Post-transplant cyclophosphamide facilitates preservation of intestinal innate lymphoid cells and intestinal stem cells. Free

Background Type 3 innate lymphoid cells (ILC3s) produce Interleukin-22 (IL-22) and contribute to epithelial maintenance and regeneration in the intestines. However, graft vs. host disease (GVHD) leads to ILC3 loss, impairing IL-22-driven intestinal regeneration. Post-transplant cyclophosphamide (PT-Cy) is administered to prevent GVHD, but its direct effects on ILCs and intestinal epithelium in bone marrow transplant (BMT) recipients are poorly understood. We sought to evaluate effects of PT-Cy on ILC3s and the intestinal stem cell (ISC) compartment after BMT to determine if PT-Cy leads to additive intestinal toxicity or protection due to mitigation of alloreactive immunity.

Methods Utilizing experimental murine BMT models, we investigated the effects of Cy, administered to normal mice and to transplant recipients, on IL-22-producing lymphocytes and to key epithelial cells in the intestines. In B10.BR –> C57BL/6 (B6) and B6 –> BALB/c BMT models, recipients received split dose radiation on day 0 (1100 cGy and 900 cGy, respectively) and then were transplanted with 5x10⁶ T-cell-depleted BM cells/mouse and 1x10⁶ T cells/mouse. Cy was administered on days +3 and +4 post-BMT at 25 mg/Kg or 50 mg/kg (and days 0 and +1 for untransplanted mice). All experiments were performed following IACUC guidelines. Each experiment included at least 3-5 mice per group.

Results To understand direct effects of Cy on intestinal epithelium, we treated untransplanted mice with escalating Cy doses. 3 days post-treatment at 0, 25, 50 and 100 mg/Kg, Cy-treated mice demonstrated a modest but statistically significant reduction in the frequency of ileal Olfm4⁺ ISCs/crypt compared to PBS-treated controls. Small intestine lamina propria (LP) ILC3 frequencies remained similar in Cy-treated mice and controls. Use of IL-22-GFP reporter mice confirmed that the frequency of IL22⁺ ILC3s remained stable despite Cy treatment at the doses tested. These results indicated that there was some direct epithelial toxicity in healthy mice due to Cy treatment, but this toxicity was relatively mild, and the treatment appeared to be tolerated by the intestinal ILC3 compartment.

We next examined the effects of Cy treatment post-transplant. Examining intestinal lymphocytes in BMT recipients (B10.BR B6, day +14), PT-Cy led to substantial reduction in donor T cells infiltrating the small intestine lamina propria as well as substantial preservation of host T cells within the intestinal lamina propria. Furthermore, use of IL-22-GFP reporter mice as BMT recipients indicated that PT-Cy treatment was also able to prevent the elimination of host IL22⁺ ILC3s within the GI tract. Therefore, in comparison to PBS-treated controls, the reduction of infiltrating GVHD-causing allogeneic T cells was associated with some preservation of the ILC compartment following PT-Cy. Moreover, despite the direct toxicity of Cy that led to ISC reductions in healthy mice, immunofluorescent (IF) staining of recipient ileum after BMT indicated preservation of Olfm4⁺ ISC frequencies by day 14 post-transplant in allogeneic BMT recipients treated with PT-Cy. While Olfm4⁺ ISC frequencies were reduced in allogeneic BMT recipients treated with PBS when compared with ISC frequencies in syngeneic BMT recipients, ISC frequencies were similar in syngeneic BMT recipients and in PT-Cy-treated allogeneic BMT recipients. Consistent with, ISC gene expression analysis post-BMT, performed by qPCR on ISC mRNA from hemagglutinin-labeled ISC ribosomes using the Cre-targeted RiboTag system, provided evidence for reduced ISC apoptosis in allogeneic BMT recipients following PT-Cy.

Conclusions These data indicate that that despite direct dose-dependent Cy-induced toxicity to intestinal epithelium, rather than exacerbating transplant-associated intestinal injury, PT-Cy facilitates protection of ISCs and IL-22-producing ILCs from GVHD. Therefore, in addition to the direct effects of reduced alloreactive injury post-transplant, another potential mechanism reducing GI GVHD in recipients of PT-Cy may be the preservation of the tissue-resident intestinal ILC compartment.