Proliferation Of Gamma Delta Research Articles

Zoledronic Acid Induces the Proliferation of Human Cord Blood Gamma Delta T Cells Ex Vivo

Cord blood (CB) T cells are known to be naïve cells, but can be activated to respond similar to adult peripheral blood (PB) T cells. Reports indicate that culture with aminobisphosphonate (NBP) stimulates CB gamma delta T cell proliferation ex vivo, specifically the TCRγ9δ2 subset, which has been extensively studied in PB gamma delta T cells. As CB gamma delta T cells are not well described, we compared CB gamma delta T cell proliferation, phenotype and genotype to PB gamma delta T cells when culturing cells with the NBP, Zometa (zoledronic acid), and IL-2. Fourteen days in culture resulted in significant fold increase in the proliferation of gamma delta T cells and in the percent of lymphocytes in both sample types. PB gamma delta T cells proliferated more robustly than CB with a 288.60 versus 21.32 fold increase, respectively. Additionally, in freshly isolated samples, CB gamma delta T cells comprised an average of 1.404% of the lymphocyte population, which was similar to PB gamma delta T cells, with an average of 2.319%. However, by day 14, PB gamma delta T cells increased to 70.15% of lymphocytes whereas CB gamma delta T cells increased to 12.49%. Phenotypically, both CB and PB had similar percent of CD45RA+ and CD45RO+ gamma delta T cell memory subsets in freshly isolated samples. Following culture, PB gamma delta T cells were mostly CD45RO+ memory cells, with significantly fewer CD45RA+ naïve cells, whereas more CB gamma delta T cells were of the intermediate CD45RA+CD45RO+ subset. Further phenotypic analysis of the memory subsets indicated that cultured PB gamma delta T cells were either effector memory cells (CD27-CD45RA-) or central memory cells (CD27+CD45RA-), while CB gamma delta T cells were mostly naïve (CD27+CD45RA+). The cytokines secreted by these cells were also assessed and the culture of PB and CB gamma delta T cells resulted in differing cytokine secretion profiles. After 14 days of culture, PB gamma delta T cells secreted more IFNγ and TNFα, while CB gamma delta T cells secreted more IL-10 and RANTES. We also examined TCRγ9 and TCRδ2 phenotypic expression and found that the TCRγ9δ2 was a common clone in freshly isolated PB gamma delta T cells, which predominated after 14 days in culture. However, while the TCRγ9δ2 variant was expressed in CB gamma delta T cells, it was low before and after culture, suggesting that Zometa may not stimulate gamma delta T cells in CB the same as PB. As limited TCRγδ phenotypic reagents are available, we developed a single cell PCR assay for genotypic analysis of the TCRγδ repertoire. PCR analysis suggests that the TCRγδ repertoire is diverse in both samples, yet TCRγ9δ2 is most prevalent. Further analysis of the variant subsets is warranted and may give insight into how each of these receptor pairings affects function. DisclosuresNo relevant conflicts of interest to declare.

Effect of Alpha 2 Macroglobulin on Luteal Cell Induced Gamma Delta T Lymphocyte Proliferation.

The paracrine factors that regulate communication between immune cells and parenchymal cells within the corpus luteum (CL) are not well defined. It has been shown that cultured luteal cells are profound stimulators of T lymphocytes and can induce T cell proliferation by cell to cell interactions and by secretory factors. By using a proteomic approach, we profiled protein expression in conditioned media from gamma delta T cell - luteal cell (LC) cocultures collected from midcycle and regressing CL. Alpha 2 macroglobulin (a2M) was one of the proteins that exhibited differential secretion. The purpose of this study was to determine if a2M mediates LC induced changes in gamma delta T cell function in fully functional, but not regressing CL. The first objective was to define if a2M secretion can be modified by luteolytic factors. Midcycle luteal cells were exposed to transforming growth factor beta(TGFb), interferon gamma/ tumor necrosis factor (IFNg/TNFa) and PGF2a. Luteal conditioned media (LCM) were collected on days 1 and 3 of cultures and protein expression was analyzed by Western Blot. Although PGF2a reduced a2M secretion in both days of collection, the effect of IFNg/TNFa was observed only on day 3 (p<0.05). The second objective was to determine if a2M mediates gamma delta T cells proliferation in response to luteal cells depending on functional status of the CL. Corpora lutea were collected from cyclic cattle on days 10–12 of the estrous cycle and 8hrs after PGF2a injection, dissociated and placed in culture. To test two different ways of cells communication, immunomagnetically isolated gamma delta T cells from the blood were placed in culture with luteal cells in contact or inside a culture insert. Cells were than incubated for 72hrs with or without a2M and proliferation of gamma delta T cells was measured. In cells collected from regressing CL, a2M increased LC stimulated gamma delta T cells proliferation only when the cells were physically separated by insert. The gamma delta T cells response to midcycle LC was not altered by a2M. The third objective was to examine if a2M can induce gamma delta T cells proliferation directly. The luteal cells were first fixed with 0.1% and 1% paraformaldehyde. Gamma delta T cells and a2M were then added to the culture system and a proliferation assay was performed after 72 hrs. This study showed that cell fixation reduced gamma delta T cells response to LC respectively to 40% and 23%. Treatment with a2M had no effect on gamma delta T cell proliferation. In conclusion, we showed that a2M protein expression varied within the estrous cycle and was regulated by factors locally secreted in the CL. The role of a2M is to entrap and bind diverse molecules secreted by luteal cells and facilitate their delivery to immune cells. Our data suggests that proteins like a2M may provide an alternative mechanism for cell communication in tissues undergoing rapid morphological changes to maintain local homeostasis. This project was supported by National Research Initiative Competitive Grant no. 2008-35203-04617 from the USDA National Institute of Food and Agriculture.

Effector γδ T cells and tumor cells as immune targets of zoledronic acid in multiple myeloma

The aim of this study was to investigate the in vitro immunomodulatory effects of zoledronic acid (Zol) on peripheral blood Vgamma9/Vdelta2 (gammadelta) T cells of normal donors and multiple myeloma (MM) patients. gammadelta T cells were stimulated with Zol and low doses of interleukin-2 (IL-2), and then analyzed for proliferation, cytokine production, and generation of effector activity against myeloma cell lines and primary myeloma cells. Proliferation of gammadelta T cells was observed in 100% of normal donors and 50% of MM patients. gammadelta T cells produced IFN-gamma, surface mobilized the CD107a and CD107b antigens, and exerted direct cell-to-cell antimyeloma activity irrespective of the ability to proliferate to Zol and IL-2. The memory phenotype was predominant in the MM gammadelta T cells that proliferated in response to Zol (responders), whereas effector cells were predominant in those that did not (nonresponders). Zol induced antimyeloma activity through the monocyte-dependent activation of gammadelta T cells and by enhancing the immunosensitivity of myeloma cells to gammadelta T cells. Mevastatin, a specific inhibitor of hydroxy-methylglutaryl-CoA reductase, completely abrogated this antimyeloma activity.

Immunisation of cattle against heartwater by infection with Cowdria ruminantium elicits T lymphocytes that recognise major antigenic proteins 1 and 2 of the agent

There is growing evidence that immunity of cattle to Cowdria ruminantium infection is mediated by T lymphocytes. C. ruminantium antigens that stimulate these responses are therefore of considerable importance to the development of a sub-unit vaccine against the disease. We have examined T cell responses against recombinant analogues of the surface-exposed C. ruminantium major antigen 1 (MAP1) a 28.8 kDa protein and MAP2 (21 kDa) antigen in cattle immunised by infection and treatment. Vigorous and sustained proliferative responses to both antigens were observed in peripheral blood mononuclear cells from immune cattle. MAP1-specific responses were predominantly restricted to cluster of differentiation four antigen positive T cells (CD4 + T cells). Reverse transcription polymerase chain reaction (RT-PCR) analysis of cytokine expression by T cell lines derived from this population revealed strong expression of interferon gamma (IFN-γ), interferon alpha (IFN-α), tumour necrosis factor alpha (TNF-α), tumour necrosis factor beta (TNF-β), interleukin-2 receptor alpha (IL-2Rα) transcripts, and weak expression of IL-2 and IL-4. Supernatants from these T cell cultures contained IFN-γ protein. CD4 + T cell clones specific for MAP1 were generated. Two of these clones proliferated in the presence of autologous infected endothelial cells. In contrast, the response to MAP2 was characterised largely by proliferation of gamma delta (γδ) T cells. RT-PCR analysis of cytokine expression by T cell lines which were dominated by γδ T cells revealed expression of IFN-γ, TNF-α, TNF-β, IL-2Rα transcripts. Supernatants of these T cell cultures also contained IFN-γ protein. Our findings indicate that immunisation of cattle by infection with C. ruminantium results in generation of MAP1- and MAP2-specific T cell responses that may play a role in protection against the pathogen.

Phenotypic and functional analysis of bovine gammadelta lymphocytes.

The studies have provided the first comprehensive comparison of the factors regulating activation and proliferation of WC1+ and WC1- gammadelta T cells. The investigation has shown that accessory molecules essential for activation and function of WC1+ and WC1- gammadelta T cells and the sources and roles of cytokines in activation of gammadelta T cells through the T cell receptor (TCR). The study has also shown that the role of cytokines in activation and function of gammadelta T cells activated indirectly through cytokines secreted by ab T cells, accessory cells and antigen presenting cells (APC). Cytokines were differentially produced by subpopulations of gammadelta T cells under different conditions of activation. The investigation obtained in this study has revealed that factors account for activation and proliferation of gammadelta T cells in cultures designed to study MHC-restricted responses to antigens. Evidence obtained here has shown there is biological relevance to activation under these culture conditions that points to potential regulatory and effector functions of gammadelta T cells. The investigations have also provided the information needed to begin identifying and characterizing antigens recognized by the TCR repertoires of WC1+ and WC1- gammadelta T cells. Finally, the investigations have provided the information needed to begin analysis of the mechanisms by which gammadelta T cells modulate MHC restricted immune responses to pathogens and derived vaccines.

Cellular requirements for the activation and proliferation of ruminant gammadelta T cells.

Requirements for the activation and proliferation of gammadelta T cells were investigated. Maximum numbers of gammadelta T cells expressed the IL-2R alpha-chain after 6-h Con A stimulation in peripheral blood, efferent lymph, and afferent lymph. In comparison, IL-2R alpha-chain expression on CD4 T cells only reached maximum levels in response to Con A stimulation in peripheral blood and afferent lymph populations. Analysis of enriched gammadelta T cells demonstrated that Con A-induced expression of the IL-2R alpha-chain was independent of APC. Together, these data suggest that the requirements for gammadelta T cell activation are less stringent than those for alphabeta T cell activation. Unfractionated peripheral blood, efferent lymph, and afferent lymph cell populations proliferated in response to Con A alone. In contrast, enriched gammadelta T cells (CD4/CD8 depleted) from efferent lymph did not proliferate in response to Con A alone, but required the addition of IL-2. This requirement for exogenous IL-2 could be overcome by the addition of dendritic cells purified from afferent lymph. These results suggested that gammadelta T cells required costimulatory signals provided by APC to ensure the production of sufficient IL-2 to drive proliferation. CD28 and CTLA-4 mRNA were detected in efferent lymph and afferent lymph populations containing CD4 and CD8 T cells stimulated with Con A and IL-2 or with Con A alone, respectively. In contrast, negligible levels of these mRNA species were detected in efferent and afferent lymph populations devoid of CD4 and CD8 T cells. These results suggest that ovine gammadelta T cells may use alternative costimulatory pathways.

Induction of gammadelta T cells during acute murine infection with Toxoplasma gondii.

The importance of the host gammadelta T cell response to microbial pathogens is not yet fully understood. We report that inbred mice infected with T. gondii developed a gammadelta T cell proliferative response to parasite Ag. Mice depleted of either the alphabeta or gammadelta TCR were found to be significantly more susceptible to infection than the parent mouse strain. Proliferation of gammadelta T cells was observed in mice deficient in the TCR-alphabeta in response to UV-irradiated parasites. These T cells lyse parasite-infected syngenic macrophages. Adoptive transfer of these gammadelta T cells into beta2 microglobulin-deficient mice that have been depleted of both CD4+ and NK cells prolongs survival against acute parasite challenge when compared with nontransferred controls. The gammadelta T cells isolated from infected alpha -/- mice express a 10-fold increase in mRNA and produce high titers of IFN-gamma. These data suggest that gammadelta T cells may play an important role in the early host response to infection with T. gondii.

Human gamma delta T cell subset-proliferative response to malarial antigen in vitro depends on CD4+ T cells or cytokines that signal through components of the IL-2R.

We examined the cellular and molecular basis of the proliferative response of human gamma delta T cells in cultures of PBMC stimulated with blood-stage Plasmodium falciparum malarial Ag. Flow cytometry revealed that maximal gamma delta T cell proliferation occurs after maximal CD4+ alpha beta T cell proliferation. Depletion of CD4+ T cells from PBMC before stimulation with malarial Ag markedly reduces the number of proliferating gamma delta T cells, which suggests that CD4+ T cells function in providing help to gamma delta T cells to respond to this parasite Ag. Removal of gamma delta T cells, however, did not alter the expansion of the CD4+ T cell subset. The addition of exogenous IL-2, IL-4, or IL-15 restored the capacity of gamma delta T cells to proliferate in Ag-stimulated cultures of PBMC depleted of CD4+ T cells. mAbs specific for the alpha- and beta-subunits of the IL-2 receptor inhibit the gamma delta T cell subset expansion in cultures stimulated with malarial Ag. Taken together, these findings suggest that the proliferation of gamma delta T cells in response to malarial Ag is dependent on the presence of CD4+ alpha beta T cells, but the requirement for CD4+ alpha beta T cells can be met by cytokines that use the IL-2R.

Selective activation of resting human γδ T lymphocytes by interleukin‐2

In rheumatoid arthritis and other inflammatory diseases we and others have found that gamma delta T cells express activation antigens, suggesting that they are involved in the pathogenesis of these disorders. In this study we have stimulated peripheral blood mononuclear cells from normal donors with recombinant interleukin-2 (rIL-2) to see whether such a stimulus alone could activate gamma delta T cells. Short-term exposure (24-96 h) to rIL-2 selectively stimulated the gamma delta but not the alpha beta T cells to express activation antigens (CD69, CD25 and HLA-DR). Long-term culture (2 weeks) in rIL-2-containing medium caused a selective increase in the proportion of the gamma delta T cells and a corresponding reduction of the fraction of alpha beta T cells. Limiting dilution analysis revealed that approximately 1/60 of the gamma delta T cells responded to IL-2 in contrast to only 1/250 of the alpha beta T cells. Comparison of the expression of the IL-2 receptor (IL-2R) alpha and beta chains showed that there was a similar expression of the alpha chain on gamma delta and alpha beta T cells whereas the relative density of the beta chain was more than twice as high on gamma delta T cells. Both the IL-2-induced proliferation of gamma delta T cells and the expression of activation antigens on these cells could be inhibited by an anti-IL-2R beta monoclonal antibody (mAb) but not by an anti-IL-2R alpha mAb. Expression of CD69 on gamma delta T cells was dependent neither on the presence of B cells, monocytes, nor alpha beta T cells. Finally, we found that the IL-2-induced expression of CD69 was inhibited by activation of cAMP-dependent protein kinase and by inhibition of the Src-family of the tyrosine protein kinase, but not by inhibition of protein kinase C or by activation of the CD45 associated tyrosine phosphatase. The ability of gamma delta T cells to be activated by IL-2 is a feature which they have in common with natural killer cells. Moreover, it may be possible that the expression of activation antigens on gamma delta T cells in inflammatory diseases is an epiphenomenon secondary to IL-2 produced by activated alpha beta T cells.