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T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-)Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3 epsilon; and have their TCR locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3 epsilon(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-T-/- mice. However, reconstituted CD3 epsilon(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications.

Heterogeneity is the hallmark feature of CD4 T cells with the ability to differentiate into effector cells of various phenotypes and perform distinct function. This diversification that is essential for optimal response has not been described in the tissue. Using influenza as an infection model, we investigated long-lived antigen-specific CD4 T cells in the tissue. We identified a heterogeneous population of T resident memory cells (TRM) in the tissue, specifically, a T follicular helper-like subset we called T resident helper (TRH) that was previously not reported. Further characterization by single cell RNA sequencing analysis revealed that TRH possess a unique tissue-specific transcriptional signature distinct from lymphoid TFH and that they can be generated independently of lymphoid replenishment. Histological analysis showed that TRH cluster closely with B cells in inducible bronchus-associated lymphoid tissue (iBALT) and require continued BCL6 signaling for their localization. Interestingly, at very late time points, signals through cognate antigen presentation were dispensable for TRH maintenance and iBALT integrity. Upon heterologous challenge, TRH cells support local antibody production highlighting a previously unexplored function of TRM.