Cytomegalovirus and tumor stress surveillance by binding of a human γδ T cell antigen receptor to endothelial protein C receptor.

T cells bearing γδ T cell antigen receptors (TCRs) function in lymphoid stress surveillance. However, the contribution of γδ TCRs to such responses is unclear. Here we found that the TCR of a human V(γ)4V(δ)5 clone directly bound endothelial protein C receptor (EPCR), which allowed γδ T cells to recognize both endothelial cells targeted by cytomegalovirus and epithelial tumors. EPCR is a major histocompatibility complex-like molecule that binds lipids analogously to the antigen-presenting molecule CD1d. However, the V(γ)4V(δ)5 TCR bound EPCR independently of lipids, in an antibody-like way. Moreover, the recognition of target cells by γδ T cells required a multimolecular stress signature composed of EPCR and costimulatory ligand(s). Our results demonstrate how a γδ TCR mediates recognition of broadly stressed human cells by engaging a stress-regulated self antigen.

Sensing of cell stress by human γδ TCR-dependent recognition of annexin A2.

Human γδ T cells comprise a first line of defense through T-cell receptor (TCR) recognition of stressed cells. However, the molecular determinants and stress pathways involved in this recognition are largely unknown. Here we show that exposure of tumor cells to various stress situations led to tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously developed to identify antigenic ligands of γδ TCRs, annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was found to be expressed on tumor cells upon the stress situations tested in a reactive oxygen species-dependent manner. Moreover, purified annexin A2 was able to stimulate the proliferation of a Vδ2neg γδ T-cell subset within peripheral blood mononuclear cells and other annexin A2-specific Vδ2neg γδ T-cell clones could be derived from peripheral blood mononuclear cells. We thus propose membrane exposure of annexin A2 as an oxidative stress signal for some Vδ2neg γδ T cells that could be involved in an adaptive stress surveillance.

γδ T cells confer protection against murine cytomegalovirus (MCMV).

Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. γδ T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 αß and/or γδ T cell-deficient mice, we here show that γδ T cells are as competent as αß T cells to protect mice from CMV-induced death. γδ T cell-mediated protection involved control of viral load and prevented organ damage. γδ T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3ε-/- mice from CMV-induced death confirming the protective antiviral role of γδ T cells. As observed in humans, different γδ T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27- γδ T cells. NK cells were the largely preponderant producers of IFNγ and cytotoxic granules throughout the infection, suggesting that the protective role of γδ T cells did not principally rely on either of these two functions. Finally, γδ T cells were strikingly sufficient to fully protect Rag-/-γc-/- mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of γδ T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new γδ T cell based therapies, especially useful in αß T cell compromised patients.

Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset.

Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.

Human NLRC4 expression promotes cancer survival and associates with type I interferon signaling and immune infiltration.

The immune system can control cancer progression. However, even though some innate immune sensors of cellular stress are expressed intrinsically in epithelial cells, their potential role in cancer aggressiveness and subsequent overall survival in humans is mainly unknown. Here, we show that nucleotide-binding oligomerization domain-like receptor (NLR) family CARD domain-containing 4 (NLRC4) is downregulated in epithelial tumor cells of patients with colorectal cancer (CRC) by using spatial tissue imaging. Strikingly, only the loss of tumor NLRC4, but not stromal NLRC4, was associated with poor immune infiltration (mainly DCs and CD4+ and CD8+ T cells) and accurately predicted progression to metastatic stage IV and decrease in overall survival. By combining multiomics approaches, we show that restoring NLRC4 expression in human CRC cells triggered a broad inflammasome-independent immune reprogramming consisting of type I interferon (IFN) signaling genes and the release of chemokines and myeloid growth factors involved in the tumor infiltration and activation of DCs and T cells. Consistently, such reprogramming in cancer cells was sufficient to directly induce maturation of human DCs toward a Th1 antitumor immune response through IL-12 production in vitro. In multiple human carcinomas (colorectal, lung, and skin), we confirmed that NLRC4 expression in patient tumors was strongly associated with type I IFN genes, immune infiltrates, and high microsatellite instability. Thus, we shed light on the epithelial innate immune sensor NLRC4 as a therapeutic target to promote an efficient antitumor immune response against the aggressiveness of various carcinomas.

Anti-metastatic potential of human Vδ1(+) γδ T cells in an orthotopic mouse xenograft model of colon carcinoma.

The role of human intraepithelial Vδ1(+) γδ T cell cytotoxic effectors in the immune surveillance against metastatic colon cancer has never been addressed, despite their reported capacity to infiltrate colon carcinomas and to kill colonic cancer cells in vitro. We previously showed that Vδ1(+) γδ T cells are enriched in blood in response to cytomegalovirus (CMV) infection, and that such increase may be protective against epithelial cancers. The objective of the present study was to investigate whether CMV-induced Vδ1(+) γδ T lymphocytes could inhibit the propagation of human colon tumors in vivo, in order to evaluate their immunotherapeutic potential in this context. Even though metastases are an important cause of death in various cancers including colorectal cancer (CRC), the anti-metastatic effect of immune effectors has been poorly analyzed. To this purpose, we set up a reliable model of metastatic colon cancer through orthotopic implantation of luciferase-expressing human HT29 cells in immunodeficient mice. Using bioluminescence imaging to follow the outcome of colonic cancer cells, we showed that a systemic treatment with CMV-induced Vδ1(+) γδ T cells could not only inhibit primary colon tumor growth but also the emergence of secondary tumor foci in the lungs and liver. Finally, our data lead to propose that Vδ1(+) γδ T lymphocytes may directly influence the appearance of metastases independently from their control of primary tumor size. These findings, which extend our previous work, pave the road for the potential manipulation of Vδ1(+) γδ T lymphocytes in novel anti-CRC immunotherapeutic protocols.

Up-regulation of cytolytic functions of human Vδ2-γ T lymphocytes through engagement of ILT2 expressed by tumor target cells.

In humans, the majority of peripheral blood γδ T cells expresses Vγ9Vδ2 T-cell receptors (TCR) and recognize nonpeptidic phosphorylated antigens. In contrast, most tissue-derived γδ T cells, which are located mainly in spleen and epithelia, preferentially use Vδ1 or Vδ3 chains paired with diverse Vγ chains to form their TCR. Our knowledge about the antigenic specificity and costimulation requirements of human Vδ2(-) γδ T cells remains limited. In an attempt to address this important issue, we characterized the specificity of a monoclonal antibody (mAb 256), screened for its ability to specifically inhibit cytolytic responses of several human Vδ2(-) γδ T-cell clones against transformed B cells. We show that mAb 256 does not target a TCR ligand but blocks key interactions between non-TCR molecules on effector γδ T cells and ILT2 molecule, expressed by tumor targets. In line with the previously reported specificity of this NK receptor for classic and nonclassic major histocompatibility complex (MHC) class I molecules, blockade of MHC class I/ILT2 interactions using MHC class I- or ILT2-specific mAbs and ILT2-Fc molecules inhibited tumor-induced activation of Vγ8Vδ3 T-cell clones. Therefore, this study describes a new cytotoxic T lymphocyte activation pathway involving MHC class I engagement on γδ T cells.

Human γδ T cell sensing of AMPK-dependent metabolic tumor reprogramming through TCR recognition of EphA2.

Human γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)-dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non-MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer.

Common features of gammadelta T cells and CD8(+) alphabeta T cells responding to human cytomegalovirus infection in kidney transplant recipients.

BACKGROUND: Kidney transplant recipients infected with cytomegalovirus (CMV) undergo a persistent gammadelta T cell expansion in their peripheral blood. The anti-CMV function of these cells was previously demonstrated by their ability to kill CMV-infected cells in vitro. METHODS: To gain insight into the role of gammadelta T cells within the antiviral immune network, we compared the expansion kinetics of these T cells with that of CMV pp65-specific CD8(+) alphabeta T cells in the peripheral blood of twenty-one kidney transplant recipients. RESULTS: Both the percentage and the absolute number of pp65-specific CD8(+) T cells and gammadelta T cells showed a concomitant increase and persistence in most of the kidney transplant recipients with CMV infection. Both cell subsets exhibited an effector/memory phenotype (CD28(-), CD27(-), and CD45RA(+)) that predominated for the entire follow-up period. CONCLUSIONS: In conclusion, CMV-specific CD8(+) alphabeta T cells and gammadelta T cells share common expansion kinetics and a common effector phenotype, suggesting that these cell types act similarly in response to CMV infection.

Ectopic expression of the ets transcription factor ER81 in transgenic mouse mammary gland enhances both urokinase plasminogen activator and stromelysin-1 transcription.

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.