An optimized cultivation method for future in vivo application of γδ T cells.

γδ T cells, with their properties of both the innate and acquired immune systems, are suitable candidates for cellular immunotherapy in cancer. Because of their non-major histocompatibility complex (MHC) binding T cell receptor, allogenic transfer is feasible without relevant graft versus host reactions. In recent years, much experience has been gained with ex vivo expansion and stimulation of γδ T cells using bisphosphonates and Interleukin 2. Unfortunately, many current stimulation protocols are based on the use of xenogenic materials and other potentially hazardous supplements, which conflicts with basic principles of Good Manufacturing Practice (GMP). Adherence to the concept and current guidelines of GMP is state of the art for production of Advanced Therapy Medicinal Products (ATMP) like cell therapeutics and a necessity for clinical use under a regulatory perspective. In this study, we developed a new stimulation protocol that induces a marked increase of γδ T cell counts and allows for an easier transition from research to clinical applications with minimized regulatory workload. It reliably leads to a cell product with a purity of more than 90% γδ T cells and improved in vitro anti-tumor activity compared to our previous standard procedure. Furthermore, by investigating correlations between properties of unstimulated γδ T cells and proliferation rate as well as degranulation ability of stimulated γδ T cells, we can draw conclusions about suitable donors. Finally, we examined if expansion can be improved by pulsing zoledronate and/or using Interleukin 15 with or without Interleukin 2. Significant improvements can be achieved with respect to intrinsic and antibody-dependent cell-mediated cytotoxicity. Our results demonstrate that the stimulation protocol presented here leads to an improved γδ T cell product for future clinical applications.

Haploidentical γδ T Cells Induce Complete Remission in Chemorefractory B-cell Non-Hodgkin Lymphoma.

The transformation of chronic lymphocytic leukemia to an aggressive lymphoma, called Richter transformation, is often accompanied by resistance to chemotherapy and high mortality. Thus, novel therapeutic strategies are required for the successful treatment of these patients. One possibility is cellular immunotherapy with chimeric antigen receptor T cells. However, the time delay until cells are available and the limited number of effector cells due to the impaired immune system of these patients potentially compromises the efficacy of this approach. Another promising attempt might be the therapy with γδ T cells. Once activated, they exhibit various antitumor effects against several types of malignancies. Furthermore, they can be safely used in an allogeneic setting and can be multiplied in vivo as already demonstrated in clinical studies. In vitro data, in addition, show that the cytotoxicity of γδ T cells can be significantly enhanced by monoclonal antibodies. Here we present a patient, who suffered from Richter transformation and did not respond to several lines of immunochemotherapy. Due to the lack of further therapy options, we conducted an individual therapy with adoptive transfer of haploidentical γδ T cells combined with the application of the monoclonal antibody obinutuzumab. A histologically confirmed complete remission was achieved through this therapy approach, whereby relevant side effects were not seen. This case highlights the potential of γδ T cells and the feasibility of this therapeutic approach for further clinical trials.

Immune activating and inhibiting effects of calcitriol on γδ T cells and NK cells.

The immune modulatory effects of vitamin D and the impact of vitamin D deficiency on various diseases are a subject of current scientific research. However, there are few data directly linking vitamin D to hard endpoints in clinical studies, apart from its well-known effects on bone health. It is therefore of interest, that the effectiveness of the therapeutic antibody rituximab is connected to the vitamin D serum level of patients with B cell lymphomas. Rituximab exerts its effect via antibody dependent cell mediated cytotoxicity (ADCC), with NK cells and γδ T cells acting as effector cells. Here we evaluated and compared how γδ T cells and NK cells are modified by long-term in vitro cultivation with calcitriol, the active metabolite of vitamin D. Peripheral blood mononuclear cells from healthy donors were stimulated in vitro with a combination of zoledronic acid (Zol) and interleukin-2 (IL-2) or with sole IL-2 and different concentrations of calcitriol were added to culture for up to 10 days. Our results confirmed the earlier reports of immune suppressive effects of calcitriol on immune cell proliferation and interferon-γ production. Under certain conditions, in vitro incubation with calcitriol led to an inhibited rituximab-mediated ADCC of Zol/IL-2 stimulated γδ T cells, but to an increased cytotoxic activity of IL-2 stimulated NK cells. This study directly shows immune modulatory activity of calcitriol on the cellular level and links vitamin D to anti-tumoral effects of NK and γδ T cells.