Tcf15 Negatively Regulates T and B Lymphopoiesis

Expression of genes associated with lymphopoiesis has been shown to be uniquely antagonistic to hematopoietic stem cell (HSC) maintenance and function. Additionally, reduced expression of lymphoid associated genes in HSCs has been shown to promote HSC expansion and self-renewal. These findings demonstrate that maintaining lymphoid developmental potential while preserving HSC function and identity requires a carefully calibrated molecular and cellular program. The transcription factor Tcf15 was recently shown to be indispensable to HSC maintenance and function. Tcf15 expression in HSCs was shown to confer long term regenerative potential. Meanwhile Tcf15 overexpression (OE) was shown to inhibit HSC proliferation and differentiation, while enriching for HSCs. Because HSC maintenance and regenerative programs are so closely linked to regulation of lymphoid potential, we hypothesized that Tcf15 could have a corollary function as a negative regulator of lymphopoiesis.

To examine the effect of Tcf15 on lymphopoiesis, we first generated Tcf15 knockout (KO) mouse models, Tcf15^fl/fl R26^CreER and Vav-iCre Tcf15^fl/fl, and a Tcf15 overexpression (OE) mouse model, Col1a1^{TetOTcf15 /TetOTcf15} R26^{M2rtTA /M2rtTA} (TetO-Tcf15). Recapitulating previously published data from our lab using lentiviral OE and KO systems, Tcf15 KO HSCs were unable to regenerate the bone marrow of recipients following secondary transplantation, while OE of Tcf15 in mice transplanted with TetO Tcf15 HSCs inhibited lineage output. These results confirmed that Tcf15 is required for HSC maintenance and regeneration.

To test whether Tcf15 OE can selectively inhibit T and B lymphopoiesis, TetO-Tcf15 mice were administered normal or doxycycline supplemented (dox) water for 10 and 30 days. Mice were then sacrificed and their bones and thymus processed for immunophenotypic analysis. Tcf15 OE mice showed a dramatic reduction in bone marrow (BM) B220⁺ cells and thymic CD4⁺CD8⁺ cells, as early as 10 days. In contrast, no significant difference was observed in the frequency of myeloid Gr-1⁺ cells. Further analysis of the B220⁺ compartment revealed a significantly reduced frequency of B220^low cells. To examine whether this lymphoid deficient phenotype was an intrinsic property of Tcf15 OE cells, we transplanted WBM cells (2x10⁶) from TetO-Tcf15 mice into lethally irradiated 45.1 recipient mice. After confirming engraftment (2 months), mice were administered normal or dox water for 1 month. Recapitulating what we observed in our native TetO-Tcf15 OE model, and confirming cell intrinsic control, dox treated mice showed a significant reduction in both B220⁺ BM and thymic CD4⁺CD8⁺ cells.

To understand the cellular mechanism by which Tcf15 OE inhibits T lymphopoiesis, we sorted MPP4s from BM and DN1 and DN2 progenitors from thymus of TetO-Tcf15 mice and differentiated these in T cell conditions on OP9 DLL4, with or without dox. While control TetO-Tcf15 MPP4s differentiated into all T cell progenitor subsets and CD4⁺CD8⁺ cells, dox treated TetO-Tcf15 MPP4s were arrested at the DN1 stage. In addition, dox treated TetO-Tcf15 DN2 and DN3 cells did not generate CD4⁺CD8⁺cells. Extending these studies to B lymphopoiesis, we sorted MPP4s from the BM of TetO-Tcf15 mice and differentiated these in B cell conditions on OP9, with or without dox. Supporting our in vivo data, dox treated TetO-Tcf15 MPP4s effectively generated myeloid cells, but no B cells. We next performed immunophenotypic analysis on MPP4s and CLPs from the BM of TetO-Tcf15 mice administered normal or dox drinking water for 30 days. Tcf15 OE resulted in a significant decrease in the frequency of MPP4s and CLPs in the BM, demonstrating that Tcf15 inhibition of lymphopoiesis extends to HSPCs. For a more mechanistic understanding, we performed single cell RNA-seq on LSK and LK cells from TetO-Tcf15 mice that were administered normal or dox drinking water for 10 days. Our initial analysis revealed that Tcf15 OE attenuates expression of DNTT, an enzyme critical to lymphoid development. Interestingly, immunophenotypic analysis of BM and thymus from Tcf15^fl/fl vs Vav-iCre Tcf15^fl/fl or tamoxifen induced Tcf15^fl/fl R26^CreER mice revealed no difference in T and B lymphoid cell frequency.

Altogether, our data suggests that Tcf15 is dispensable for lymphoid development from HSCs, but that elevated Tcf15 expression may repress the activity of regulatory networks that promote T and B lymphopoiesis.