Richard D. Lopez

Protective Immunosurveillance and Therapeutic Antitumor Activity of γδ T Cells Demonstrated in a Mouse Model of Prostate Cancer

In contrast to Ag-specific αβ T cells, γδ T cells can kill malignantly transformed cells in a manner that does not require the recognition of tumor-specific Ags. Although such observations have contributed to the emerging view that γδ T cells provide protective innate immunosurveillance against certain malignancies, particularly those of epithelial origin, they also provide a rationale for developing novel clinical approaches to exploit the innate antitumor properties of γδ T cells for the treatment of cancer. Using TRAMP, a transgenic mouse model of prostate cancer, proof-of-concept studies were performed to first establish that γδ T cells can indeed provide protective immunosurveillance against spontaneously arising mouse prostate cancer. TRAMP mice, which predictably develop prostate adenocarcinoma, were backcrossed with γδ T cell-deficient mice (TCRδ−/− mice) yielding TRAMP × TCRδ−/− mice, a proportion of which developed more extensive disease compared with control TRAMP mice. By extension, these findings were then used as a rationale for developing an adoptive immunotherapy model for treating prostate cancer. Using TRAMP-C2 cells derived from TRAMP mice (C57BL/6 genetic background), disease was first established in otherwise healthy wild-type C57BL/6 mice. In models of localized and disseminated disease, tumor-bearing mice treated i.v. with supraphysiological numbers of syngeneic γδ T cells (C57BL/6-derived) developed measurably less disease compared with untreated mice. Disease-bearing mice treated i.v. with γδ T cells also displayed superior survival compared with untreated mice. These findings provide a biological rationale for clinical trials designed to adoptively transfer ex vivo expanded autologous γδ T cells for the treatment of prostate cancer.

CD58/LFA-3 and IL-12 provided by activated monocytes are critical in the in vitro expansion of CD56+ T cells

Abstract A small proportion of human CD3+ T lymphocytes are known to co-express CD56, an antigen usually restricted in its expression to natural killer (NK) cells. Whereas the in vivo function of CD3+ CD56+ T cells remains unknown, we and others have previously shown that both in vitro and in vivo, these cells can mediate a significantly greater degree of MHC-unrestricted cytotoxicity against a variety of human tumor cells when compared to either CD3+ CD56− T cells or lymphokine activated killer (LAK) cells. While the mechanisms regulating the in vivo expansion of CD56+ T cells are not known, here we demonstrate the importance of CD2-mediated IL-12-dependent signals in the in vitro expansion of CD56+ T cells. Specifically, we show that activated monocytes provide a contact dependent factor (CD58/LFA-3) and a soluble factor (IL-12), both critical for the in vitro expansion of CD56+ T cells. The biological and therapeutic implications of these findings are discussed.

Alemtuzumab for the prevention and treatment of graft-versus-host disease

Alemtuzumab is a humanized monoclonal antibody against the CD52 antigen, which is expressed on the surface of various hematopoietic cells such as B and T lymphocytes, and has been widely used for preventing acute graft-versus-host disease (GVHD) in allogeneic stem cell transplantation (SCT). Administration of 100 mg alemtuzumab before transplantation has resulted in a low incidence of acute GVHD in HLA-matched and mismatched transplantation from either related or unrelated donors. However, because alemtuzumab could remain in the blood at the lympholytic level 1–2 months after transplantation, immune reconstitution was substantially delayed, leading to a high incidence of viral infection and relapse. A dose reduction of alemtuzumab was attempted in a reduced-intensity conditioning setting to facilitate immune reconstitution, and this resulted in earlier immune reconstitution, but the clinical benefits were unclear. The dose of alemtuzumab and the timing of its administration should be optimized to maximize the benefit of acute GVHD suppression and minimize the risk of infection and relapse. Another strategy to facilitate immune reconstitution and augment anti-tumor effects is donor cell infusion of T and NK cells. Although there is accumulating evidence regarding the use of alemtuzumab for acute GVHD prevention, information on the salvage treatment for steroid-refractory acute and chronic GVHD is still limited.

STAT6-independent production of IL-4 by mast cells.

The acquisition of an IL‐4‐producing phenotype in Th2 cells requires IL‐4 signaling through the STAT6 pathway during T cell differentiation. In this study we demonstrate that, unlike in naive T cells, IL‐4 is not necessary for the development of an IL‐4‐producing phenotype in mast cells. Bone marrow‐derived mast cell precursors from STAT6–/– mice can differentiate into mature cells that express IL‐4 levels comparable to those of wild‐type mast cells. In differentiated mast cells, activation in the presence of neutralizing anti‐IL‐4 antibodies or mutation of the consensus STAT6 sites does not diminish IL‐4 promoter activity, indicating thatIL‐4 is not required for active transcription. Taken together, these data suggest that mast cell IL‐4 production is not STAT6 dependent, providing evidence that these cells could generate IL‐4 needed for the initiation and amplification of an effective Th2 immune response.

Characterization and immunotherapeutic potential of γδ T-cells in patients with glioblastoma

Classical immunotherapeutic approaches to glioblastoma multiforme (GBM) have shown mixed results, and therapies focused on innate lymphocyte activity against GBM have not been rigorously evaluated. We examined peripheral blood lymphocyte phenotype, gammadelta T-cell number, mitogenic response, and cytotoxicity against GBM cell lines and primary tumor explants from GBM patients at selected time points prior to and during GBM therapy. Healthy volunteers served as controls and were grouped by age. T-cell infiltration of tumors from these patients was assessed by staining for CD3 and T-cell receptor gammadelta. Our findings revealed no differences in counts of mean absolute T-cells, T-cell subsets CD3+CD4+ and CD3+CD8+, and natural killer cells from healthy volunteers and patients prior to and immediately after GBM resection. In contrast, gammadelta T-cell counts and mitogen-stimulated proliferative response of gammadelta T-cells were markedly decreased prior to GBM resection and throughout therapy. Expanded/activated gammadelta T-cells from both patients and healthy volunteers kill GBM cell lines D54, U373, and U251, as well as primary GBM, without cytotoxicity to primary astrocyte cultures. Perivascular T-cell accumulation was noted in paraffin sections, but no organized T-cell invasion of the tumor parenchyma was seen. Taken together, these data suggest that gammadelta T-cell depletion and impaired function occur prior to or concurrent with the growth of the tumor. The significant cytotoxicity of expanded/activated gammadelta T-cells from both healthy controls and selected patients against primary GBM explants may open a previously unexplored approach to cellular immunotherapy of GBM.

Human γδ-T cells in adoptive immunotherapy of malignant and infectious diseases

Human γδ-T cells are capable of mediating both innate antitumor and antiviral activity, functions that theoretically might be exploitable in the treatment of a variety of malignant or infectious diseases. Nonetheless, experimental therapies incorporating the adoptive transfer of human γδ-T cells have remained unfeasible to date owing largely to the difficulty of isolating or expanding sufficient numbers of γδ-T cells. It is in this context that recent discoveries from our laboratory are presented. By identifying specific signaling pathways that selectively inhibit activation-induced apoptosis in apoptosis-prone γδ-T cells, we have been able to expand large numbers of viable and functional human γδ-T cells, an undertaking until now not possible. As important, these apoptosis-resistant γδ-T cells appear to retain major histocompatibility complex-unrestricted (innate) anti-tumor activity against a wide variety of human tumor cells both in vitro and in vivo. Moreover, apoptosis-resistant γδ-T cells also display potent innate antiviral activity in vitro against human immunodeficiency virus-1. Both the biologic and practical implications of these findings are considered and discussed particularly as they relate to the development of future adoptive immunotherapy strategies.

Adoptively transferred ex vivo expanded γδ-T cells mediate in vivo antitumor activity in preclinical mouse models of breast cancer

In contrast to antigen-specific αβ-T cells (adaptive immune system), γδ-T cells can recognize and lyse malignantly transformed cells almost immediately upon encounter in a manner that does not require the recognition of tumor-specific antigens (innate immune system). Given the well-documented capacity of γδ-T cells to innately kill a variety of malignant cells, efforts are now actively underway to exploit the antitumor properties of γδ-T cells for clinical purposes. Here, we present for the first time preclinical in vivo mouse models of γδ-T cell-based immunotherapy directed against breast cancer. These studies were explicitly designed to approximate clinical situations in which adoptively transferred γδ-T cells would be employed therapeutically against breast cancer. Using radioisotope-labeled γδ-T cells, we first show that adoptively transferred γδ-T cells localize to breast tumors in a mouse model (4T1 mammary adenocarcinoma) of human breast cancer. Moreover, by using an antibody directed against the γδ-T cell receptor (TCR), we determined that localization of adoptively transferred γδ-T cells to tumor is a TCR-dependant process. Additionally, biodistribution studies revealed that adoptively transferred γδ-T cells traffic differently in tumor-bearing mice compared to healthy mice with fewer γδ-T cells localizing into the spleens of tumor-bearing mice. Finally, in both syngeneic (4T1) and xenogeneic (2Lmp) models of breast cancer, we demonstrate that adoptively transferred γδ-T cells are both effective against breast cancer and are otherwise well-tolerated by treated animals. These findings provide a strong preclinical rationale for using ex vivo expanded adoptively transferred γδ-T cells as a form of cell-based immunotherapy for the treatment of breast cancer. Additionally, these studies establish that clinically applicable methods for radiolabeling γδ-T cells allows for the tracking of adoptively transferred γδ-T cells in tumor-bearing hosts.

Innate Anti-breast Cancer Immunity of Apoptosis-resistant Human γδ-T cells

SummaryWe previously identified a CD2-initiated signaling pathway which inhibits activation-induced cell death in mitogen-stimulated human γδ-T cells permitting the large-scale expansion of these cells. Here we report the innate anti-tumor activity of expanded human γδ-T cells against human breast cancer cells. Apoptosis-resistant human γδ-T cells which were expanded in vitro from cultured human peripheral blood mononuclear cells displayed lytic activity against breast cancer cell lines MDA-MB-231, MCF-7 and T-47D, but failed to kill normal human skin fibroblasts and normal human liver cells. Monoclonal antibodies (mAb) directed against the γδ-T cell receptor (TCR) or mAb directed against either the Vγ9 or the Vδ2 TCR chains were able to block γδ-T cell-mediated lysis of MDA-MB-231 cells. In addition, mAb against intercellular adhesion molecules-1 (ICAM-1/CD54) or CD18 (β subunit of ICAM-1 counter–receptor) also blocked γδ-T cell-mediated killing of MDA-MB-231 cells. Ex vivo expanded human γδ-T cells are thus able to innately recognize and kill human breast cancer cells in a γδ-TCR-dependent manner; ICAM-1 and CD18 also appear to be involved in the interactions between sensitive breast cancer cells and cytolytic γδ-T cells. As apoptosis-resistant human γδ-T cells can now readily be expanded to large numbers (clinical scale), these findings must be considered in the context of developing adoptive immunotherapy strategies to exploit γδ-T cell innate immune responses for the primary or adjuvant treatment of breast cancer.

Expression of intercellular adhesion molecule (ICAM)‐1 or ICAM‐2 is critical in determining sensitivity of pancreatic cancer cells to cytolysis by human γδ‐T cells: Implications in the design of γδ‐T‐cell‐based immunotherapies for pancreatic cancer

Background and Aims:  γδ‐T cells can recognize and kill malignant cells, particularly those of epithelial origin, through mechanisms which do not require the recognition of tumor‐specific antigens (innate immune response). This natural ability of γδ‐T cells to kill tumor cells in a tumor antigen‐independent manner provides a strong rationale for developing clinical trials designed to exploit the innate antitumor properties of γδ‐T cells.

Down-regulation of IL-2 receptor α (CD25) characterizes human γδ-T cells rendered resistant to apoptosis after CD2 engagement in the presence of IL-12

Abstract. We recently identified a CD2-mediated, IL-12-dependent signaling pathway that inhibits apoptosis in mitogen-stimulated human γδ-T cells. Here we show that γδ-T cells which acquire resistance to mitogen-induced apoptosis upregulate IL-12 receptor beta 1 subunit (IL-12Rβ1); in contrast, γδ-T cells which remain sensitive to mitogen-induced apoptosis fail to express IL-12Rβ1. Next we show that γδ-T cells which are rendered resistant to mitogen-induced apoptosis attenuate their expression of the IL-2 receptor α chain (IL-2Rα/CD25), this in part accounting for their acquired resistance to IL-2-induced death. In contrast, apoptosis-sensitive γδ-T cells are shown to persist in their expression of IL-2Rα/CD25, thus remaining sensitive to IL-2-induced death. Moreover, we show that apoptosis-resistant, but not apoptosis-sensitive, γδ-T cells display an enhanced responsiveness to IL-15, a finding in keeping with the known function of IL-15 as a growth and survival factor. Finally, we present evidence to suggest that this differential responsiveness to IL-15 occurs in part by the increased expression of the IL-15Rα chain on apoptosis-resistant γδ-T cells, compared to apoptosis-sensitive γδ-T cells. The biological and clinical implications of these findings are discussed.