BackgroundHuman γδ T cells expressing Vγ2Vδ2 T cell receptors monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and tumors. Adoptive immunotherapy with Vγ2Vδ2 T cells has been used to treat cancer patients with partial and complete remissions. Most clinical trials and preclinical studies have used continuous zoledronate exposure to expand Vγ2Vδ2 cells where zoledronate is slowly diluted over the course of the culture. Zoledronate inhibits farnesyl diphosphate synthase (FDPS) in monocytes causing isopentenyl pyrophosphate to accumulate that then stimulates Vγ2Vδ2 cells. Because zoledronate inhibition of FDPS is also toxic for T cells, we hypothesized that a short period of exposure would reduce T cell toxicity but still be sufficient for monocytes uptake. Additionally, IL-15 increases the anti-tumor activity of murine αβ T cells in mice but its effect on the in vivo anti-tumor activity of human Vγ2Vδ2 cells has not been assessed.MethodsHuman Vγ2Vδ2 T cells were expanded by pulse or continuous zoledronate stimulation with IL-2 or IL-15. Expanded Vγ2Vδ2 cells were tested for their expression of effector molecules and killing of tumor cells as well as their in vivo control of human prostate cancer tumors in immunodeficient NSG mice.ResultsPulse zoledronate stimulation with either IL-2 or IL-15 resulted in more uniform expansion of Vγ2Vδ2 cells with higher purity and cell numbers as compared with continuous exposure. The Vγ2Vδ2 cells had higher levels of CD107a and perforin and increased tumor cytotoxicity. Adoptive immunotherapy with Vγ2Vδ2 cells derived by pulse stimulation controlled human PC-3 prostate cancer tumors in NSG mice significantly better than those derived by continuous stimulation, halting tumor growth. Although pulse zoledronate stimulation with IL-15 preserved early memory subsets, adoptive immunotherapy with IL-15-derived Vγ2Vδ2 cells equally inhibited PC-3 tumor growth as those derived with IL-2.ConclusionsPulse zoledronate stimulation maximizes the purity, quantity, and quality of expanded Vγ2Vδ2 cells for adoptive immunotherapy but there is no advantage to using IL-15 over IL-2 in our humanized mouse model. Pulse zoledronate stimulation is a simple modification to existing protocols that will enhance the effectiveness of adoptively transferred Vγ2Vδ2 cells by increasing their numbers and anti-tumor activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-017-0209-6) contains supplementary material, which is available to authorized users.

Tissue macrophages and their precursors-the blood monocytes-respond rapidly to a bacterial infection with the release of inflammatory mediators. These mediators are involved in the recruitment of phagocytic cells, principally neutrophils, from the blood to the site of infection. To initiate this process macrophages and monocytes must be able to detect the presence of bacteria in a reliable, but nevertheless nonspecific, fashion. It is thought that this is achieved by means of receptors on the cell surface which recognize structures common to many different bacteria. One candidate for such a "pattern recognition element" is the cell surface glycoprotein CD14. CD14 has been shown to bind components of the Gram-positive cell wall and it also binds soluble lipopolysaccharide released from Gram-negative bacteria. In both cases the interaction with CD14 leads to an activation of the cell. Here we show that human peripheral blood monocytes can, in addition, bind intact Gram-negative bacteria in the presence of serum and this process involves CD14. When CD14 expression is induced on the myelomonocytic cell line U937 by treatment with vitamin D3 the cells concomittently acquire the capacity to bind bacteria. Furthermore, a non-monocytic cell line which does not bind bacteria acquires the capacity to do so when transfected with either the human or mouse CD14 gene. This binding can be inhibited by blocking the CD14 receptor with anti-CD14 antibody or by blocking the ligand on the bacteria with soluble CD14. Finally we demonstrate binding of sCD14 to Escherichia coli. We conclude that in the presence of serum both membrane-bound and soluble forms of CD14 can bind to Gram-negative bacteria. This suggests that CD14 may play a role in the detection and elimination of intact bacteria in vivo.

T lymphocytes bearing the γδ-TCR accumulate during wound healing and inflammation. However, the role of γδ-T lymphocytes in fibrogenic tissue reactions is not well understood. Therefore, we addressed the question of whether human γδ-T cells express and synthesize connective tissue growth factor (CTGF), a factor known to regulate fibrogenesis and wound healing. In addition, the lymphoblastic leukemia T cell line (Loucy) that possesses characteristics typical of γδ-T cells was used as a model to evaluate the regulation of CTGF gene expression. Blood γδ-T cells isolated from healthy donors were grown in the presence of IL-15/TGF-β1 for 48 h and assessed for the expression and synthesis of CTGF. Nonstimulated human blood γδ-T cells and Loucy γδ-T cells expressed low levels of CTGF mRNA. Costimulation of the cells with IL-15 and TGF-β1 resulted in a substantially increased level of CTGF mRNA expression within 4–8 h, and it remained elevated for at least 48 h. In contrast, no CTGF mRNA was detected when nonstimulated and stimulated human CD4+ αβ-T cells were analyzed. In addition, Western blot analysis of human γδ-T cell lysates prepared 4 days following stimulation with IL-15 and TGF-β1 revealed a 38-kDa CTGF protein in cell lysates of human γδ-T cells. Detection was confirmed using Colo 849 fibroblasts, which can constitutively express high levels of CTGF. In conclusion, we herein present novel evidence that in contrast to CD4+ αβ-T cells human γδ-T cells are capable of expressing CTGF mRNA and synthesizing its corresponding protein, which supports the concept that γδ-T cells may contribute to wound healing or tissue fibrotic processes.

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