Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration.

Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell–mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ–deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell–deficient organoids, IFNγR-deficient Paneth cells, IFNγR–deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell–mediated pathology.

Graft-versus-host disease (GVHD) and posttransplant immunodeficiency are frequently related complications of allogeneic hematopoietic transplantation. Alloreactive donor T cells can damage thymic epithelium, thus limiting new T-cell development. Although the thymus has a remarkable capacity to regenerate after injury, endogenous thymic regeneration is impaired in GVHD. The mechanisms leading to this regenerative failure are largely unknown. Here we demonstrate in experimental mouse models that GVHD results in depletion of intrathymic group 3 innate lymphoid cells (ILC3s) necessary for thymic regeneration. Loss of thymic ILC3s resulted in deficiency of intrathymic interleukin-22 (IL-22) compared with transplant recipients without GVHD, thereby inhibiting IL-22-mediated protection of thymic epithelial cells (TECs) and impairing recovery of thymopoiesis. Conversely, abrogating IL-21 receptor signaling in donor T cells and inhibiting the elimination of thymic ILCs improved thymopoiesis in an IL-22-dependent fashion. We found that the thymopoietic impairment in GVHD associated with loss of ILCs could be improved by restoration of IL-22 signaling. Despite uninhibited alloreactivity, exogenous IL-22 administration posttransplant resulted in increased recovery of thymopoiesis and development of new thymus-derived peripheral T cells. Our study highlights the role of innate immune function in thymic regeneration and restoration of adaptive immunity posttransplant. Manipulation of the ILC-IL-22-TEC axis may be useful for augmenting immune reconstitution after clinical hematopoietic transplantation and other settings of T-cell deficiency.

2016-11-02T18:49:00

No abstract

Mechanisms regulating host tissue recovery from immune-mediated damage in gastrointestinal graft vs. host disease (GI GVHD) remain incompletely understood. Prophylactic strategies selectively promoting epithelial regeneration after allogeneic hematopoietic stem/progenitor cell transplantation (allo-HCT) have the potential to reduce GVHD without limiting therapeutic graft vs. leukemia/lymphoma (GVL) responses. We have previously shown that IL-22 produced by recipient-derived innate lymphoid cells (ILCs) provides a critical signal for epithelial recovery following experimental allo-HCT. IL-22-deficient recipients demonstrated increased GVHD mortality and significantly worse loss of crypt base intestinal stem cells (ISCs) during GVHD. Paradoxically, GVHD led to reduced GI IL-22 levels in wild-type (WT) recipients due to the elimination of radioresistant intestinal ILCs. We therefore sought to determine if IL-22 administration after allo-HCT could negate the effect of ILC elimination and reduce GVHD pathology without impairing GVL. We utilized a clinically modeled LP into C57BL/6 (B6) minor antigen mismatched model with T cell-depleted marrow and MACS-purified T cells transplanted into lethally irradiated mice. Recipients were treated daily with PBS or 4ug murine recombinant (r)IL-22 delivered via intraperitoneal (IP) injection starting day 7 post-HCT. This schedule was based on the results of rIL-22 pharmacokinetics tested in untransplanted mice. We found that daily IP administration with rIL-22 led to decreased GVHD pathology in recipient small intestine, large intestine, and liver three weeks post-HCT (Figure 1, p<.001). No differences were observed in skin histopathology, consistent with our previous finding that IL-22-deficient recipients demonstrated equivalent skin GVHD. Further assessment of the intestinal pathology indicated that recipients of rIL-22 had decreased intestinal crypt apoptosis in both small and large intestine (p<.01) with no difference in intestinal lymphocytic infiltration, suggesting that the decrease in GVHD was due to direct effects of IL-22 on the epithelium. Furthermore, no differences were observed in splenic T cell expansion or in GI cytokine expression, including a multiplex panel of inflammatory cytokines. To assess the effects of IL-22 administration on the ISC compartment, we performed LP into B6 allo-HCT using Lgr5-LacZ ISC reporter mice. Recipients treated with rIL-22 demonstrated increased numbers of Lgr5+ ISC three weeks post-HCT during active GVHD with no immunosuppression (Figure 2, p<.05). Preliminary evidence with Lgr5-GFP reporter mice suggested increased ISC Ki-67 staining and thus increased ISC proliferation following IL-22 administration. Small intestine qPCR after IL-22 treatment demonstrated increased expression of Reg3γ (p<.001) and Reg3β (p<.01), suggesting a potential antimicrobial benefit of IL-22 administration. However, there was no difference in Wnt3 or EGF expression, arguing that the stem cell benefit after IL-22 administration was not due to improvement in ISC niche function. Given the lack of IL-22R expression in hematopoietic cells, we hypothesized that IL-22 administration would not limit GVL. This was confirmed by monitoring luciferase+ A20 bioluminescence in B6 into BALB/c tumor challenge recipients treated with rIL-22. Finally, we have previously shown that rIL-22 administration can increase the number of double positive thymocytes post-HCT by protecting thymic epithelium from radiation injury and from GVHD. We hypothesized that this could translate into improved peripheral T cell reconstitution even during active GVHD. Indeed, FVB into BALB/c MHC-mismatched transplant with Rag2-GFP marrow and WT T cells indicated that IL-22 administration increased the development of donor marrow-derived CD4 and CD8+ thymic emigrants four weeks post-HCT (Figure 3, p<.01). In summary, we found that IL-22 administration could reduce intestinal pathology, improve ISC recovery, and promote donor marrow-derived T cell development during GVHD. Importantly, IL-22 administration did not impair GVL. These results suggest that post-transplant IL-22 administration represents a novel strategy to protect intestinal epithelium and improve immune reconstitution after allo-HCT. Disclosures: No relevant conflicts of interest to declare.

with high troughs 2 mcg/mL had enhanced platelet recovery (p¼0.005). In a competing risk 2-week landmark analysis, there were no differences grade II-IV aGVHD incidences (61% vs 57%, p¼0.52) according to troughs. However, patients with a low MPA trough early post-CBT had nearly triple the incidence of grade III-IV aGVHD (27.8% vs 9.5%, p¼0.06, Figure). Conclusions: Higher total MPA troughs are safe and may protect against severe aGVHD. The platelet benefit could be explained by the lower severe aGVHD incidence. Prospective investigation of MPA troughs, and ultimately intervention based on drug monitoring in CBT recipients is warranted.

No abstract