Lentiviral Engineered T Regulatory Cells Effectively Inhibit Lymphocytes Alloreactivity across Major HLA Barriers
Authors: Sangiolo Dario , Jordaney Noela , Mesiano Giulia , Circosta Paola , Elia Angela , Stella Stefania , Todorovic Maja , Carnevale-Schianca Fabrizio , Aglietta Massimo , Cignetti Alessandro 
Published: October 2019

Abstract
Adoptive immunotherapy strategies enrolling T regulatory cells (Tregs) might have a great potential in modulating donor T cells alloreactivity after Hematopoetic Cell Transplant (HCT). In murine models of HCT Tregs were shown to promote engraftment and contribute controlling graft versus host disease (GVHD) while still not conclusive data are available on humans. Ex-vivo engineering conventional CD4+ T cells to over-express the transcription factor FOXP3 is an intriguing approach to overcome the main difficulty of obtaining large amount of Tregs for experimental studies. Reports of retrovirus-mediated expression of FOXP3 not consistently resulted in functional Tregs while, recently, a lentivirus-mediated strategy was successfully reported to result in homogeneous and stable expression of FOXP3. Lentiviral transduced Tregs were able to suppress a polyclonal proliferation of CD4 purified lymphocytes stimulated with soluble Ab anti-CD3. In our study we generated lentiviral engineered Tregs (eng-Tregs) and investigated their inhibitory effect on unselected lymphocytes alloreactivity across major HLA barriers. Within the bulk lymphocytes population we separately tracked the suppressive influence of eng-Tregs on both CD4+ and CD8+ T cells. To obtain eng-Tregs, CD4+ T cells were purified from healthy donors and transduced with a bidirectional lentiviral vector encoding for FOXP3 and the truncated Nerve Growth Factor Receptor (ΔNGFR). Prior to transduction CD4+ cells were activated for 72 hours with IL2 (100U/ml), IL7 (20ng/ml) and soluble Ab anti-CD3 (200 ng/ml, only IL2 was added to the culture medium after transduction.
The lentiviral transduction efficiency ranged from 8 to 25%, ΔNGFR+ T cells were positively selected and tested for their ability to suppress a mixed lymphocyte reaction across major HLA barriers. Effector peripheral blood mononuclear cells (PBMC) were collected from the same donors from whom eng-Tregs were generated. Effector PBMC were stained with CFSE in oder to separately track the alloreactive proliferation of both CD8+ and CD4+ subsets of T cells. Eng-Tregs were added on day 0 and HLA-mismatched irradiated PBMC were used as stimulators; both eng-Tregs and irradiated stimulators were used in a 1:1 ratio with the effectors. No cytokines or additional soluble stimulators were added to the MLR culture medium. The alloreactive proliferation of T cell subsets was determined by evaluating the logarithmic decrease of CFSE fluorescence intensity. The flow cytometry analysis on day +7 showed that alloreactive proliferation of both CD4+ and CD8+ effector cells was significantly inhibited (>75%) by the addition of eng-Tregs compared to controls. In order to rule out a possible role played by the naturally present Tregs (nat-Tregs), the effectors were depleted of the CD4+CD25high subpopulation before the MLR started. The observed alloreactive proliferation was higher after the depletion of nat-Tregs but still it could be significantly inhibited by the addition of eng-Tregs. Eng-Tregs did not significantly expanded when cultured in vitro (up to 2 weeks) with IL2 (100U/ml) but maintained a stable expression of the transgene and retained their suppressive capacity. Our data show that lentiviral engineered Tregs can efficiently down-modulate both CD4+ and CD8+ T cell alloreactivity across major HLA barriers. The observed independence from the presence of nat-Tregs might be important in future experimental HCT settings where the adoptive infusion of eng-Tregs might encounter a great variability in the number and activity of recipient’s nat-Tregs. The possibility of transducing a potentially unlimited number of CD4+ cells makes this strategy appealing for future pre-clinical studies to control GVHD in HCT settings.