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.
Highlights d The crypt base region is the primary intestinal location invaded by T cells after BMT d T cell infiltration does not correlate with overall intestinal vascular architecture d Allo donor T cells rapidly access the ISC compartment and directly interact with ISCs d MAdCAM-1 localizes to crypt region vessels and directs T cells to the ISC compartment
55 years, range 4-72) with moderate-severe CGVHD following an allogeneic HCT for a primary hematologic malignancy. NIH consensus criteria (2015) were used for CGVHD grading. Patients were divided into 2 cohorts, a) those receiving an extended course of azithromycin (14 days) for CGVHD management (cohort 1, n=86) and b) those who did not (cohort 2, n=153). Patients in cohort 2 either did not receive any azithromycin (n=122) or had received an abbreviated (<14 day) course (n=31) of azithromycin post-HCT. For cohort 1, the median time to initiation of azithromycin therapy was 15 months post-HCT (range 3-68), with a 26 month median duration of azithromycin therapy (range 1-77). All patients in cohort 1 met NIH consensus criteria for BOS. Patients in cohort 2 did not exhibit BOS, but still met NIH criteria for moderatesevere CGVHD. Hematologic conditions included acute leukemia (n=139), MDS/MPD (n=44), malignant lymphomas (n=26), chronic leukemia (n=24), and multiple myeloma (n=6). Ninetyone patients exhibited moderate and 148 patients exhibited severe CGVHD. The two cohorts were balanced for CGVHD severity, with severe CGVHD noted in 69% and 63% of patients in cohorts 1 and 2 respectively. RESULTS: Decreased rates of relapse and improved survival were noted for patients treated with azithromycin for CGVHD. At 2 years, the cumulative incidence of relapse was 2% (95% CI:1-9%) for patients in cohort 1 vs 16% (95% CI: 11-23%) in cohort 2, p=0.001. Overall survival (2-year OS) in cohort 1 was 93% (95%CI: 88-99%) vs 78% (95% CI:72-85%) for cohort 2, p=0.003. Overall, 7 of 86 (8.1%) CGVHD patients treated with an extended course of azithromycin (cohort 1) have relapsed, the median time to relapse 876 days (range 379-1303) post-HCT. In comparison, 28 of 153 (18.3%) in cohort 2 have relapsed, a median 371 days (range 98-1252) post-HCT. CONCLUSION: The use of azithromycin for the management of moderate to severe CGVHD was not associated with an increased risk of relapse in patients undergoing HCT for a hematologic malignancy. Azithromycin therapy for patients with CGVHD should not be contra-indicated in this patient population.
Corticosteroids (CS) represent first-line treatment for gastrointestinal graft vs host disease (GI GVHD), and CS failure is associated with severe morbidity and mortality. While the immune system is the intended target of CS treatment, the glucocorticoid receptor (GR) is widely expressed, and there is limited understanding of the direct effects of CS on intestinal epithelium following immune-mediated damage. We thus investigated how CS treatment could impact intestinal homeostasis and regeneration following experimental bone marrow transplantation (BMT). In healthy C57BL/6 (B6) mice, in vivo administration of clinically relevant CS doses reduced Ki67 + epithelial proliferation in the ileum (p<0.001; Fig. 1A) without inducing crypt loss or overt pathology. Given the numerous potential effects of systemic administration, we next utilized ex vivo small intestine (SI) organoid cultures to explore direct effects of CS on murine and human epithelium. Assessing a variety of clinically relevant CS agents, we found that methylprednisolone (MP), dexamethasone, and budesonide all decreased murine organoid size without affecting organoid number (p<0.05; only MP shown; Fig. 1B). We also identified that GR-deficient (Nr3c1 -/-) organoids were significantly resistant to growth inhibition by MP (p<0.05), indicating a direct GR-mediated effect of CS on intestinal epithelium leading to reduced growth. Furthermore, MP treatment significantly decreased the size of human organoids generated from primary duodenal tissue without affecting organoid numbers (p<0.001). Organoid culture models were thus highly consistent with the findings from in vivo CS treatment. We next investigated CS effects on epithelial cells during immune-mediated damage. Pre-treatment of mice with 2 mg/kg MP x 7 days in vivo prior to crypt harvest and organoid culture increased organoid sensitivity to T-cell-mediating killing ex vivo (p<0.05). Additionally, modeling steroid-refractory disease, GR-deficient (Nr3c1 -/-) T cells mediated greater killing of SI organoids if co-cultures were performed in the presence of MP (p<0.01). We next investigated CS-mediated effects on epithelial damage in vivo, treating with MP x 7 days starting on day 7 after MHC-mismatched BMT, once GVHD had already been established. Vehicle-treated mice demonstrated GVHD-associated T cell activation, lymphocytic tissue infiltration, and ileal crypt loss compared to BM only controls, as well as increased height and Ki67 + cell frequency in residual crypts reflecting damage-induced regeneration (p<0.001, Fig. 2A-C). Modeling steroid-refractory disease, systemic CS treatment failed to reduce T cell activation or lymphocytic infiltration. However, MP treatment appeared to attenuate regeneration and worsen intestinal pathology, as evidenced by exacerbated crypt loss in association with reduced crypt height and Ki67 + cell frequency (p<0.01; Fig. 2A-C). Despite potential harmful side effects, CS are frequently necessary for treatment of clinical GVHD. We hypothesized that CS-mediated epithelial suppression could be mitigated by concurrent administration of agents capable of inducing tissue regeneration. Interleukin-(IL)-22 has been shown to promote epithelial proliferation and recovery following GI damage. We thus investigated whether IL-22 treatment could counterbalance CS-induced impairment of epithelial recovery in GVHD. Indeed, addition of IL-22 to MP-treated organoids promoted organoid growth without inducing toxicity/organoid loss in both murine and human SI organoid cultures (p<0.001; Fig. 3A and B). Moreover, IL-22 administration in vivo with F-652, a clinical grade recombinant human IL-22 dimer, reversed MP-mediated crypt loss and reduction of crypt height and Ki67 + cell frequency in mice with GVHD (p<0.001; Fig. 3C). In summary, these findings indicate that CS treatment can suppress epithelial proliferation in the intestines and exacerbate GI damage if it fails to control the pathologic immune response. However, deleterious CS side effects can be counterbalanced by promotion of epithelial regeneration, providing rationale for combining immunosuppression with tissue-supporting therapeutics such as IL-22 to optimize intestinal recovery in GVHD. Figure 1 Figure 1. Disclosures Blazar: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rheos Medicines: Research Funding; Equilibre Pharmaceuticals Corp: Research Funding; Carisma Therapeutics, Inc: Research Funding; Tmunity Therapeutics: Other: Co-founder. Hanash: Evive Biotech: Ended employment in the past 24 months.
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