🎗Engineering tacrolimus-resistant A2-CAR Tregs for use in solid organ transplantation

Introduction: Tacrolimus (TAC), a calcineurin inhibitor, forms the backbone of conventional immunosuppressive therapy following solid organ transplantation (SOT). TAC blocks the nuclear translocation of NFAT, a transcription factor critical for all T cells, including regulatory T cells (Treg). Chimeric antigen receptor (CAR) Treg therapy is a promising adjunct to conventional immunosuppressive drug therapy following SOT but how TAC affects CAR Tregs was unknown. Using CRISPR-Cas9 we developed FKBP12KO CAR Tregs recognizing HLA-A2 (A2-CAR Tregs) and investigated how TAC affects their phenotype and function and whether FKBP12KO cells were resistant to possible inhibitory effects.
Methods: TAC-resistant FKBP12KO human and mouse A2-CAR Tregs were generated by CRISPR-Cas9 targeting FKBP1A, the gene encoding the intracellular TAC target, FKBP12. Control cells were electroporated with Cas9 alone. WT and KO human and mouse Treg activation (CD25, CD69), function (CTLA4), stability (FOXP3), CAR expression (Myc), and proliferation (Ki-67) were assessed by flow cytometry following 72-hour activation with HLA-A2+ cells, in the absence or presence of TAC. To test in vivo effects, we used an HLA-A2-mismatched skin transplant model. Male B6 mice received a skin transplant from male HLA-A2 transgenic B6 mice. At the same time mice were injected with 250K KO or WT A2-CAR Tregs followed by TAC 5 mg/kg IP daily for 14 days. Tissue was harvested on day 14 for flow cytometric analysis. Statistical comparisons used ANOVA and Student’s t test.
Results: FKBP12 KO efficiency ranged from 62-87% in mouse A2-CAR Tregs, with optimized conditions yielding consistent KO efficiency close to 80%. Mouse KO A2-CAR Tregs were protected from the anti-proliferative effects of TAC (Ki-67) and expressed significantly higher levels of CTLA-4 compared to WT cells. In the skin transplant model, KO A2-CAR Tregs exhibited four-fold greater engraftment compared to WT. KO A2-CAR Tregs also expressed higher levels of functional (Ki-67, CD25, CTLA4) and tissue-residency (CD103) markers. TAC was also associated with reduced FOXP3 expression in WT but not KO cells. Human FKBP12 KO efficiency ranged from 45-73% depending on the sgRNA and culture conditions. In the presence of TAC, compared to WT, human KO A2-CAR Tregs expressed significantly higher levels of the A2-CAR, were protected from TAC’s anti-activation/proliferative effects (CD69, Ki-67), and had reduced susceptibility to tacrolimus-mediated FOXP3 destabilization. KO A2-CAR Tregs retained susceptibility to cyclosporine A. 
Conclusions: TAC is deleterious to A2-CAR Tregs, leading to low proliferation, activation and FOXP3 expression, in vitro and in vivo. In contrast, FKBP12 KO CAR Tregs engraft efficiently and retain high levels of functional markers in the presence of clinically relevant doses of TAC. These data demonstrate that functional resilience to TAC can be achieved in A2-CAR Tregs through CRISPR-Cas9 FKBP12 KO.