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T cells could be engineered to overcome the aberrant metabolic milieu of solid tumors and tip the balance in favor of a long‐lasting clinical response. Here, we explored the therapeutic potential of stably overexpressing cystathionine‐gamma‐lyase (CTH, CSE, or cystathionase), a pivotal enzyme of the transsulfuration pathway, in antitumor CD8+ T cells with the initial aim to boost intrinsic cysteine metabolism. Using a mouse model of adoptive cell transfer (ACT), we found that CTH‐expressing T cells showed a superior control of tumor growth compared to control T cells. However, contrary to our hypothesis, this effect was not associated with increased T cell expansion in vivo or proliferation rescue in the absence of cysteine/cystine in vitro. Rather than impacting methionine or cysteine, ACT with CTH overexpression unexpectedly reduced glycine, serine, and proline concentration within the tumor interstitial fluid. Interestingly, in vitro tumor cell growth was mostly impacted by the combination of serine/proline or serine/glycine deprivation. These results suggest that metabolic gene engineering of T cells could be further investigated to locally modulate amino acid availability within the tumor environment while avoiding systemic toxicity.
Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. Here we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in RORgt + cells and conventional dendritic cells (cDCs) using germline and conditional double knock-out (DKO) mice. While Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells (ILC3s) in the intestinal mucosa, we found that they were required in cDCs for optimal antigen processing and presentation to CD4 + T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC II compartment (MIIC) of DCs for the fine regulation of antigen presentation and naive CD4 + T cell priming.Lancien et al.
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Introduction:We have demonstrated the therapeutic potential of human polyclonal and antigen-specific CAR-modified CD8 + Tregs cell therapy to prevent allogeneic human skin transplantation rejection and xenogeneic GvHD in humanized NSG mice (Bézie et al., Front Immunol.2017, Blood Adv.2019). However, their potential has never been evaluated in a clinical trial. We are thus preparing the launch of a phase I/IIa human clinical trial using polyclonal CD8 + Tregs cell therapy in living donors kidney transplant patients. Method: CD8 + cells were isolated from blood of healthy volunteers and patients with kidney failure by Clinimacs System then CD8+CD4-CD45RClow/-CD56 -T cells were sorted by MACSQuant Tyto cell sorter. Cells were stimulated with anti-CD3 and CD28 mAbs every week and cultured in presence of rapamycin, IL-2 and IL-15. Cytotoxicity against allogeneic PBMCs was assessed by Annexin V/DAPI staining and suppression capacity was assessed in vitro on syngeneic CD4 + T cells proliferation in response to allogeneic APCs and in vivo on 1.5Gy-irradiated NSG mice coinjected with human PBMCs. Results: We developed a new GMP-compatible cell manufacturing process. First, we determined a new method of isolation of CD8 + Tregs from peripheral blood using a safe closed system. Next, we identified the optimal clinically compatible culture medium, refined cytokines doses and stimulation methods to preserve a high proliferation rate, phenotypic profile and suppressive function of CD8 + Tregs. Using this process, we were able to efficiently expand CD8 + Tregs from peripheral blood of patients with renal failure, with high purity, while preserving their regulatory function. We confirmed the absence of cytotoxicity. In addition, we showed that CD8 + Tregs were phenotypically and functionally stable for 4h after conditioning, which is important for the logistic delay between cell harvest and patient infusion. Finally, we showed that CD8 + Tregs persist for more than 90 days in NSG mice without inducing any signs of xenogeneic GVHD, and could withstand combined immunosuppressive drug therapy administered to the patient. Conclusion: We designed a clinically compatible manufacturing process to isolate and culture CD8 + Tregs from renal failure patients that preserves their function and patient safety.
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