendritic cells. Although mice deficient in BTLA do not show lymphocyte developmental defects, T cells from these animals are hyperresponsive to anti-CD3 Ab stimulation. Conversely, anti-BTLA Ab can inhibit T cell activation. These results implicate BTLA as a negative regulator of the activation and/or function of various hemopoietic cell types.

CD8+ cytotoxic and CD4+ helper/inducer T cells develop from common thymocyte precursors that express both CD4 and CD8 molecules. Upon T cell receptor signaling, these cells initiate a differentiation program that includes complex changes in CD4 and CD8 expression, allowing identification of transitional intermediates in this developmental pathway. Little is known about regulation of these early transitions or their specific importance to CD4 and CD8 T cell development. Here, we show a severe block at the CD4loCD8lo transitional stage of positive selection caused by loss of the nuclear HMG box protein TOX. As a result, CD4 lineage T cells, including regulatory T and CD1d-dependent natural killer T cells, fail to develop. In contrast, functional CD8+ T cells develop in TOX-deficient mice. Our data suggest that TOX-dependent transition to the CD4+CD8lo stage is required for continued development of class II major histocompatibility complex–specific T cells, regardless of ultimate lineage fate.

Thymocyte selection-associated HMG box factor (TOX) is a DNA-binding factor required for development of CD4 T cells, natural killer T cells, and T regulatory cells. Here we document that both NK cell development and lymphoid tissue organogenesis are inhibited in the absence of TOX. We find that development of lymphoid tissue inducer cells, a rare subset of specialized cells that plays an integral role in lymphoid tissue organogenesis, requires TOX. Tox is highly upregulated in immature NK cells in the bone marrow, consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus, multiple cell lineages in the immune system share a TOX-dependent step for development.

In the thymus, pre-T cell receptor (pre-TCR)--mediated signaling and then TCR-mediated signaling initiate changes in gene expression that result in the maturation of CD4 and CD8 lineage T cells from common precursors. Using gene chip technology, we isolated a murine gene, designated Tox, that encodes a member of the HMG (high-mobility group) box family of DNA-binding proteins. TOX expression is up-regulated by both pre-TCR and TCR activation of immature thymocytes but not by TCR activation of mature naïve T cells. Transgenic mice that express TOX show expanded CD8+ and reduced CD4+ single positive thymocyte subpopulations. We present evidence here that this phenotype results from a perturbation in lineage commitment due to reduced sensitivity to TCR-mediated signaling. This molecular marker of thymic selection events may therefore play a role in establishing the activation threshold of developing T cells and patterning changes in gene expression.

The proliferation of helper T cells and the T-dependent induction of B cell activation are events that are initiated by T cell recognition of antigen in association with Ia glycoproteins. However, the molecular basis of helper T cell antigen recognition remains unknown. Like several other laboratories, we have approached this problem by raising antibodies that are individually specific for functionally characterized cloned T cell lines. Clone-specific antibodies raised against alloreactive Ia-recognizing T cell clones (1), antigen-Ia-specific helper T cell hybrids (2), a human cytotoxic HLA-A recognizing T cell clone (3), and a murine H-2 recognizing cytotoxic T cell clone (4) have already been described. These clone-specific antibodies are either stimulatory themselves, as is the case of antisera raised to alloreactive T cell clones, or inhibit antigen-driven T cell activation, as are antibodies raised against helper T cell hybrids. However, stimulatory antibodies specific for major histocompatibility complex (MHC) 1 restricted, antigen-specific cloned helper T cell lines have not been described. In addition, the question of whether a monoclonal antibody of this specificity can activate a T cell has never been addressed.In this paper, we describe antisera that both activate the T cells themselves and induce T cell-dependent B cell activation. In addition, we have produced a monoclonal antibody specific for a single cloned helper cell line that can also activate the T cells of this line. The anti-clone antibodies we have produced appear to completely replace the initial antigen-Ia recognition event by the T