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11. NKG2D activation drives Th17 response in Crohn's disease

B. Pariente1, I. Mocan1, M. Camus1, J. Ettersperger1, C. Baudry2, J. Gornet2, M. Lémann2, N. Dulphy1, A. Toubert1, M. Allez2

1INSERM U940, Saint-Louis Hospital, Paris, France; 2Department of Gastroenterology, Saint-Louis Hospital, Paris, France

IL17 has been identified has a key cytokine in the physiopathology of Crohn's disease (CD). We have previously identified a subset of CD4+ T cells exhibiting cytotoxic and pro-inflammatory properties, characterized by the expression of the Natural Killer activating receptor 2D (NKG2D).

Aims: To better characterize the functional properties of CD4+NKG2D+ T cells and their relation with the Th17 pro-inflammatory lymphocytes.

Methods: Lamina propria lymphocytes (LPL) were isolated from 18 CD patients undergoing surgery. Peripheral blood lymphocytes (PBL) were isolated from 42 CD patients, 12 ulcerative colitis (UC) patients and 10 healthy controls. Phenotypic analysis was performed by eight-color flow cytometry and by Reverse-Transcription Polymerase Chain Reaction (Q-RT-PCR). Lymphocytes were incubated with brefeldin A and stimulated for 4 hours with PMA and ionomycin before intracellular staining (IL17, IFNγ and TNFα). To study the effect of NKG2D stimulation, fresh PBLs or LPLs from CD patients were stimulated with P815 cell line transfected or not with NKG2D ligands (MICA/B, ULPB 1/2/3), coated with an agonist anti-CD3 antibody (TCR stimulation) or control isotype followed by cytokine staining as above. To assess the Th1 and Th17 cytokine polarization of CD4+NKG2D+ and CD4+NKG2D− T cells, LPMCs were cultured with anti-CD3/anti-CD28 activation beads with different cocktails of cytokines (IL12 for Th1 and IL1β and IL23 Th17 polarization). After 3 days of culture, IL17 and IFNγ secretions were studied.

Results: LP CD4+NKG2D+ T cells were highly positive for IL17 intracellular staining (34.5±25.3%) and expressed significantly higher levels of IL17 than their LP CD4+NKG2D− counterparts (8.8±6%, p = 0.001). NKG2D expression on PB CD4+ T cells correlated significantly with IL17 production (p = 0.01). In Q-RT-PCR, messenger RNAs of IL-17 and RORC (the transcription factor for Th17) were expressed preferentially in LP CD4+NKG2D+ T cells compared to the CD4+NKG2D− subset (p = 0.02). CCR6 and IL23R were significantly more expressed on LP and PB CD4+NKG2D+ T cells as compared to CD4+NKG2D− T cells (p < 0.05). A high proportion of IL17 producing T cells co-expressed both NKG2D and CD161, as compared to cells not producing IL17 (52.5±23.7% vs. 4.5±3.2%, p = 0.003). Targeting NKG2D by its ligands expressed on P815 cell lines in co-stimulation with the TCR significantly increased the production of IL17 by CD4+ T cells (4.5%±2.1) compared to the stimulation through the TCR alone (1.3%±1.1, p < 0.05). LP CD4+NKG2D+ population secreted two times more IL17 in the presence of IL1β and/or IL23 as compared to the IL15 condition, whereas the CD4+NKG2D− showed almost no stimulation under these conditions.

Conclusion: The subset of CD4+ T cells expressing NKG2D represents a major source of IL17 in CD, and has typical features of Th17 cells. Interactions between NKG2D and its ligands may strongly influence IL17 production in CD. NKG2D pathway could represent a promising therapeutic target in CD.