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Supplementary Figures 1-5 and tables 1-3

Acute myeloid leukemia (AML) remains a clinical challenge due to frequent chemotherapy resistance and deadly relapses. We are exploring the immunotherapeutic potential of peripheral blood Vδ1+ T cells, which associate with improved long-term survival of stem-cell transplant recipients but have not yet been applied as adoptive cell therapy. Using our clinical-grade protocol for expansion and differentiation of “Delta One T” (DOT) cells, we found DOT cells to be highly cytotoxic against AML primary samples and cell lines, including cells selected for resistance to standard chemotherapy. Unlike chemotherapy, DOT-cell targeting did not select for outgrowth of specific AML lineages, suggesting a broad recognition domain, an outcome that was consistent with the polyclonality of the DOT-cell T-cell receptor (TCR) repertoire. However, AML reactivity was only slightly impaired upon Vδ1+ TCR antibody blockade, whereas it was strongly dependent on expression of the NKp30 ligand, B7-H6. In contrast, DOT cells did not show reactivity against normal leukocytes, including CD33+ or CD123+ myeloid cells. Adoptive transfer of DOT cells in vivo reduced AML load in the blood and target organs of multiple human AML xenograft models and significantly prolonged host survival without detectable toxicity, thus providing proof-of-concept for DOT-cell application in AML treatment.

S1. LDL-cholesterol does not affect the viability of human γÎ' T cells. S2. Effects of LDL-cholesterol on Vï�¤2+ T cells after 36 or 48 hours of culture. S3. Effects of LDL-cholesterol on additional immune functions of γÎ' T cells. S4. Effect of LDL-cholesterol on expanded Vγ9VÎ'2 T cells cultured in the presence of human plasma. S5. Effects of receptor blockade on breast cancer cell targeting by γÎ' T lymphocytes. S6: Cholesterol-rich diet reduces multifunctional anti-tumour γÎ' infiltrating lymphocytes.

Vγ9Vδ2 T cells, the main subset of γδ T lymphocytes in human peripheral blood, are endowed with antitumor functions such as cytotoxicity and IFNγ production. These functions are triggered upon T-cell receptor–dependent activation by non-peptidic prenyl pyrophosphates (“phosphoantigens”) that are selective agonists of Vγ9Vδ2 T cells, and which have been evaluated in clinical studies. Because phosphoantigens have shown interindividual variation in Vγ9Vδ2 T-cell activities, we asked whether metabolic resources, namely lipids such as cholesterol, could affect phosphoantigen-mediated Vγ9Vδ2 T-cell activation and function. We show here that Vγ9Vδ2 T cells express the LDL receptor upon activation and take up LDL cholesterol. Resulting changes, such as decreased mitochondrial mass and reduced ATP production, correlate with downregulation of Vγ9Vδ2 T-cell activation and functionality. In particular, the expression of IFNγ, NKG2D, and DNAM-1 were reduced upon LDL cholesterol treatment of phosphoantigen-expanded Vγ9Vδ2 T cells. As a result, their capacity to target breast cancer cells was compromised both in vitro and in an in vivo xenograft mouse model. Thus, this study describes the role of LDL cholesterol as an inhibitor of the antitumor functions of phosphoantigen-activated Vγ9Vδ2 T cells. Our observations have implications for therapeutic applications dependent on Vγ9Vδ2 T cells. Cancer Immunol Res; 6(4); 448–57. ©2018 AACR.

Supplemental Methods, Reagents and Materials for DOT-cell production and testing; detailed DOT-cell phenotyping and yields; and complementary in vivo analyses.

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.

Chimeric Antigen Receptor (CAR)-T cells have emerged as a breakthrough treatment for relapse/refractory (r/r) hematological tumors, showing impressive complete remission rates in B-cell malignancies. However, around 50% of the patients relapse before 1-year post-treatment. T-cell “fitness” is critical to prolong the persistence and activity of the adoptively transferred product. Allogeneic T cells from healthy donors are less dysfunctional or exhausted than autologous patient-derived T cells, enabling a very attractive and cost-effective “off-the-shelf” therapy option. In this context, Delta One T cells (DOTs), a recently described cellular product based on MHC/HLA-independent Vδ1+ γδ T cells generated from the peripheral blood of healthy donors, represent a robust platform of allogeneic effector T cells. Here we generated and pre-clinically validated 4-1BB-based CAR-DOTs directed against the IL-3α chain receptor (CD123), a target antigen widely expressed on acute myeloid leukemia (AML) blasts. CD123CAR-DOTs showed vigorous, superior to control DOTs, cytotoxicity against AML cell lines and primary samples both in vitro and in vivo. Continuous administration of IL-15 supported the long-term persistence of a single-dose CD123CAR-DOTs in patient-derived xenograft models, sustaining their anti-leukemic efficacy as demonstrated in a re-challenge assay in vivo. Our results provide proof-of-concept for an allogeneic next-generation therapy based on CD123CAR-DOTs for r/r AML patients.KEY POINTS- CD123CAR-DOTs exert specific and robust cytotoxicity in vitro and anti-leukemic activity in vivo against AML cell lines and primary cells.- CD123CAR-DOTs show IL-15-dependent long-term persistence in vivo and vigorous anti-leukemic activity upon re-challenge.

Acute myeloid leukemia (AML) remains a clinical challenge due to frequent chemotherapy resistance and deadly relapses. We are exploring the immunotherapeutic potential of peripheral blood Vd1 þ T cells, which associate with improved long-term survival of stem-cell transplant recipients but have not yet been applied as adoptive cell therapy. Using our clinical-grade protocol for expansion and differentiation of "Delta One T" (DOT) cells, we found DOT cells to be highly cytotoxic against AML primary samples and cell lines, including cells selected for resistance to standard chemotherapy. Unlike chemotherapy, DOT-cell targeting did not select for outgrowth of specific AML lineages, suggesting a broad recognition domain, an outcome that was consistent with the polyclonality of the DOT-cell T-cell receptor (TCR) repertoire. However, AML reactivity was only slightly impaired upon Vd1 þ TCR antibody blockade, whereas it was strongly dependent on expression of the NKp30 ligand, B7-H6. In contrast, DOT cells did not show reactivity against normal leukocytes, including CD33 þ or CD123 þ myeloid cells. Adoptive transfer of DOT cells in vivo reduced AML load in the blood and target organs of multiple human AML xenograft models and significantly prolonged host survival without detectable toxicity, thus providing proof-of-concept for DOT-cell application in AML treatment., SCOPUS: ar.j, info:eu-repo/semantics/published

Vγ9Vδ2 T cells, the main subset of γδ T lymphocytes in human peripheral blood, are endowed with antitumor functions such as cytotoxicity and IFNγ production. These functions are triggered upon T-cell receptor–dependent activation by non-peptidic prenyl pyrophosphates (“phosphoantigens”) that are selective agonists of Vγ9Vδ2 T cells, and which have been evaluated in clinical studies. Because phosphoantigens have shown interindividual variation in Vγ9Vδ2 T-cell activities, we asked whether metabolic resources, namely lipids such as cholesterol, could affect phosphoantigen-mediated Vγ9Vδ2 T-cell activation and function. We show here that Vγ9Vδ2 T cells express the LDL receptor upon activation and take up LDL cholesterol. Resulting changes, such as decreased mitochondrial mass and reduced ATP production, correlate with downregulation of Vγ9Vδ2 T-cell activation and functionality. In particular, the expression of IFNγ, NKG2D, and DNAM-1 were reduced upon LDL cholesterol treatment of phosphoantigen-expanded Vγ9Vδ2 T cells. As a result, their capacity to target breast cancer cells was compromised both in vitro and in an in vivo xenograft mouse model. Thus, this study describes the role of LDL cholesterol as an inhibitor of the antitumor functions of phosphoantigen-activated Vγ9Vδ2 T cells. Our observations have implications for therapeutic applications dependent on Vγ9Vδ2 T cells. Cancer Immunol Res; 6(4); 448–57. ©2018 AACR.

© 2014 by The American Association of Immunologists, Inc., 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., This work was supported by Fundação para a Ciência e Tecnologia Grant EXPL/BIM-ONC/0490/2012 and by the Young Investigator Program of the European Molecular Biology Organization.

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.

Copyright © 2007 European Molecular Biology Organization, 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., We thank Fundação para a Ciência e Tecnologia of the Portuguese Ministry of Science and Technology [PTDC/SAU-MII/71662/2006] for support. B.S.-S. is a European Molecular Biology Organization (EMBO) Installation Grantee and acknowledges the EMBO's Young Investigator Programme.

γδ T cells are innate-like lymphocytes that typically account for 1–5 % of human peripheral blood lymphocytes. Several properties of γδ T cells make them attractive targets for cancer immunotherapy, namely, their MHC-unrestricted recognition of tumors, potent cytotoxicity, and abundant production of interferon-γ (IFN-γ) and tumor necrosis factor (TNF). Moreover, seminal work with mice genetically deficient for γδ T cells has clearly shown they constitute a key, nonredundant component of cancer immunosurveillance in vivo.

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.

© 2011 by The American Society of Hematology, 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., This work was supported by the Young Investigator Program of the European Molecular Biology Organization and Fundação Calouste Gulbenkian SDH Oncologia 2008/99293 (B.S.-S.); and by the Italian Ministry of Health (Ricerca Finalizzata, ICH-2007-643769), the European Union (Marie Curie International Reintegration Grant, 204188), and Istituto Clinico Humanitas (Intramural Research Program 20090917; D.M.). B.S.-S. is also funded by the European Research Council (StG260352), and D.V.C. is a PhD fellow of Fundação para a Ciência e Tecnologia (SFRH/BD/37898/2007).

© 2010 by The American Society of Hematology, On the path to successful immunotherapy of hematopoietic tumors, γδ 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 γδ T cells remain unclear. Here we show that the expression levels of UL16-binding protein 1 (ULBP1) determine lymphoma susceptibility to γδ T cell–mediated cytolysis. Consistent with this, blockade of NKG2D, the receptor for ULBP1 expressed on all Vγ9+ 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 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 γδ T cell–based immunotherapy, and therefore propose ULBP1 to be used as a leukemia/lymphoma biomarker in upcoming clinical trials., This work was distinguished with the Pfizer Award for ClinicalResearch 2009. This work was further supported by Fundação para a Ciência e Tecnologia (PTDC/SAU-MII/71662/2006) and Fundação Calouste Gulbenkian (SDH Oncologia 2008; Projecto 99293). L.F.M. is a Young Investigator from the Human Frontier Science Program and was supported by Fundação Luso-Americana para o Desenvolvimento and Fundação para a Ciência e Tecnologia (FCT; PTDC/SAU-MII/69280/2006 and PTDC/SAU-MII/78333/2006). T.L. and D.V.C. received PhD fellowships (47342/2008 and 37898/2007) from FCT.

© 2010 WILEY, 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., This work was supported by grant PTDC/SAU-MII/71662/2006 from Fundação para a Ciência e a Tecnologia (FCT), who also provided individual fellowships to J.C.R., A.dB., D.V.C. and I.C., and by an Installation Grant from the European Molecular Biology Organization (YIP Project 1440).

BackgroundThe unique responsiveness of Vgamma9Vdelta2 T-cells, the major gammadelta subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current gammadelta T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human gammadelta 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 Vgamma9Vdelta2 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 gammadelta T-cell activation and anti-tumor properties.Methodology/principal findingsWe have performed a comparative study between HMB-PP and the anti-CD3epsilon monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular gammadelta 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 Vgamma9(+) T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including gammadelta T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vgamma9(+) TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains gammadelta T-cell activation upon re-stimulation. This ultimately translates in potent human gammadelta T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice,Conclusions/significanceThe 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 Vgamma9(+) T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials.

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.