M. D'Asaro

Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer

The increasing evidence that gammadelta T cells have potent antitumor activity suggests their value in immunotherapy, particularly in areas of unmet need such as metastatic carcinoma. To this end, we initiated a phase I clinical trial in metastatic hormone-refractory prostate cancer to examine the feasibility and consequences of using the gammadelta T-cell agonist zoledronate, either alone or in combination with low-dose interleukin 2 (IL-2), to activate peripheral blood gammadelta cells. Nine patients were enlisted to each arm. Neither treatment showed appreciable toxicity. Most patients were treated with zoledronate + IL-2, but conversely only two treated with zoledronate displayed a significant long-term shift of peripheral gammadelta cells toward an activated effector-memory-like state (T(EM)), producing IFN-gamma and perforin. These patients also maintained serum levels of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), consistent with a parallel microarray analysis showing that TRAIL is produced by gammadelta cells activated via the T-cell receptor and IL-2. Moreover, the numbers of T(EM) gammadelta cells showed a statistically significant correlation with declining prostate-specific antigen levels and objective clinical outcomes that comprised three instances of partial remission and five of stable disease. By contrast, most patients treated only with zoledronate failed to sustain either gammadelta cell numbers or serum TRAIL, and showed progressive clinical deterioration. Thus, zoledronate + IL-2 represents a novel, safe, and feasible approach to induce immunologic and clinical responses in patients with metastatic carcinomas, potentially providing a substantially increased window for specific approaches to be administered. Moreover, gammadelta cell phenotypes and possibly serum TRAIL may constitute novel biomarkers of prognosis upon therapy with zoledronate + IL-2 in metastatic carcinoma.

Efficient Killing of Human Colon Cancer Stem Cells by γδ T Lymphocytes

Colon cancer comprises a small population of cancer stem cells (CSC) that is responsible for tumor maintenance and resistant to cancer therapies, possibly allowing for tumor recapitulation once treatment stops. We previously demonstrated that such chemoresistance is mediated by autocrine production of IL-4 through the up-regulation of antiapoptotic proteins. Several innate and adaptive immune effector cells allow for the recognition and destruction of cancer precursors before they constitute the tumor mass. However, cellular immune-based therapies have not been experimented yet in the population of CSCs. Here, we show that the bisphosphonate zoledronate sensitizes colon CSCs to Vγ9Vδ2 T cell cytotoxicity. Proliferation and production of cytokines (TNF-α and IFN-γ) and cytotoxic and apoptotic molecules (TRAIL and granzymes) were also induced after exposure of Vγ9Vδ2 T cells to sensitized targets. Vγ9Vδ2 T cell cytotoxicity was mediated by the granule exocytosis pathway and was highly dependent on isoprenoid production by of tumor cells. Moreover, CSCs recognition and killing was mainly TCR mediated, whereas NKG2D played a role only when tumor targets expressed several NKG2D ligands. We conclude that intentional activation of Vγ9Vδ2 T cells by zoledronate may substantially increase antitumor activities and represent a novel strategy for colon cancer immunotherapy.

In vivo manipulation of Vgamma9Vdelta2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients.

The potent anti‐tumour activities of γδ T cells have prompted the development of protocols in which γδ‐agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vγ9Vδ2 T cell agonist, plus low‐dose interleukin (IL)‐2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vγ9Vδ2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vγ9Vδ2 T cell numbers emerged, as seven patients who failed to sustain Vγ9Vδ2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vγ9Vδ2 cell populations showed declining CA15‐3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vγ9Vδ2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low‐dose IL‐2 offers a novel, safe and feasible approach to enhance this in a subset of treatment‐refractory patients with advanced breast cancer.

Increase of CCR7 − CD45RA + CD8 T cells (T EMRA ) in chronic graft-versus-host disease

Among the late effects of hematopoietic stem cell transplantation (HSCT), chronic graft-vs-host disease (cGVHD) still remains as the major determinant of long-term outcome and quality of life. The disease typically appears between 3 months to 1.5 years following an allogeneic transplantation and is characterized by symptoms similar to those of autoimmune disease.

In Vitro Effects of Aminobisphosphonates on Vγ9Vδ2 T Cell Activation and Differentiation

In this study we have evaluated the in vitro effects of four different aminobisphosphonates, alendronate, risedronate, neridronate and zoledronate, on Vγ9Vδ2 T cell activation and differentiation. All tested aminobisphosphonates induce an IL-2-dependent activation and expansion of Vγ9Vδ2 T lymphocytes in primary PBMC cultures of healthy donors. Most notably, they also determine a different distribution of Vγ9Vδ2 T cell subsets, with decrease of Tnaive and TCM cells and increase of TEM and TEMRA Vγ9Vδ2 cells, indicating that in vitro treatment with aminobisphosphonates induces Vγ9Vδ2 T lymphocytes to differentiate towards an effector/cytotoxic phenotype. Accordingly, Vγ9Vδ2 T lymphocytes cultured with aminobisphosphonates and IL-2 showed a major content of IFN-γ and acquired the ability to kill tumor target cells.

Increase of CCR7− CD45RA+ CD8 T cells (TEMRA) in chronic graft-versus-host disease

Among the late effects of hematopoietic stem cell transplantation (HSCT), chronic graft-vs-host disease (cGVHD) still remains as the major determinant of long-term outcome and quality of life. The disease typically appears between 3 months to 1.5 years following an allogeneic transplantation and is characterized by symptoms similar to those of autoimmune disease.

Analysis of Memory and Effector CD8+ T Cell Subsets in Chronic Graft-versus-Host Disease

In humans, the selective depletion of CD8+ cells may prevent GVHD after allogeneic transplantation. These cells can infiltrate and damage target tissues. It is of interest to investigate the phenotypical characteristics and cytotoxic properties of the different CD8+ subsets in cGVHD patients. In a preliminary study we found that patients with cGVHD had a markedly elevated percentage of peripheral blood CCR7−/CD45RA+ cells compared to patients without cGVHD; conversely, the CCR7+/CD45RA+ subsets of CD8+ cells was significantly decreased. In this study, we report in depth on the phenotype of effector T cell subsets in cGVHD patients, as well as their proliferative capability, cytotoxic properties and cellular turnover. We confirm a predominance of effector T cell subsets in cGVHD patients and show that a large fraction of these cells down-regulate CCR7 and re-express CD45RA, thus approaching end-stage differentiation. Moreover CD8+ cells of cGVHD patients have low CD8 coreceptor expression, reduced proliferative potential and a high content of perforin and granzyme A. They also have a lower cell turnover and have more propensity to apoptosis, as demonstrated by BrdU incorporation. Taken together, our findings indicate a perturbation of the balance between naive/memory and effector/CD45RA+ CD8+ T cells, and suggest an involvement of the latter compartment characterized by a high content of cytotoxic equipment, in the pathogenesis of cGVHD.

Increase of CCR7|[minus]| CD45RA|[plus]| CD8 T cells (TEMRA) in chronic graft-versus-host disease

Among the late effects of hematopoietic stem cell transplantation (HSCT), chronic graft-vs-host disease (cGVHD) still remains as the major determinant of long-term outcome and quality of life. The disease typically appears between 3 months to 1.5 years following an allogeneic transplantation and is characterized by symptoms similar to those of autoimmune disease.