IPSC-Derived HLA-A2 CAR Tregs Showed Suppression of GvHD in a Xenograft Model

Abstract

Overcoming pathologies caused by excessive immunity, such as graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation and autoimmune diseases such as type1 diabetes and inflammatory bowel disease, is a challenge for medical research. It has been reported that CD4 single positive helper T cells can be induced from iPS cells (iPS-CD4Ts) by organoid culture using Notch ligand-expressing cells. However, these cells are conventional helper T cells (Tconvs) that secrete inflammatory cytokines such as IFN-γ or IL-4 if they are not treated in any way. Now, we report that we have succeeded in inducing FOXP3, a marker of immunosuppressive regulatory T cells (Tregs), in these iPS-CD4Ts at a high rate by combining compounds and cytokines. We used CDK8/19 inhibitor(A), TNFR2 antibody(M), Rapamycin(R), and TGF-β(T). Therefore, we named this process the AMRT. AMRT-treated iPS-CD4Ts didn't secrete inflammatory cytokines even under strong stimulus conditions. And, these cells were accompanied by demethylation of the Treg-specific demethylation region (TSDR), which suggested stable conversion to immunosuppressive cells. Furthermore, the combination of compounds and cytokines that induced FOXP3 expression in iPSC-derived Tconvs and converted them to FOXP3+ cells can also be used to amplify primary natural Tregs (nTregs) while retaining FOXP3. Comprehensive gene expression analysis (Ampliseq) showed close profiling of primary nTregs and AMRT-treated iPS-CD4Ts. These AMRT-treated iPS-CD4Ts were assessed functionally by CAR-mediated immunosuppressive ability. We evaluated the ability of these iPSC-derived HLA-A2 CAR-transduced FOXP3+ T cells to inhibit CTL division in mixed lymphocyte reaction (MLR) assays with HLA-A2+ allo CTLs. AMRT-treated iPS-CD4Ts showed CTL division suppression ability beyond primary nTregs. Surprisingly, this suppressive capacity was independent of the induction of CAR. If proximity is guaranteed in advance, the bystander suppression effect may be sufficient. We then assessed the immunosuppressive capacity of these cells through the extent to which they could suppress the xenograft GvHD that human HLA-A2+ PBMCs cause in NSG mice. AMRT-treated HLA-A2 CAR-transduced iPS-CD4Ts showed a survival benefit comparable to that of primary nTregs. In contrast to the invitro results, the invivo immunosuppressive capacity was CAR-specific. In summary, we have successfully generated large numbers of immune-suppressing regulatory T cells from iPS cells to suppress GvHD. This is a groundbreaking achievement that paves the way for curative therapies for GvHD and autoimmune diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal