Ex Vivo High Salt Activated Tumor-Primed CD4+T Lymphocytes Exert a Potent Anti-Cancer Response.

Venkataswarup Tiriveedhi,Roy Zent,Michael T Ivy,Jens Titze,Jeffrey C Rathmell,Elbert L Myles

doi:10.3390/cancers13071690

Abstract

Simple SummaryCell based immunotherapy is rapidly emerging as a promising cancer treatment. Salt (sodium chloride) treatment to immune cell cultures is known to induce inflammatory activation. In our current study, we analyzed the anti-cancer ability of salt treatment on immune cells outside the host followed by reinfusion into the host. Using a pre-clinical breast cancer model, we demonstrated that external salt treatment on T-cell subset of immune cells produced a viable anti-cancer response, which may have future human clinical application.Cell based immunotherapy is rapidly emerging as a promising cancer treatment. A modest increase in salt (sodium chloride) concentration in immune cell cultures is known to induce inflammatory phenotypic differentiation. In our current study, we analyzed the ability of salt treatment to induce ex vivo expansion of tumor-primed CD4 (cluster of differentiation 4)+T cells to an effector phenotype. CD4+T cells were isolated using immunomagnetic beads from draining lymph nodes and spleens from tumor bearing C57Bl/6 mice, 28 days post-injection of Py230 syngeneic breast cancer cells. CD4+T cells from non-tumor bearing mice were isolated from splenocytes of 12-week-old C57Bl/6 mice. These CD4+T cells were expanded ex vivo with five stimulation cycles, and each cycle comprised of treatment with high salt (Δ0.035 M NaCl) or equimolar mannitol controls along with anti-CD3/CD28 monoclonal antibodies for the first 3 days, followed by the addition of interleukin (IL)-2/IL-7 cytokines and heat killed Py230 for 4 days. Ex vivo high salt treatment induced a two-fold higher Th1 (T helper type 1) expansion and four-fold higher Th17 expansion compared to equimolar mannitol treatment. Importantly, the high salt expanded CD4+T cells retained tumor-specificity, as demonstrated by higher in vitro cytotoxicity against Py230 breast cancer cells and reduced in vivo syngeneic tumor growth. Metabolic studies revealed that high salt treatment enhanced the glycolytic reserve and basal mitochondrial oxidation of CD4+T cells, suggesting a role of high salt in enhanced pro-growth anabolic metabolism needed for inflammatory differentiation. Mechanistic studies demonstrated that the high salt induced switch to the effector phenotype was mediated by tonicity-dependent transcription factor, TonEBP/NFAT5. Using a transgenic murine model, we demonstrated that CD4 specific TonEBP/NFAT5 knock out (CD4cre/creNFAT5flox/flox) abrogated the induction of the effector phenotype and anti-tumor efficiency of CD4+T cells following high salt treatment. Taken together, our data suggest that high salt-mediated ex vivo expansion of tumor-primed CD4+T cells could induce effective tumor specific anti-cancer responses, which may have a novel cell-based cancer immunotherapeutic application.

Highlights

Adoptive T cell transfer (ATCT) with the reinfusion of isolated and ex vivo activatedT lymphocytes is emerging as a novel cell-based cancer immunotherapeutic strategy [1].Tumor-specific T cells are highly enriched within tumors and in the draining lymph nodes [2]
Studies by Wu et al [19] demonstrated that treatment of CD4+T cells isolated from splenocytes of wild-type mice when cultured for 3 days at 40 mM excess NaCl induced inflammatory phenotype switch
The CD4+T cells from a single cell suspension of draining lymph nodes (DLNs) and spleen were isolated at greater than 95% purity

Summary

Introduction

Adoptive T cell transfer (ATCT) with the reinfusion of isolated and ex vivo activatedT lymphocytes is emerging as a novel cell-based cancer immunotherapeutic strategy [1].Tumor-specific T cells are highly enriched within tumors and in the draining lymph nodes [2]. Adoptive T cell transfer (ATCT) with the reinfusion of isolated and ex vivo activated. De novo expansion of naïve T cells might limit antigen specificity and anti-tumor efficiency [3]. ATCT has a unique advantage over other active immunization strategies in that it does not require in vivo activation and expansion of antigen specific T cell responses by a cancer patient with an otherwise weak immune system. Specific in vitro immune depletion strategies prior to infusion have met with limited success due to CD4 (cluster of differentiation)+T cell plasticity [5,6,7,8]. The optimal protocols for ex vivo T cell activation to evoke a favorable effector anti-tumor response for clinical application are still unclear

Methods

Results

Conclusion