Pierre Vantourout

Six-of-the-best: unique contributions of γδ T cells to immunology

γδ T cells are a unique and conserved population of lymphocytes that have been the subject of a recent explosion of interest owing to their essential contributions to many types of immune response and immunopathology. But what does the integration of recent and long-established studies really tell us about these cells and their place in immunology? The time is ripe to consider the evidence for their unique and crucial functions. We conclude that whereas B cells and αβ T cells are commonly thought to contribute primarily to the antigen-specific effector and memory phases of immunity, γδ T cells are distinct in that they combine conventional adaptive features (inherent in their T cell receptors and pleiotropic effector functions) with rapid, innate-like responses that can place them in the initiation phase of immune reactions. This underpins a revised perspective on lymphocyte biology and the regulation of immunogenicity.

Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells

In this study we demonstrate a new form of immunoregulation: engagement on CD4+ T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (TH1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4+ T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4+ T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-γ (IFN-γ). Finally, γδ T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) γδ and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.

Epithelia Use Butyrophilin-like Molecules to Shape Organ-Specific γδ T Cell Compartments

Summary Many body surfaces harbor organ-specific γδ T cell compartments that contribute to tissue integrity. Thus, murine dendritic epidermal T cells (DETCs) uniquely expressing T cell receptor (TCR)-Vγ5 chains protect from cutaneous carcinogens. The DETC repertoire is shaped by Skint1, a butyrophilin-like (Btnl) gene expressed specifically by thymic epithelial cells and suprabasal keratinocytes. However, the generality of this mechanism has remained opaque, since neither Skint1 nor DETCs are evolutionarily conserved. Here, Btnl1 expressed by murine enterocytes is shown to shape the local TCR-Vγ7+ γδ compartment. Uninfluenced by microbial or food antigens, this activity evokes the developmental selection of TCRαβ+ repertoires. Indeed, Btnl1 and Btnl6 jointly induce TCR-dependent responses specifically in intestinal Vγ7+ cells. Likewise, human gut epithelial cells express BTNL3 and BTNL8 that jointly induce selective TCR-dependent responses of human colonic Vγ4+ cells. Hence, a conserved mechanism emerges whereby epithelia use organ-specific BTNL/Btnl genes to shape local T cell compartments.

Immunological Visibility: Posttranscriptional Regulation of Human NKG2D Ligands by the EGF Receptor Pathway

Environmental changes induce human NKG2D ligand expression—immunological visibility—through the EGFR stress response. Attracting Big Brother It is increasingly acknowledged that immune cells serve as the surveillance system of the body—constantly on the lookout for things that are out of place, such as infections or cancer. Indeed, researchers and clinicians are now trying to harness the immune system either by overcoming inhibitions or by pointing them at particular targets. But what happens when the target tries to hide? Vantourout et al. now look at the mechanisms that regulate immunological visibility. The authors stressed human epithelial cells by various means and found that up-regulation of ligands that activate NKG2D, a lymphocyte-activating receptor, was dependent on epithelial growth factor receptor (EGFR). They looked further into the mechanism and discovered that EGFR activation resulted in relocalization of AUF1 proteins, which normally destabilize NKG2D ligands, and resulted in increased expression of these ligands—and improved immunological visibility. What’s more, EGFR activation is one of the most common forms of dysregulation underpinning human carcinomas, and primary tumors with high EGFR expression showed higher NKG2D ligand expression levels. Conversely, NKG2D ligand expression was reduced by erlotinib and cetuximab, two EGFR inhibitors commonly used in the clinic. These data suggest that the effects of therapeutics that limit cancer growth should be considered with immunotherapy. Human cytolytic T lymphocytes and natural killer cells can limit tumor growth and are being increasingly harnessed for tumor immunotherapy. One way cytolytic lymphocytes recognize tumor cells is by engagement of their activating receptor, NKG2D, by stress antigens of the MICA/B and ULBP families. This study shows that surface up-regulation of NKG2D ligands by human epithelial cells in response to ultraviolet irradiation, osmotic shock, oxidative stress, and growth factor provision is attributable to activation of the epidermal growth factor receptor (EGFR). EGFR activation causes intracellular relocalization of AUF1 proteins that ordinarily destabilize NKG2D ligand mRNAs by targeting an AU-rich element conserved within the 3′ ends of most human, but not murine, NKG2D ligand genes. Consistent with these findings, NKG2D ligand expression by primary human carcinomas positively correlated with EGFR expression, which is commonly hyperactivated in such tumors, and was reduced by clinical EGFR inhibitors. Therefore, stress-induced activation of EGFR not only regulates cell growth but also concomitantly regulates the cells’ immunological visibility. Thus, therapeutics designed to limit cancer cell growth should also be considered in terms of their impact on immunosurveillance.

Heteromeric interactions regulate butyrophilin (BTN) and BTN-like molecules governing γδ T cell biology

Significance Although gamma delta (γδ) T cells compose an evolutionarily conserved third lineage of diversified lymphocytes, alongside αβ T cells and B cells, they can seem overtly different across species and tissues. Thus, human blood γδ cells show butyrophilin (BTN)3A1-dependent responses to metabolites (“phosphoantigens”) not seen by rodent γδ cells, whereas some rodent, γδ-rich compartments, notably in the skin, lack obvious human counterparts. Recently, however, mouse and human intraepithelial gut γδ cells were found to be regulated by pairings of BTN-like genes. This study now shows that BTN3A1 also functions as a pairing, with its subcellular trafficking and optimal activity both regulated by BTN3A2. Hence, seemingly diverse γδ cell biologies across species and tissues are underpinned by conserved mechanisms. The long-held view that gamma delta (γδ) T cells in mice and humans are fundamentally dissimilar, as are γδ cells in blood and peripheral tissues, has been challenged by emerging evidence of the cells’ regulation by butyrophilin (BTN) and butyrophilin-like (BTNL) molecules. Thus, murine Btnl1 and the related gene, Skint1, mediate T cell receptor (TCR)-dependent selection of murine intraepithelial γδ T cell repertoires in gut and skin, respectively; BTNL3 and BTNL8 are TCR-dependent regulators of human gut γδ cells; and BTN3A1 is essential for TCR-dependent activation of human peripheral blood Vγ9Vδ2+ T cells. However, some observations concerning BTN/Btnl molecules continue to question the extent of mechanistic conservation. In particular, murine and human gut γδ cell regulation depends on pairings of Btnl1 and Btnl6 and BTNL3 and BTNL8, respectively, whereas blood γδ cells are reported to be regulated by BTN3A1 independent of other BTNs. Addressing this paradox, we show that BTN3A2 regulates the subcellular localization of BTN3A1, including functionally important associations with the endoplasmic reticulum (ER), and is specifically required for optimal BTN3A1-mediated activation of Vγ9Vδ2+ T cells. Evidence that BTNL3/BTNL8 and Btnl1/Btnl6 likewise associate with the ER reinforces the prospect of broadly conserved mechanisms underpinning the selection and activation of γδ cells in mice and humans, and in blood and extralymphoid sites.

A long-playing CD about the γδ TCR repertoire.

In this issue of Immunity, a study by Luoma et al. (2013) provides structural evidence for direct interactions of human Vδ1(+) T cell receptors with CD1d, capping a long trail of evidence that CD1 might be a major influence on γδ T cell biology.

Abstract IA20: Targeting molecules mediating the dialogue between epithelial cells and local T cells

Some extraordinary clinical successes of immunotherapy have had game-changing effects on the way we view cancer biology and the interaction of the immune system with our tissues. The conventional context for considering cancer immunology is one in which dendritic cells and/or monocytes present cancer antigens, particularly neo-antigens generated by somatic mutation, to systemic T cells within cancer-draining lymph nodes, as a result of which an expansive adaptive, cancer-specific T lymphocyte-driven immune response develops. Furthermore, cancer-specific T cells may become tissue-associated, maintaining a so-called Tissue-Resident Memory (TRM) pool. However, although this perspective readily explains many basic biological and clinical data, it leaves several issues unanswered. For example, does the immune system recognize tumor cells that do not carry high mutational loads, many of which cancers have shown disappointing responses to immunotherapy? Related to this, what is the nature of cancer immunogenicity that is required to first activate the immune system, thereby promoting the presentation of cancer antigens by DC and/ or monocyte? We have addressed these issues by considering a separate T cell compartment that is constitutively tissue-resident, rather than being primed in the lymphoid circulation. Prominent within this T cell compartment are intraepithelial gamma delta T cells that have been particularly well characterized in mice, wherein they have been shown to limit sensitivity to carcinogens, and to promote tissue integrity 1,2 . Moreover, a recent bio-informatic analysis showed a tumor-associated gamma delta T cell signature to be the strongest gene expression correlate with overall survival in over 18,000 patients across 39 tumor types 3 . Furthermore, another very strong correlate was the expression of NKG2D, a cell surface receptor expressed on tissue-resident gamma delta T cells and whose ligands are upregulated on tumor cells by dysregulation of the EGF-receptor pathway 4 . Given these observations, we have asked several questions: [1] what is the nature of tissue-resident gamma delta T cell compartments in human skin, gut, and breast tissue? [2] do such cells become activated solely by the upregulation of NKG2D ligands, thereby offering an explanation for tumor immunogenicity? [3] what are the effector functions and potentials of such cells? [4] what are the molecular checkpoints by which the cells are regulated? [5] can the cells be explanted, grown, and cultured in a manner that preserves their function(s) and that can provide a source of such cells to be used for adoptive immunotherapy? [6] might such cells be applied “off-the-shelf” rather than requiring autologous transfusions? Emerging answers to these questions will be provided. Thus, by examining over 100 donors, we have found that human skin, gut, and breast reproducibly harbor large and complex T cell subsets that include substantive gamma delta T cell compartments clearly distinct from most blood-borne gd T cells. These tissue-resident gamma delta T cells show strong, innate-like responses to NKG2D-ligands and to cytokines that were not shown by CD8 + TRM cells harvested in parallel from the same sites. Additionally, however, tissue-resident gamma delta T cells are uniquely and powerfully regulated at steady-state by novel, CD80/B7/PDL1-like molecules known as butyrophilin-like (BTNL) proteins that are expressed by epithelial cells in a tissue-specific fashion, and that de facto constitute local checkpoints. These emerging insights create a new context in which to consider the interaction of immune cells with developing cancers, and possibly to explain the patient benefit of a strong gamma delta T cell signature. At the same time, we have developed novel methods for maintaining and expanding tissue-derived gamma delta T cells that offer a practical off-the-shelf approach to tissue-targeted cellular immunotherapy, and that may be particularly suited to adenocarcinomas of low mutation load or with high rates of tumor evolution and antigenic drift. Citation Format: Oliver Nussbaumer, Yin Wu, Fernanda Kyle, Rick Woolf, Robin Dart, Deborah Enting, Pierre Vantourout, Adrian Hayday. Targeting molecules mediating the dialogue between epithelial cells and local T cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA20.