Tissue-Derived IL-33 Is a Critical Local Signal That Targets Th1 Cells in the Small Intestine to Sustain Graft Versus Host Disease in the Absence of IL-12
Authors: Dwyer Gaelen K , Mathews Lisa R , Lucas Anna , Blazar Bruce R. , Shlomchik Warren D , Turnquist Heth 
Published: February 2021

Background: Graft vs. host disease (GVHD) remains a major complication of allogeneic stem cell transplantation (alloSCT). To create space for donor stem cells and prevent their rejection, alloSCT protocols rely on conditioning regimens involving chemotherapy, frequently combined with radiation. Interleukin (IL)-33 is a nuclear protein that is found in the secondary lymphoid organs and barrier tissues. IL-33 is augmented in recipient barrier tissues by conditioning. We have established that IL-33 is a clinically-relevant signal. While we have further elucidated that IL-33 is acting directly on donor T cells to augment GVHD, neither the critical place nor timing of IL-33 stimulation of donor T cells during the alloimmune response mediating GVHD have been defined.
Methods: We used both IL-33 knockout mice as recipients and ST2 (IL33 receptor) knockout mice as T cell donors in MHC mismatch alloSCT models to define the spatial and temporal importance of IL-33 in GVHD tissue damage. More specifically, to establish the role of IL-33 in T cell effector function in the small intestine during GVHD, irradiated Il33-/- and Il33+/+ C57BL/6 (B6) recipients were given BALB/c bone marrow (BM) and T cells. Additionally, irradiated BALB/c mice were given St2+/+ B6 BM and Cd4 Cre x St2fl/fl or St2-/- and St2+/+ T cells. To determine the role of IL-33 on T cell persistence in GVHD, we used an MHCII-disparate model. Irradiated Il33-/- and Il33+/+ bm12 recipients were reconstituted with B6 BM, with or without CD90.1+ B6 T cells.
Results: When donor T cell responses were characterized by flow cytometry at days 7, 14, and 28 post alloSCT, the bulk of these data suggest that IL-33 sustains CD4+ T helper Type 1 (Th1) cells in the barrier tissues at late points following GVHD initiation. Specifically, B6 Il33-/- recipients had fewer BALB/c CD4+Tbet+ cells in the small intestine lamina propria (SI LP; WT 15.9 ± 1.31, KO 9.53 ± 1.32; p=0.03; n=3/group), but not in the spleen (WT 54.8 ± 7.37, KO 57.68 ± 4.90; p=0.75; n=4/group) at day 7. Additionally, we found that when we used therapeutic doses of anti-IL-12p40 to neutralize IL-12 there was no impact on the CD4+Tbet+ cells in the SI of B6 il33-/- recipients (KO control IgG 7.29 ± 0.63, KO anti-IL-12p40 6.61 ± 0.75; p=0.52; n=4/group) suggesting that the decrease in the frequency of CD4+Tbet+ T cells in Il33-/- in the SI is independent of IL-12. In the CD4-dependent B6 to bm12 GVHD model, we find that IL-33-deficient recipients have reduced overall CD4+ donor T cells in the SI LP at both day 14 and day 28 (d14: WT 16.15 ± 2.55, KO 0.35 ± 0.07; p=0.003; n=3/group; d28: WT 33.07 ± 7.17, KO 6.27 ± 1.59; p=0.022; n=3/group). Consistent with our B6 recipients of BALB/c T cells above, this decrease was not observed in the spleen or lymph nodes. Reduced clusters of CD3+ cells were evident in immunohistochemistry of bm12 Il33-/- relative to bm12 Il33+/+ at day 28 post-alloSCT. In corroboration with our IL-33-deficient recipient results, B6 CD4+ T cells lacking the ST2 receptor when co-transferred with ST2 competent T cells were not sustained in the SI LP at day 7 (ST2 WT 18.55 ± 0.77, ST2 KO 6.35 ± 1.42; p=0.0003; n=4/group). ST2 deficient CD4+ donor cells were of equal frequency to the ST2 competent donor cells in the spleen of BALB/c recipients (ST2 WT 54.58 ± 1.914, ST2 KO 50 ± 2.53; p=0.19; n=4/group).
Conclusions: Our data reveal that IL-33 is not required for donor Th1 responses in the spleen and LN, but IL-33 is critical for Th1 cells to persist in barrier tissues like the small intestine after alloSCT in the absence of IL-12 stimulation. These data suggest that targeting IL-33 signaling may be an effective therapy to diminish GVHD in target tissues.

Disclosures
Blazar: Fate Therapeutics Inc.: Research Funding; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeutics: Research Funding; BlueRock Therapeuetic: Consultancy; Magenta Therapeutics: Consultancy; KidsFirst Fund: Research Funding; Tmunity: Other: Co-founder. Shlomchik:Bluesphere Bio: Consultancy, Other: shareholder.