Conventional CD4⁺CD25^- and Regulatory CDCD4⁺25⁺ t Cells Have Opposite Effects on Progenitor Cells and Hematopoietic Reconstitution Following Stem Cell Transplantation

The bone marrow (BM) is a complex microsystem to support lifelong blood production. At steady-state most hematopoietic stem cells (HSC) are quiescent. However, in situations of increased demand, their activation is triggered by an array of signals, such as cytokines. Following hematopoietic cell transplantation (HCT) in the phase of hematopoietic reconstitution maximal blood production is needed. In an HCT donor HSC are given together with immune cells in the belief that T cells support HSC engraftment and regeneration of the blood system. Yet, states of immune mediated BM insufficiency, hypoplasia, and cytopenias are often observed. Moreover, with increased use of reduced intensity conditioning (RIC) engraftment failure has reemerged as a serious problem.

Here, we studied the effects of distinct T cell subsets on hematopoietic reconstitution following HCT; specifically, we examined how conventional CD4⁺CD25^- T cells (CD4_conv) vs regulatory T cells (CD4⁺CD25⁺, Treg) modify the BM environment and influence donor-HSC activity and engraftment. We used minor-mismatched mouse models, non-myeloablative total body irradiation (TBI) conditioning and transplantations of purified HSC (KTLS; cKit⁺Thy1.1^loLin^–Sca-1⁺) plus selected T cell subsets. Recipients of HSC, HSC+Treg or HSC+CD8⁺ - but not HSC+CD4_conv- demonstrated prompt donor engraftment with mixed chimerism in all lineages. Transplantation of HSC+Treg resulted in significantly faster lymphocyte recovery and higher levels of donor chimerism compared with recipients of HSC, HSC+CD8⁺ or HSC+CD4_conv. Particularly B-cell regeneration was markedly higher in HSC+Treg-recipients compared with all other groups. In contrast, (B-) lymphopoiesis was severely impaired in recipients of HSC+CD4_conv; when lymphocytes recovered eventually they were of host type. Moreover, in BM and spleens of HSC+CD4_conv recipients pronounced hypocellularity was observed. This suppression of hematopoiesis was due to IFNg secretion of donor CD4_conv cells, which were activated by dendritic cells via IL-12. High cytokine levels (of both IL-12 and IFNg) were only detectable in the BM (and not the spleen) of HSC+CD4_conv recipients, where they resulted in an arrest of early hematopoiesis at the stage of short-term HSC and decreased cell-cycle activity within the progenitor compartment. As a consequence more mature multipotent progenitors were lacking. The key role of IFNg in halting hematopoietic maturation was confirmed by using CD4_conv cells from IFNg-/- mice, which had no suppressive effects on BM cellularity and maturation of blood cells; rather, recipients of HSC+IFNg^-/-CD4_conv cells had equivalent cell numbers and subset distributions as mice given HSC alone. We hypothesized that differences of hematopoietic regeneration and donor engraftment relate to cell cycle activity of HSC in presence of CD4_conv vs Tregs. To study the influence of these CD4-subsets on HSC cycling in more detail FACS-purified Treg vs CD4_conv cells were infused into congenic mice following low-dose TBI-stimulation. In fact, on d+8 post-infusion HSC in the BM of Treg-recipients had increased cell-cycling activity in long-term-HSC and multipotent-progenitor fractions compared with mice given CD4_conv cells or radiation only. These data lead us to speculate that Tregs promote, directly or indirectly, HSC proliferation; in the context of an allogeneic HCT this increased cycling of host HSC my open-up the niche space required to allow donor HSC to engraft. In contrast, CD4_convinhibited HSC cycling activity, resulting in BM hypoplasia and cytopenias; at the same time donor HSC engraftment was impaired due to HSC-niche occupation by quiescent host HSC.

Our findings underscore the critical role of T cells in regulating hematopoiesis under physiologic conditions and even more following allogeneic HCT. While donor T cells are generally believed to improve regeneration of the blood post-HCT and to be required to overcome host barriers, our data suggest Treg facilitate engraftment and hematopoiesis by increasing HSC cycling-activity and thereby making marrow sites available. CD4_conv appear to have the opposite effect, resulting in decreased HSC proliferation and maturation - thus occupation of HSC niches. Our studies are of particular relevance to allogeneic HCT settings using RIC, where host HSC persist and grafts can be rejected by residual host immune cells.