Restoration of T-cell Effector Function, Depletion of Tregs, and Direct Killing of Tumor Cells: The Multiple Mechanisms of Action of a-TIGIT Antagonist Antibodies.

TIGIT is an immune checkpoint inhibitor expressed by effector CD4+ and CD8+ T cells, NK cells, and regulatory T cells (Tregs). Inhibition of TIGIT-ligand binding using antagonistic anti-TIGIT mAbs has shown in vitro potential to restore T-cell function and therapeutic efficacy in murine tumor models when combined with an anti-PD(L)-1 antibody. In the current work, we demonstrate broader TIGIT expression than previously reported in healthy donors and patients with cancer with expression on γδ T cells, particularly in CMV-seropositive donors, and on tumor cells from hematologic malignancies. Quantification of TIGIT density revealed tumor-infiltrating Tregs as the population expressing the highest receptor density. Consequently, the therapeutic potential of anti-TIGIT mAbs might be wider than the previously described anti-PD(L)-1-like restoration of αß T-cell function. CD155 also mediated inhibition of γδ T cells, an immune population not previously described to be sensitive to TIGIT inhibition, which could be fully prevented via use of an antagonistic anti-TIGIT mAb (EOS-448). In PBMCs from patients with cancer, as well as in tumor-infiltrating lymphocytes from mice, the higher TIGIT expression in Tregs correlated with strong antibody-dependent killing and preferential depletion of this highly immunosuppressive population. Accordingly, the ADCC/ADCP-enabling format of the anti-TIGIT mAb had superior antitumor activity, which was dependent upon Fcγ receptor engagement. In addition, the anti-TIGIT mAb was able to induce direct killing of TIGIT-expressing tumor cells both in human patient material and in animal models, providing strong rationale for therapeutic intervention in hematologic malignancies. These findings reveal multiple therapeutic opportunities for anti-TIGIT mAbs in cancer therapeutics.

αßT cell receptors expressed by CD4(-)CD8αß(-) intraepithelial T cells drive their fate into a unique lineage with unusual MHC reactivities.

Coreceptor CD4 and CD8αß double-negative (DN) TCRαß(+) intraepithelial T cells, although numerous, have been greatly overlooked and their contribution to the immune response is not known. Here we used T cell receptor (TCR) sequencing of single cells combined with retrogenic expression of TCRs to study the fate and the major histocompatibility complex (MHC) restriction of DN TCRαß(+) intraepithelial T cells. The data show that commitment of thymic precursors to the DN TCRαß(+) lineage is imprinted by their TCR specificity. Moreover, the TCRs they express display a diverse and unusual pattern of MHC restriction that is nonoverlapping with that of CD4(+) or CD8αß(+) T cells, indicating that they sense antigens that are not recognized by the conventional T cell subsets. The new insights indicate that DN TCRαß(+) T cells form a third lineage of TCRαß T lymphocytes expressing a variable TCR repertoire, which serve nonredundant immune functions.

Themis sets the signal threshold for positive and negative selection in T-cell development.

Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate 'positive selection', causing maturation of CD4- or CD8αß-expressing 'single-positive' thymocytes from CD4(+)CD8αß(+) 'double-positive' precursors. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in 'negative selection' by activation-induced apoptosis or 'agonist selection' of functionally differentiated self-antigen-experienced T cells. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens.

Transcriptional reprogramming of mature CD4⁺ helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes.

TCRαß thymocytes differentiate into either CD8αß(+) cytotoxic T lymphocytes or CD4(+) helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II-restricted CD4(+) thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4(+) T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4(+) T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II-restricted CD4(+) cytotoxic T lymphocytes.

Following the fate of one insulin-reactive CD4 T cell: conversion into Teffs and Tregs in the periphery controls diabetes in NOD mice.

In diabetic patients and susceptible mice, insulin is a targeted autoantigen. Insulin B chain 9-23 (B:9-23) autoreactive CD4 T cells are key for initiating autoimmune diabetes in NOD mice; however, little is known regarding their origin and function. To this end, B:9-23-specific, BDC12-4.1 T-cell receptor (TCR) transgenic (Tg) mice were studied, of which, despite expressing a single TCR on the recombination activating gene-deficient background, only a fraction develops diabetes in an asynchronous manner. BDC12-4.1 CD4 T cells convert into effector (Teff) and Foxp3(+)-expressing adaptive regulatory T cells (aTregs) soon after leaving the thymus as a result of antigen recognition and homeostatic proliferation. The generation of aTreg causes the heterogeneous diabetes onset, since crossing onto the scurfy (Foxp3) mutation, BDC12-4.1 TCR Tg mice develop accelerated and fully penetrant diabetes. Similarly, adoptive transfer and bone marrow transplantation experiments showed differential diabetes kinetics based on Foxp3(+) aTreg's presence in the BDC12-4.1 donors. A single-specificity, insulin-reactive TCR escapes thymic deletion and simultaneously converts into aTreg and Teff, establishing an equilibrium that determines diabetes penetrance. These results are of particular importance for understanding disease pathogenesis. They suggest that once central tolerance is bypassed, autoreactive cells arriving in the periphery do not by default follow solely a pathogenic fate upon activation.

Protein kinase C η is required for T cell activation and homeostatic proliferation.

Protein kinase C η (PKCη) is abundant in T cells and is recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell; however, its function in T cells is unknown. We showed that PKCη was required for the activation of mature CD8+ T cells through the T cell receptor. Compared with wild-type T cells, PKCη-/- T cells showed poor proliferation in response to antigen stimulation, a trait shared with T cells deficient in PKCθ, which is the most abundant PKC isoform in T cells and was thought to be the only PKC isoform with a specific role in T cell activation. In contrast, only PKCη-deficient T cells showed defective homeostatic proliferation, which requires self-antigen recognition. PKCη was dispensable for thymocyte development; however, thymocytes from mice doubly deficient in PKCη and PKCθ exhibited poor development, indicating some redundancy between the PKC isoforms. Deficiency in PKCη or PKCθ had opposing effects on the relative numbers of CD4+ and CD8+ T cells. PKCη-/- mice had a higher ratio of CD4+ to CD8+ T cells compared to that of wild-type mice, whereas PKCθ-/- mice had a lower ratio. Mice deficient in both isoforms exhibited normal cell ratios. Together, these data suggest that PKCη shares some redundant roles with PKCθ in T cell biology and also performs nonredundant functions that are required for T cell homeostasis and activation.

The light and dark sides of intestinal intraepithelial lymphocytes.

The intraepithelial lymphocytes (IELs) that reside within the epithelium of the intestine form one of the main branches of the immune system. As IELs are located at this critical interface between the core of the body and the outside environment, they must balance protective immunity with an ability to safeguard the integrity of the epithelial barrier: failure to do so would compromise homeostasis of the organism. In this Review, we address how the unique development and functions of intestinal IELs allow them to achieve this balance.

The thymus chapter in the life of gut-specific intra epithelial lymphocytes.

The intestinal intraepithelial lymphocytes (IEL) represent multi-lineage T cell populations. In addition to a major gammadeltaTCR(+) T cell subset, many IEL express alphabetaTCRs and they can be separated into alphabeta sublineages. Some TCRalphabeta(+)IEL have characteristics in common with conventional TCRalphabeta(+)T cells whereas others share an unconventional phenotype with their TCRgammadelta(+) counterparts. Because the latter are enriched for autoreactive TCRs and can be generated in the absence of a thymus, it has long been postulated that some IEL subsets develop locally in the intestine. Several new data however, indicate that under physiological conditions, IEL require a thymic education that directs lineage commitment and functional differentiation. This review will discuss the contributions of the thymus in shaping the various intestinal IEL sublineages.

Doubting the TCR coreceptor function of CD8alphaalpha.

"The beginning of wisdom is found in doubting; by doubting we come to question, and by seeking we may come upon the truth." -Pierre Abélard. CD8 is a glycoprotein expressed on hematopoietic cells. Two isoforms of CD8, CD8alphabeta and CD8alphaalpha, have been identified that are distinct in their expression and function. Whereas CD8alphabeta serves as a T cell receptor (TCR) coreceptor to enhance the functional avidity and is constitutively expressed on MHC class I-restricted T cells, CD8alphaalpha marks T cells that are distinct from the conventional thymus-selected and MHC-restricted CD4(+) or CD8alphabeta(+) T cells. Inconsistent with a coreceptor function, CD8alphaalpha decreases antigen sensitivity of the TCR, and it can be transiently or permanently expressed on T cells, regardless of the MHC restriction of the TCR or the presence of conventional coreceptors. Together, these observations indicate that CD8alphaalpha on T cells marks a differentiation stage and that it likely functions as a TCR corepressor to negatively regulate T cell activation.

Thymic differentiation of TCR alpha beta(+) CD8 alpha alpha(+) IELs.

Intraepithelial lymphocytes (IELs) contain several subsets, but the origin of the T-cell receptor (TCR)alphabeta(+) CD8 alpha alpha(+) IELs has been particularly controversial. Here we provide a synthesis, based on recent work, that attempts to unify the divergent views. The intestine has a primordial function in lymphopoiesis, and precursors with the potential to differentiate into T cells are found both in the epithelium and underlying lamina propria. Moreover, the thymus has been reported to export cells to the intestine that are not fully differentiated. TCR alpha beta(+) CD8 alpha alpha(+) IELs can differentiate in the intestine from each of these sources, but in normal euthymic mice, the thymus appears to be the major source for TCR alpha beta(+) CD8 alpha alpha(+) IELs. This unique IEL subset is a self-reactive population that requires exposure to self-agonists for selection in the thymus, similar to other regulatory T-cell populations. IELs transition through a double-positive (DP) intermediate in the thymus, but they originate from a subset of the DP cells that can be identified by its expression of CD8 alpha alpha homodimers. The agonist-selected cells in the thymus are TCRbeta(+) but CD4 and CD8 double negative. The evidence suggests that reacquired expression of CD8 alpha alpha and downregulation of CD5 occur after thymus export, perhaps in the intestine under the influence of interleukin-15. As a result of agonist exposure, a new gene expression program is activated. Therefore, the increased understanding of the developmental origin of TCR alpha beta(+) CD8 alpha alpha(+) IELs may help us to understand how they participate in immune regulation and protection in the intestine.

Identification of pre- and postselection TCRalphabeta+ intraepithelial lymphocyte precursors in the thymus.

The immune system preserves and makes use of autoreactive lymphocytes with specialized functions. Here we showed that one of these populations, CD8alphaalpha(+)TCRalphabeta(+) intestinal intraepithelial lymphocytes (IELs), arose from a unique subset of double-positive thymocytes. This subset of cells was precommitted to preferentially give rise to CD8alphaalpha(+)TCRalphabeta(+) IELs, but they required exposure to self-agonist peptides. The agonist-selected TCRalphabeta(+) thymocytes are CD4 and CD8 double-negative, and their final maturation, including the induction of CD8alphaalpha expression, appeared to occur only after thymus export in the IL-15-rich environment of the gut. These developmental steps, including precommitment of immature thymocytes, TCR-mediated agonist selection, and postthymic differentiation promoted by cytokines, define a unique pathway for the generation of CD8alphaalpha(+)TCRalphabeta(+) IEL.

The thymus exports long-lived fully committed T cell precursors that can colonize primary lymphoid organs.

Thymic export of cells is believed to be restricted to mature T cells. Here we show that the thymus also exports fully committed T cell precursors that colonize primary lymphoid organs. These precursor cells exited the thymus before T cell receptor rearrangements and colonized lymphoid organs such as the thymus and the gut. Migration of the thymic T cell-committed precursors led to permanent colonization of the gut precursor compartment, improved the capacity of gut precursors to further differentiate into T cells and was sufficient for the generation of 'euthymic like' CD8alphaalpha(+) intraepithelial lymphocytes. These data demonstrate a new function for the thymus in peripheral seeding with T cell precursors that become long lived after thymus export.

Extrathymic hemopoietic progenitors committed to T cell differentiation in the adult mouse.

The role of the thymus in T cell commitment of hemopoietic precursor is yet controversial. We previously identified a major T cell progenitor activity in precursor cells isolated from bone marrow-derived spleen colonies. In this study, we characterize the properties of these pre-T cells. We demonstrate that they have unique phenotype and can be generated in a total absence of any thymic influence. Indeed, even when studied at the single-cell level, extrathymic T cell-committed precursors express T cell-specific genes. Moreover, these cells are not committed to a particular T cell differentiation pathway because they can generate both extrathymic CD8alphaalpha+ intraepithelial lymphocytes and thymus-derived conventional thymocytes. We also compared these pre-T cells with fully T cell-committed thymic progenitors. When tested in vitro or by direct intrathymic transfer, these cells have a low clonogenic activity. However, after i.v. transfer, thymus repopulation is efficient and these precursors generate very high numbers of peripheral T cells. These results suggest the existence of extra steps of pre-T cell maturation that improve thymus reconstitution capacity and that can be delivered even after full T cell commitment. Consequently, our studies identify a source of extrathymic progenitors that will be helpful in defining the role of the thymus in the earliest steps of T cell differentiation.

Tolerance induction to self antigens by peripheral dendritic cells.

It has been suggested, but not formally demonstrated, that peripheral dendritic cells (DC) alone are capable of tolerance induction by clonal deletion and/or anergy. To resolve such an issue, it is important to develop in vivo systems where DC are the only cells capable of presenting antigen and where a T cell population with a known antigen specificity can be followed. Here we use a transgenic murine model, which expresses the influenza virus hemagglutinin (HA) on B cells and on CD8alpha(+) and CD8alpha(-) DC but not on macrophages. If these mice are on a RAG(-/-) background, one has a model in which only DC present the HA antigen. In these mice, HA-specific T cells are deleted very efficiently in the thymus and those remaining in the periphery cannot respond to further antigenic stimulation in vitro and cannot eliminate antigen in vivo. By performing adoptive transfers, we show for the first time that self-antigen presentation exclusively by peripheral DC results in very efficient clonal deletion of the majority of antigen-specific T cells with the remaining ones in an anergic state. This model will permit us to further address the mechanisms by which DC tolerize or prime T cells and to investigate whether anergy induction by DC is similar to anergy induction by B cells.

Major T cell progenitor activity in bone marrow-derived spleen colonies.

Common lymphoid progenitors (CLP) are generated in adult bone marrow (BM), but the intermediate steps leading to T cell commitment are unknown, and so is the site at which this commitment occurs. Here, we show that colonies arising in the spleen 12 days after BM injection harbor T cell precursors that are undetectable in BM. These precursors did not generate myeloid cells in vivo but repopulated the thymus and the peripheral T cell compartment much faster than did CLP. Two lineage negative (Lin(-)) subpopulations were distinguished, namely CD44(+) Thy1(-) cells still capable of natural killer generation and transient low-level B cell generation, and T cell-restricted CD44(-) Thy1(+) cells. At a molecular level, frequency of CD3epsilon and preTalpha mRNA was very different in each subset. Furthermore, only the CD44(-) Thy1(+) subset have initiated rearrangements in the T cell receptor beta locus. Thus, this study identifies extramedullary T cell progenitors and will allow easy approach to T cell commitment studies.

Naive T cells proliferate strongly in neonatal mice in response to self-peptide/self-MHC complexes.

Adult naive T cells, which are at rest in normal conditions, proliferate strongly when transferred to lymphopenic hosts. In neonates, the first mature thymocytes to migrate to the periphery reach a compartment devoid of preexisting T cells. We have extensively analyzed the proliferation rate and phenotype of peripheral T cells from normal C57BL/6 and T cell antigen receptor transgenic mice as a function of age. We show that, like adult naive T cells transferred to lymphopenic mice, neonatal naive T cells proliferate strongly. By using bone-marrow transfer and thymic-graft models, we demonstrate that the proliferation of the first thymic emigrants reaching the periphery requires T cell antigen receptor-self-peptide/self-MHC interactions and is regulated by the size of the peripheral T cell pool.

Characterization of T cell differentiation in the murine gut.

Gut intraepithelial CD8 T lymphocytes (T-IEL) are distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. The intermediate stages of differentiation between CP and mature T-IEL were not identified, and the local differentiation process was not characterized. We identified and characterized six phenotypically distinct lineage-negative populations in the CP and the gut epithelium: (a) we determined the kinetics of their generation from bone marrow precursors; (b) we quantified CD3-epsilon, recombination activating gene (Rag)-1, and pre-Talpha mRNAs expression at single cell level; (c) we characterized TCR-beta, -gamma, and -alpha locus rearrangements; and (d) we studied the impact of different mutations on the local differentiation. These data allowed us to establish a sequence of T cell precursor differentiation in the gut. We also observed that the gut differentiation varied from that of the thymus by a very low frequency of pre-Talpha chain mRNA expression, a different kinetics of Rag-1 mRNA expression, and a much higher impact of CD3 epsilon/delta and pre-Talpha deficiencies. Finally, only 3% of CP cells were clearly involved in T cell differentiation, suggesting that these structures may have additional physiological roles in the gut.