Peripheral T cell transcriptomic profile is altered in non-human primate kidney transplant recipients treated with ECDI-fixed donor B cell infusions

Introduction

Immunologic tolerance to allogeneic tissue without compromising host defences against infection or malignancy is a major goal in clinical transplantation. Apoptotic donor leukocytes are a promising therapy for the induction of donor-specific immunological tolerance in allogeneic transplantation.

Methods

In an NHP model of complete MHC mismatch kidney transplantation, ex vivo expanded donor B cells were treated with the chemical cross-linker ECDI to induce apoptosis and infused at POD -7 and +1 peri-transplant, combined with temporary immunosuppression (day -8 to day +28). Recipient PBMCs from day +35 post-transplant were cell surface stained before FACS-sorting of live CD3+ T cells. Single cell RNA sequencing was performed using a 10x Genomics system. Raw sequencing data were aligned to the RM reference Mmul_10 with Cell Ranger 1.3 (10x Genomics), generating expression count matrix files. Quality control removed cells with excess mitochondrial, ribosomal RNA or haemoglobin genes and doublets. A graph-based clustering approach in Seurat was then used to cluster cells, with manual annotation based on differential gene expression patterns. 

Results

ECDI-B treated animals had significantly longer median rejection-free survival time (133 vs. 42 days, p = 0.04). The transcriptome of peripheral T cells at day +35 after transplant revealed significant differences between ECDI-B treated and control samples. Cells segregated into 12 clusters (Figure 1A). CD4+ T regulatory cells (Tregs) have been identified as potential key mediators of ECDI tolerance induction, and examination of this population revealed 5 distinct subclusters (Figure 1B) with differences in distribution between ECDI-B treated and control samples (Figure 1C). ECDI-B treated samples had higher proportion of highly suppressive "effector Tregs" which expressed CTLA4, PDLIM1 (PD-L1) and CSF1 which exerts regulatory effects on myeloid cells (Figure 1D). "IFN-stimulated" Tregs, also expressing PDLIM1 and CSF1, were exclusive to ECDI-B treated samples, and expressed high levels of IFN-induced genes including ISG15 that maintain Treg function and survival during inflammatory responses (Figure 1D).

Additionally, MAIT-like/GV9 T cells, associated with attenuation alloreactive responses were increased in ECDI-B treated samples (3.47% vs. 1.72%, p <0.001). Within the CD4+ compartment there was broad reduction in inflammatory signalling pathways and downregulation of key Th1 differentiation markers including GADD45B, SLC7A5 and SLC2A3. 

Conclusions

In summary, ECDI-B infusions lead to prolonged immunosuppression-free survival in non human primate kidney transplantation. The peripheral T cell transcriptional profile demonstrates a regulatory immunophenotype with enhanced Treg functionality, increased numbers of suppressive MAIT-like/GV9 T cells and a shift away from Th1 responses.