Preparation of ILC2s for co-transplantation
Bruce et al. have previously reported the beneficial effects of WT ILC2s on gut manifestations of aGVHD by using cells isolated from C57BL6 mice. Both Id1tg/tg and dKO mice, also on the C57BL6 background, generate a large amount of ILC2s in the thymus due to down-regulation of E protein transcription factors, which suppress the ILC2 fate. However, ILC2s from Id1tg/tg and dKO mice arise from multipotent progenitors and committed T cell precursors, respectively [18, 19]. To prepare ILC2s for transplant, we followed the experimental schema reported by Bruce et al. (Fig. 1A) and sorted lineage-negative and Thy1+ cells from the mesenteric lymph nodes after the mice were treated with 400 ng of IL-25 per day for 4 days (Fig. 1B) [14]. These sorted cells were then propagated in 10 ng/ml IL-7 and IL-33 for 7 days. This resulted in approximately 10-fold expansion of ILC2s. It is of note that we were usually able to obtain fewer than 20,000 Lin−Thy1+ cells per treated WT mouse whereas about 105 and 106 such cells from Id1tg/tg and dKO mice, respectively. Therefore, we could easily set up cohorts of at least 5 recipients of 106 Id1tg/tg or dKO ILC2s in each transplant session. However, we only had enough WT ILC2s from 10 treated donor mice for 1–2 recipients (106 cells per recipient), thus making the transplant cohort difficult to establish.
In addition to ILC2s, we sorted B220−CD25− splenocytes from C57BL6 mice to enrich for T cells while removing B cells and T regulatory cells. Whole bone marrow cells (BM) were used for each recipient to rescue hematopoiesis. We reasoned that T cell depletion from the bone marrow was unnecessary because most of the transplant cohorts received 106 donor T cells and the BM alone group did not have long-term engraftment to cause GVHD (supplemental Fig. 1). Unfortunately, due to the lack of a donor-specific marker, we were not able to localize donor ILC2s in the recipients.
ILC2s from Id1tg/tg and dKO mice can be activated by IL-25 and IL-33
To assess the purity of our ILC2 preparations, we stained the cells for the expression of Thy1 and ST2 markers at the end of the 7-day culture (Fig. 2A). Although Thy1+ cells were placed in culture, incubation for 7 days led to the down-regulation of Thy1 expression, which is not unusual. Consistent with their response to IL-33, the majority of the cells expressed ST2, a component of the IL-33 receptor.
To determine the functionality of the ILC2 preparations, we tested their ability to produce ILC2 signature cytokines upon stimulation with PMA and ionomycin in the presence of a Golgi blocker, monensin, for 3 h. Intracellular staining for the expression IL-5 and IL-13 were then carried out. Cells from both Id1tg/tg and dKO mice produced these cytokines as avidly as WT ILC2 controls (Fig. 2B), suggesting that these cells possess a key feature of ILC2s. This is consistent with our previous findings using lung ILC2s from WT, Id1tg/tg and dKO mice [19]. Although PMA is a strong stimulator that might elicit non-physiological responses, our previous experience showed that stimulation with PMA and ionomycin only increased the magnitude of IL5/IL-13 expression [19].
ILC2s from Id1tg/tg mice are beneficial in GVHD
The aGVHD model was established by transplanting 106 BM cells with or without equal numbers of C57BL/6 T cells plus or minus ILC2s into lethally irradiated BalB/c mice. Because C57BL/6 and BalB/c mice carry MHC haplotypes of b and d, respectively, GVHD was readily detectable in the transplant recipients. However, over 75% of the recipients survived up to 45–60 days and no difference in survival rates were observed among different groups of recipients, which comprise the recipients receiving BM only (BM), BM and mismatched T cells (BM + T) or BM and mismatched T cells plus Id1 ILC2s (BM + T + ILC2). WT ILC2s were also used in parallel co-transplantation experiments as controls.
Weight changes were quantified as the percent of the initial weight. While the BM only group regained weight after overcoming the effect of irradiation, the BM + T group continued to exhibit weight loss with a mean weight of 85.6% of the original (p < 0.0001) (Fig. 3A). Co-transplantation with Id1 ILC2s alleviated the weight loss to 94.3% (p < 0.0001) of the initial weight. In contrast, WT ILC2s slightly improved weight loss (by 1.2%) compared to BM + T but the difference was statistically insignificance due to a small cohort of mice receiving WT ILC2s.
The GVHD score consists of 5 criteria: activity, posture, fur texture, and skin integrity as described by van den Brink et al. [26]. Within each category, a score of 1 or 2 is assigned based on the degree of alteration. For example, a weight loss by 10–25% is deemed a score of 1 and that of over 25% is quantified as 2. The maximum GVHD score is therefore 10, which represents the worst disease. When analyzing the GVHD scores of the three groups mentioned above, a similar pattern emerged where the mean score of the BM only group was 0.08 compared to the BM + T group which was 3.04 (p < 0.0001) and the BM + T + Id1 ILC2 group was reduced to 1.58 (p < 0.0001) (Fig. 3B). Co-transplantation of WT ILC2s also significantly decreased the mean score to 2.58 (p = 0.0013). Taken together, our data support the notion that Id1 transgenic ILC2s have protective effects on aGVHD.
ILC2s from dKO mice have complex effects on GVHD
dKO mice have their E protein genes ablated at the committed T cell precursor stages and a large number of ILC2s accumulated throughout the body including mesenteric lymph nodes (MLNs). We were therefore able to evaluate the effects of these ILC2s from MLNs with or without pre-treatment with IL-25, which was intended to expand ILC2s in vivo. As described for Id1tg/tg ILC2s, the mesenteric lymph nodes were processed and ILC2s were cultured for 7 days. The majority of the transplant recipients survived and no significant differences were observed among the different recipient groups (data not shown). Comparing to mice receiving BM only, both the BM + T and BM + T + dKO ILC2 groups showed significant weight loss (around 84–86% of the initial weight, p < 0.0001) (Fig. 4A). Unlike Id1 ILC2s, dKO ILC2s isolated from mice with or without IL-25 pre-treatment did not significantly alleviate weight loss relative to the BM + T group.
When analyzing the overall GVHD scores, dKO ILC2s from mice without IL-25 pre-treatment improved the scores compared to the BM + T group (mean scores of 2.53 vs. 3.41, p < 0.0001) (Fig. 4B). However, dKO ILC2s from IL-25 treated mice slightly worsened the scores (means scores of 3.80 vs. 3.41, p = 0.004). Together, the results show that dKO ILC2s have limited beneficial effects in aGVHD compared to Id1 ILC2s and strikingly, IL-25 treatment of dKO mice generated ILC2s with adverse impacts in the setting of aGVHD.
Alleviation of skin lesions by Id1 transgenic ILC2s
One of major score-driving phenotypes was the development of severe skin lesions as evidenced by areas of denudation, dermatitis and multiple areas of tail hemorrhages in recipients of MHC-mismatched T cells (Fig. 5A). It should be noted that the conventional scoring scale for skin lesions. As shown in Fig. 5A, this is not quantitative enough to accurately reflect the varying degree of skin defects. Nevertheless, w compared the maximum scores of skin integrity in mice which survived the first three weeks after transplant in different cohorts. Consistent with the impact of ILC2s on the overall GVHD scores, ILC2s from Id1tg/tg mice alleviated the skin lesion significantly compared to the B + T group (mean scores of 0.97 versus 0.33, p = 0.02) (Fig. 5B). WT ILC2s slightly reduced the mean skin scores by 0.22 but the difference was deemed statistically insignificant. In contrast, co-transplantation with ILC2s from IL-25 treated dKO mice did not significantly reduce the skin integrity score. ILC2s from dKO mice without IL-25 treatment also did not have any protective effects (data not shown).