Daniel V. Correia

Differentiation of human peripheral blood Vδ1+ T cells expressing the natural cytotoxicity receptor NKp30 for recognition of lymphoid leukemia cells

The success of cancer immunotherapy depends on productive tumor cell recognition by killer lymphocytes. γδ T cells are a population of innate-like lymphocytes endowed with strong, MHC-unrestricted cytotoxicity against tumor cells. This notwithstanding, we recently showed that a large proportion of human hematologic tumors is resistant to γδ peripheral blood lymphocytes (PBLs) activated with specific agonists to the highly prevalent Vγ9Vδ2 TCR. Although this probably constitutes an important limitation to current γδ T cell-mediated immunotherapy strategies, we describe here the differentiation of a novel subset of Vδ2(-) Vδ1(+) PBLs expressing natural cytotoxicity receptors (NCRs) that directly mediate killing of leukemia cell lines and chronic lymphocytic leukemia patient neoplastic cells. We show that Vδ1(+) T cells can be selectively induced to express NKp30, NKp44 and NKp46, through a process that requires functional phosphatidylinositol 3-kinase (PI-3K)/AKT signaling on stimulation with γ(c) cytokines and TCR agonists. The stable expression of NCRs is associated with high levels of granzyme B and enhanced cytotoxicity against lymphoid leukemia cells. Specific gain-of-function and loss-of-function experiments demonstrated that NKp30 makes the most important contribution to TCR-independent leukemia cell recognition. Thus, NKp30(+) Vδ1(+) T cells constitute a novel, inducible and specialized killer lymphocyte population with high potential for immunotherapy of human cancer.

The MHC class Ib protein ULBP1 is a nonredundant determinant of leukemia/lymphoma susceptibility to γδ T-cell cytotoxicity

On the path to successful immunotherapy of hematopoietic tumors, gammadelta T cells offer great promise because of their human leukocyte antigen (HLA)-unrestricted targeting of a wide variety of leukemias/lymphomas. However, the molecular mechanisms underlying lymphoma recognition by gammadelta T cells remain unclear. Here we show that the expression levels of UL16-binding protein 1 (ULBP1) determine lymphoma susceptibility to gammadelta T cell-mediated cytolysis. Consistent with this, blockade of NKG2D, the receptor for ULBP1 expressed on all Vgamma9(+) T cells, significantly inhibits lymphoma cell killing. Specific loss-of-function studies demonstrate that the role of ULBP1 is nonredundant, highlighting a thus far unique physiologic relevance for tumor recognition by gammadelta T cells. Importantly, we observed a very wide spectrum of ULBP1 expression levels in primary biopsies obtained from lymphoma and leukemia patients. We suggest this will impact on the responsiveness to gammadelta T cell-based immunotherapy, and therefore propose ULBP1 to be used as a leukemia/lymphoma biomarker in upcoming clinical trials.

Non-classical major histocompatibility complex proteins as determinants of tumour immunosurveillance

Tumours develop in vertebrate organisms endowed with immune systems that are potentially able to eradicate them. Nevertheless, our ever‐increasing understanding of the complex interactions between lymphocytes and tumour cells fuels the long‐standing hope of developing efficient immunotherapies against cancer. This review focuses on a versatile family of proteins, the major histocompatibility complex class Ib, which has been recently implicated in both the establishment of anti‐tumour immune responses and in tumour immune response evasion. We focus on a subset of class Ib proteins, human leukocyte antigen (HLA)‐G, Qa‐2, CD1d and NKG2D ligands, which bind to either stimulatory or inhibitory receptors expressed on T, natural killer (NK) and NKT lymphocytes, and thereby modulate their anti‐tumour activity.

Identification of a panel of ten cell surface protein antigens associated with immunotargeting of leukemias and lymphomas by peripheral blood γδ T cells

Background Vγ9Vδ2 T lymphocytes are regarded as promising mediators of cancer immunotherapy due to their capacity to eliminate multiple experimental tumors, particularly within those of hematopoietic origin. However, Vγ9Vδ2 T-cell based lymphoma clinical trials have suffered from the lack of biomarkers that can be used as prognostic of therapeutic success. Design and Methods We have conducted a comprehensive study of gene expression in acute lymphoblastic leukemias and non-Hodgkin’s lymphomas, aimed at identifying markers of susceptibility versus resistance to Vγ9Vδ2 T cell-mediated cytotoxicity. We employed cDNA microarrays and quantitative real-time PCR to screen 20 leukemia and lymphoma cell lines, and 23 primary hematopoietic tumor samples. These data were analyzed using state-of-the-art bioinformatics, and gene expression patterns were correlated with susceptibility to Vγ9Vδ2 T cell mediated cytolysis in vitro. Results We identified a panel of 10 genes encoding cell surface proteins that were statistically differentially expressed between “γδ-susceptible” and “γδ-resistant” hematopoietic tumors. Within this panel, 3 genes (ULBP1, TFR2 and IFITM1) were associated with increased susceptibility to Vγ9Vδ2 T-cell cytotoxicity, whereas the other 7 (CLEC2D, NRP2, SELL, PKD2, KCNK12, ITGA6 and SLAMF1) were enriched in resistant tumors. Furthermore, some of these candidates displayed a striking variance of expression among primary follicular lymphomas and T-cell acute lymphoblastic leukemias. Conclusions Our results suggest that hematopoietic tumors display a highly variable repertoire of surface proteins that can impact on Vγ9Vδ2 cell-mediated immunotargeting. The prognostic value of the proposed markers can now be evaluated in upcoming Vγ9Vδ2 T cell-based lymphoma/leukemia clinical trials.

Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human γδ T-Cells

Background The unique responsiveness of Vγ9Vδ2 T-cells, the major γδ subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current γδ T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human γδ T-cells remain unclear. In particular, previous reports have described a very slow kinetics of activation of T-cell receptor (TCR)-associated signal transduction pathways by isopentenyl pyrophosphate and bromohydrin pyrophosphate, seemingly incompatible with direct binding of these antigens to the Vγ9Vδ2 TCR. Here we have studied the most potent natural phosphoantigen yet identified, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by Eubacteria and Protozoa, and examined its γδ T-cell activation and anti-tumor properties. Methodology/Principal Findings We have performed a comparative study between HMB-PP and the anti-CD3ε monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular γδ T-cell activation events. We show that HMB-PP activates MEK/Erk and PI-3K/Akt pathways as rapidly as OKT3, and induces an almost identical transcriptional profile in Vγ9+ T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including γδ T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vγ9+ TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains γδ T-cell activation upon re-stimulation. This ultimately translates in potent human γδ T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice, Conclusions/Significance The development of efficient cancer immunotherapy strategies critically depends on our capacity to maximize anti-tumor effector T-cell responses. By characterizing the intracellular mechanisms of HMB-PP-mediated activation of the highly cytotoxic Vγ9+ T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials.

Engagement of NKp30 on Vδ1 T-cells induces the production of CCL3, CCL4 and CCL5 and suppresses HIV-1 replication

Natural cytotoxicity receptors (NCRs) were originally identified as specific natural killer cell activating receptors that, on binding to their endogenous ligands, trigger the killing of tumor cell targets. We recently described the differentiation of a novel subset of NCR(+) Vδ1 T cells characterized by a remarkably high cytolytic potential against cancer cells. Here we demonstrate that the engagement of NKp30, one of the NCRs expressed de novo on Vδ1 T cells after stimulation, triggers the production of high levels of CCL3/MIP-1α, CCL4/ MIP-1β, and CCL5/RANTES but not of CXCL12/SDF-1. In turn, this NKp30-induced secretion of cc-chemokines is able to significantly suppress the replication of a CCR5 tropic strain of HIV-1 in CD4(+)/CCR5(+) infected PM1 cell lines. This experimental evidence disclosing an unanticipated antiviral function of NCR(+) Vδ1 T cells opens new avenues for understanding the pathogenic role and for manipulating the function of γδ T cells in HIV-1 infection.

Human γδ Thymocytes Are Functionally Immature and Differentiate into Cytotoxic Type 1 Effector T Cells upon IL-2/IL-15 Signaling

Cytotoxicity and IFN-γ production by human γδ T cells underlie their potent antitumor functions. However, it remains unclear where and how human γδ T cells acquire these key effector properties. Given the recent disclosure of a major contribution of the thymus to murine γδ T cell functional differentiation, in this study we have analyzed a series of human pediatric thymuses. We found that ex vivo–isolated γδ thymocytes produced negligible IFN-γ and lacked cytolytic activity against leukemia cells. However, these properties were selectively acquired upon stimulation with IL-2 or IL-15, but not IL-4 or IL-7. Unexpectedly, TCR activation was dispensable for these stages of functional differentiation. The effects of IL-2/IL-15 depended on MAPK/ERK signaling and induced de novo expression of the transcription factors T-bet and eomesodermin, as well as the cytolytic enzyme perforin, required for the cytotoxic type 1 program. These findings have implications for the manipulation of γδ T cells in cancer immunotherapy.

Inhibition of murine γδ lymphocyte expansion and effector function by regulatory αβ T cells is cell-contact-dependent and sensitive to GITR modulation

γδ T cells are highly cytolytic lymphocytes that produce large amounts of pro‐inflammatory cytokines during immune responses to multiple pathogens. Furthermore, their ability to kill tumor cells has fueled the development of γδ‐T‐cell‐based cancer therapies. Thus, the regulation of γδ‐T‐cell activity is of great biological and clinical relevance. Here, we show that murine CD4+CD25+ αβ T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of γδ T cells, namely the production of the pro‐inflammatory cytokines, IFN‐γ and IL‐17, cytotoxicity, and lymphocyte proliferation in vitro and in vivo. We further show that suppression is dependent on cellular contact between Treg and γδ T cells, results in the induction of an anergic state in γδ lymphocytes, and can be partially reversed by manipulating glucocorticoid‐induced TNF receptor‐related protein (GITR) signals. Our data collectively dissect a novel mechanism by which the expansion and pro‐inflammatory functions of γδ T cells are regulated.

Tumor cell recognition by γδ T lymphocytes: T-cell receptor vs. NK-cell receptors

The dissection of the molecular mechanisms underlying tumor-cell recognition by γδ T-cells is crucial to improve their performance in cancer immunotherapy. Here, we discuss the controversy around the relative contributions of the γδ T-cell receptor (TCR) and natural killer receptors (NKRs) to tumor-cell targeting by γδ T cells.

Delta One T cells for immunotherapy of chronic lymphocytic leukemia: clinical-grade expansion/ differentiation and preclinical proof-of-concept

Purpose: The Vδ1+ subset of γδ T lymphocytes is a promising candidate for cancer immunotherapy, but the lack of suitable expansion/differentiation methods has precluded therapeutic application. We set out to develop and test (preclinically) a Vδ1+ T-cell–based protocol that is good manufacturing practice compatible and devoid of feeder cells for prompt clinical translation. Experimental design: We tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (more than 2–3 weeks) Vδ1+ T cells from the peripheral blood of healthy donors and patients with chronic lymphocytic leukemia (CLL). We characterized the phenotype and functional potential of the final cellular product, termed Delta One T (DOT) cells, in vitro and in vivo (xenograft models of CLL). Results: We describe a very robust two-step protocol for the selective expansion (up to 2,000-fold in large clinical-grade cell culture bags) and differentiation of cytotoxic Vδ1+ (DOT) cells. These expressed the natural cytotoxicity receptors, NKp30 and NKp44, which synergized with the T-cell receptor to mediate leukemia cell targeting in vitro. When transferred in vivo, DOT cells infiltrated tumors and peripheral organs, and persisted until the end of the analysis without showing signs of loss of function; indeed, DOT cells proliferated and produced abundant IFNγ and TNFα, but importantly no IL17, in vivo. Critically, DOT cells were capable of inhibiting tumor growth and preventing dissemination in xenograft models of CLL. Conclusions: We provide a clinical-grade method and the preclinical proof of principle for application of a new cellular product, DOT cells, in adoptive immunotherapy of CLL. Clin Cancer Res; 22(23); 5795–804. ©2016 AACR.