Immunophenotypic profiling reveals novel T cell subset associated with “tolerance” in pediatric transplant recipients

Abstract

Abstract Achieving allograft health remains a challenge in pediatric organ transplantation. Recently. we performed multilineage, single-cell analysis of the immune cell composition in pediatric solid-organ transplant recipients who received a heart, liver, or kidney allograft (n=52); 24 children had stable graft function while 28 children had a rejection episode. Hierarchical clustering of the mean cell type proportion across allograft types and health states demonstrated a distinct subpopulation of CD4 T cells (CD45RA-,CD25+,CD5+,CD38-) was significantly (p<0.05) associated with stable graft function and were termed TOT cells. The cosine similarity analysis of proteomics data showed that TOT cells are phenotypically most like Tregs and least like CD4+ TEMRA. To characterize the TOT cells, we developed an 8-marker flow cytometry panel to specifically sort Treg and TOT populations and performed 10x single-cell RNA-seq and TCR-seq with subsequent analysis in R and Loupe Browser. Differentially expressed genes that were specifically associated with TOT cells included ANXA1, IL7R, TCF7, THEMIS, NELL2, KLRB1 and CD40LG while Treg expressed FOXP3, IKZF2, CTLA4, IL2RA, and RTKN2, consistent with their known function in immune suppression. TCR analysis indicates unique expansions of T cell clonotypes in TOT cells not seen in Treg. Further investigation into the immunoregulatory capacity of TOT cells could be instrumental in developing new strategies to prolong graft survival.