Inflammatory bowel disease (IBD), an umbrella term for Crohn's disease and ulcerative colitis, affects almost 1 in 150 Australians. Current treatments cause side-effects and are unable to reverse tissue-remodeling and scarring. New therapeutics are urgently needed. Type 1 regulatory cells (Tr1 cells), a subset of CD4+ regulatory T cells, have been shown to suppress inflammation and promote wound healing in vitro. We hypothesized that Tr1 cellular therapy could prevent gut inflammation and damage.
We tested our hypothesis in a DSS-induced colitis mouse model. First, CD4+ T cells were isolated from Thy1.1+ IL-10-GFP reporter mice and stimulated ex vivo for 48-hours with anti-CD3/CD28 antibodies and IL-27 with or without all-trans retinoic acid (ATRA), and the IL-10+ Tr1 cells were isolated by cell sorting. We confirmed these Tr1 cells had significantly higher expression of the co-inhibitory marker ICOS than endogenous Tr1 cells, they retained high levels of IL-10 secretion, and those treated with ATRA had increased expression of the gut homing integrin α4β7. Next, 10-week-old C57BL/6J mice were sub-lethally irradiated and injected (I.V.) with 1x106 Tr1 cells (N = 10) or PBS (controls; N = 11). Following a 7-day engraftment period, mice were administered drinking water containing 1.5-2% DSS for another 7-days. Mice were then given normal drinking water for 7-days, allowing for recovery, at which point blood, spleen, mesenteric lymph nodes (MLN), and colonic cross-sections were acquired for flow cytometric analysis and histology.
Mice receiving Tr1 cellular therapy retained significantly more weight at day 7 compared to controls (p = 0.0163) and had improved colon length, width, and histologic scores (p = 0.0406, 0.0234, 0.0229 respectively). Our most striking finding was that mice treated with ATRA-conditioned ICOS+ Tr1 cells retained more weight than controls and had decreased levels of the inflammatory marker lipocalin-2 in stool samples. RNA-sequencing revealed ATRA-conditioned Tr1 cells upregulated Icos, Il10, Ahr, and Maf transcripts, all associated with functional Tr1 cells in both mice and human studies.
Our data show that Tr1 cellular therapy prevents weight loss and colonic damage in a DSS-induced murine model of colitis. Preliminary data suggests ATRA-induced ICOS+ Tr1 cells are superior at preventing murine colitis and may be a suitable cellular therapy for IBD.