Runx1/Cbfb2 Complexes Are Required for Lymphoid Tissue Inducer Cell Differentiation at Two Developmental Stages

Hematopoietic lymphoid tissue inducer (LTi) cells are essential for the development of secondary lymphoid tissues including lymph nodes and Peyer's patches. Two transcription factors, the helix-loop-helix inhibitor Id2 and the retinoic acid-related orphan receptor gt (Rorgt), have been shown to be crucial for LTi cell development. However, it remains unclear how the specification of multipotent hematopoietic progenitor cells toward the LTi lineage is programmed. In this study, we report impaired lymphoid tissue organogenesis in mice in which the function of Runx1/Cbfb transcription factor complexes was attenuated by the loss of either the distal promoter-derived Runx1 or Cbfb2 variant protein. We found that LTi progenitors in fetal liver, defined previously as a lineage marker-negative a4b7 integrin (a4b7) + IL-7R a-chain (IL-7Ra) + population, can be subdivided into Rorgt-expressing IL-7Ra high cells and nonexpressing IL-7Ra mid cells. Whereas Id2 and Rorgt are required to direct a4b7 + IL-7Ra mid cells to become a4b7 + IL-7Ra high cells, Runx1/Cbfb2 complexes are necessary for the emergence of a4b7 + IL-7Ra mid cells. In addition, the loss of Cbfb2, but not P1-Runx1, resulted in an inefficient upregulation of Rorgt in residual a4b7 + IL-7Ra + LTi cells at anlagen. Our results thus revealed that Runx1/Cbfb2 complexes regulate the differentiation of LTi cells at two stages: an early specification of hematopoietic progenitors toward the LTi lineage and a subsequent activation of Rorgt expression at anlagen. T he development of the immune system involves the differentiation of effecter cells and lymphoid organogenesis. Peripheral secondary lymphoid tissues, such as lymph nodes (LNs) and Peyer's patches (PPs), are essential sites at which the immune responses take place and are regulated. The formation of anlagen of these tissues is initiated during embryogenesis via the interaction of hematopoietic lymphoid tissue inducer (LTi) cells with mesenchymal lymphoid tissue organizer (LTo) cells (1, 2). Although previous studies have provided molecular insights into how LTi cells migrate correctly and communicate with LTo cells to initiate anlagen formation (3–5), the early differentiation pathway of LTi cells from multipotent hematopoietic progenitor cells remains to be characterized. CD4 + CD3 2 cells that accumulate in neonatal lymph nodes were first described by Kelly and Scollay (6) and are found as cell clusters in the intestines of 17.5 d postconception (dpc) embryos (7). These CD4 + CD3 2 cells express molecules involved in lym-phoid organogenesis such as lymphotoxin-b and chemokine receptor CXCR5 (8). Adoptive transfer of a CD4 …