Extrathymic Sites Drive Thymus-Independent Development of Functional T Cells After Bone Marrow Transplantation

T cell deficiency after bone marrow transplantation (BMT) leads to significant morbidity and mortality due to infection and malignant relapse. We have previously shown that CD4-CD8- T cell precursors (preT) generated via OP9-DL1 coculture improve thymic and peripheral T cell reconstitution when administered at the time of BMT. While the thymus supports T cell development into old age, adult BMT recipients have limited thymic function due to age-related involution and additional injury caused by transplant conditioning. We therefore hypothesized that T cell reconstitution after BMT may depend to some extent on extrathymic T cell development, which has previously been described as present but abnormal. We found that, in addition to the thymus, preT cells engrafted in extrathymic sites, including mesenteric lymph nodes (MLN), peripheral lymph nodes (PLN) and spleen, within 24 hours of transfer into euthymic BMT recipients. We detected CD4+CD8+ (double positive, DP) thymocyte-like cells of both preT and bone marrow (BM) origin in MLN, but not PLN, BM, or spleen, suggesting T cell development in MLN. Using a novel dual reporter for bioluminescence imaging we confirmed that preT trafficked to gut-associated tissues, where they underwent NFAT activation, consistent with T cell development. In competitive reconstitution studies we found that engraftment of preT in thymic and extrathymic sites required PSGL-1, while CCR9 may be dispensable. To better study extrathymic T cell development, we transferred preT into thymectomized or athymic BMT recipients. We demonstrated that preT and BM cells develop into both TCRαβ and TCRγδ T cells in athymic BMT recipients. As in euthymic recipients, preT engraft in extrathymic gut-associated sites and MLN support DP early after transfer into athymic BMT recipients. We found more CD4+, CD8αβ+, and CD8αα+ T cells in the spleen, PLN, MLN, and Peyer's patches of athymic BMT recipients that received preT than in nontransplanted or BM only recipients. Extrathymic-derived CD4+ and CD8αβ+ T cells are naive CD44- cells with a diverse T cell receptor (TCR) repertoire. In addition, extrathymic-derived T cells proliferated to a similar extent as thymic-derived T cells and produced IFNγ and TNFα following in vitro stimulation. In conclusion, using adoptively transferred preT, we have demonstrated that extrathymic T cell development occurs in BMT recipients, especially MLN, and results in functional T cells with a broad TCR repertoire.