NOD2^−/− allo-BMT recipients have increased systemic and target organ GVHD because of enhanced activation and proliferation of alloreactive donor T cells. (A) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2^−/− allo-BMT recipients received 5 × 10⁶ B10BR TCD-BM or 5 × 10⁶ LP TCD-BM with or without 10⁶ B10BR T cells or 2 × 10⁶ LP T cells. Combined data from four independent experiments are shown; n = 45/group. (B-E) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2^−/− allo-BMT recipients received 5 × 10⁶ B10BR TCD-BM + 10⁶ B10BR T cells. Organs were harvested at day 21 after allo-BMT. Combined data from two independent experiments are shown; n = 10/group. (B) Histopathologic GVHD scores in target organs showing increased GVHD in NOD2^−/− allo-BMT recipients. (C) CD3⁺ histochemistry images showing increased infiltration of T cells (brown) in liver tissue of NOD2^−/− allo-BMT recipients. Bars, 50 µm. (D) Quantification of T cell infiltration in GVHD target organs by CD3⁺ histochemistry. (E) Increased thymic GVHD in NOD2^−/− allo-BMT recipients demonstrated by reduced numbers of CD4⁺/CD8⁺ double-positive thymocytes. Shown are flow cytometric plots and the absolute numbers of DP (CD4⁺/CD8⁺), DN (CD4⁻/CD8⁻), CD4⁺, and CD8⁺ thymocytes. (F) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2^−/− allo-BMT recipients received 5 × 10⁶ CFSE-labeled B10BR CD5⁺ T cells. Recipient spleens were harvested 96 h after transfer of T cells; n = 8/group; combined data from two independent experiments are shown. CFSE histogram overlays of donor T cells in WT allo-BMT recipients versus NOD2^−/− allo-BMT recipients (left); ratio of proliferating donor T cells (right, top) and absolute numbers of donor T cells (right, bottom) showing increased donor T cell proliferation in NOD2^−/− allo-BMT recipients. (G and H) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2^−/− allo-BMT recipients were transplanted with 5 × 10⁶ B10BR TCD-BM + 10⁶ B10BR T cells. Organs were harvested at day 14 after allo-BMT. Combined data from two independent experiments are shown; n = 10/group. (G) Absolute numbers and activation status (CD25⁺) of donor CD3⁺ T cells in spleen and MLNs. Percentage of FoxP3⁺ cells in CD4⁺ splenocytes (top right) and expression of the gut homing marker LPAM in MLNs (bottom right) are shown. (H) Absolute numbers of donor B cells, NK cells, monocytes, DCs, and granulocytes in spleen. Error bars indicate SEM.

NOD2^−/− DCs have a higher activation status and increased ability to induce T cell proliferation during GVHD. Lethally irradiated (11 Gy) B6 WT versus B6 NOD2^−/− allo-BMT recipients were transplanted with 5 × 10⁶ B10BR TCD-BM + 2 × 10⁶ B10BR T cells. Organs were harvested at day 7 after allo-BMT. Donor and host-derived DCs were distinguished by MHC class I disparity in flow cytometry (H2^k vs. H2^b). Combined data from two independent experiments are shown; n = 8/group. (A) Quantification of host DC subsets by flow cytometry. (B) Quantification of activation marker expression of host DCs by flow cytometry before allo-BMT and at day 7 after allo-BMT. (C) Histogram overlays of activation marker expression of host DCs during GVHD. (D) MLR using 10⁵ splenic DCs (CD11⁺ selection) from B6 WT allo-BMT recipients versus B6 NOD2^−/− allo-BMT recipients as stimulators and 10⁵ B10BR T cells (CD5⁺ selection). Shown is the thymidine H³ uptake (cpm) between 48 and 72 h of MLR. Error bars indicate SEM.

NOD2 deficiency in donor BM cells and/or donor T cells does not affect the development of GVHD. (A–C) Lethally irradiated (8.5 Gy) BALB/c allo-BMT recipients received 5 × 10⁶ CFSE-labeled B6 WT or B6 NOD2^−/−CD5⁺ T cells. Recipient spleens were harvested 96 h after transfer of T cells; n = 8/group; combined data from two independent experiments are shown. (A) Identical CFSE histogram overlays of WT and NOD2^−/− donor T cells (left), as well as similar total number of CFSE^high and CFSE^low donor T cells. (B) Similar up-regulation of the alloactivation marker CD25 on WT B6 donor T cells versus NOD2^−/− donor T cells. (C) Similar expression of the gut-homing marker LPAM on WT B6 donor T cells versus NOD2^−/− donor T cells. (D) MLR with WT versus NOD2^−/−CD5⁺ T cells and irradiated BALB/c stimulators. Combined data of three independent experiments are shown; n = 12/group. (E) Lethally irradiated BALB/c (8.5 Gy) or LP (11Gy) allo-BMT recipients were transplanted with 5 × 10⁶ B6 WT TCD-BM versus B6 NOD2^−/− TCD-BM and 10⁶ B6 WT T cells versus B6 NOD2^−/− T cells. Combined data from four independent experiments are shown; n = 15–45/group. Error bars indicate SEM.

NOD2 deficiency of the hematopoietic system of the allo-BMT recipient aggravates GVHD. (A–D) Chimeric mice with NOD2 deficiency either in the hematopoietic system or in the nonhematopoietic system were created by syngeneic BMT (B6 WT→B6 NOD2^−/− or B6 NOD2^−/−→B6 WT). After 90 d, lethally irradiated (11 Gy) B6 WT versus B6 NOD2^−/− versus chimeric allo-BMT recipients were transplanted with 5 × 10⁶ B10BR TCD-BM or 5 × 10⁶ LP TCD-BM + 2 × 10⁶ B10BR T cells or 3 × 10⁶ LP T cells. (A) Survival curves. Combined data from two independent experiments are shown; n = 16/group. (B-E) Lethally irradiated (11 Gy) B6 WT→B6 NOD2^−/− or B6 NOD2^−/−→B6 WT chimeric allo-BMT recipients were transplanted with 5 × 10⁶ B10BR TCD-BM + 10⁶ B10BR T cells. Organs were harvested at day 14 after allo-BMT. Combined data from two independent experiments are shown; n = 8/group. (B) Histopathologic GVHD scores in GVHD target organs. (C) Quantification of T cell infiltration in GVHD target organs by CD3⁺ histochemistry. (D) Absolute number and activation status (CD25⁺) of donor CD3⁺ T cells in spleen and MLNs, percentage of FoxP3⁺ regulatory T cells (top right), and expression of the gut-homing marker LPAM in MLNs (bottom right). Error bars indicate SEM.

NOD2 deficiency in the hematopoietic system regulates experimental colitis. (A) TNBS colitis (5 mg TNBS in 50% ethanol) was induced in B6 WT or B6 NOD2^−/− mice. Higher weight loss of NOD2^−/− mice during colitis. Combined data from three independent experiments are shown; n = 12/group. (B–D) First, chimeric mice with NOD2 deficiency either in the hematopoietic system or in the nonhematopoietic system were created by syngeneic BMT (B6 WT→B6 NOD2^−/− or B6 NOD2^−/−→B6 WT). After 90 d, TNBS colitis (5 mg TNBS in 50% ethanol) was induced. Colons were harvested at day 3 after induction of colitis. Combined data from two independent experiments are shown; n = 8/group. (B) Higher weight loss of NOD2^−/−→WT chimera versus WT→NOD2^−/− chimera during colitis. (C) Hematoxylin and eosin–stained images of the colon at day 3 after induction of colitis. Bar, 200 µm. (D) Histological scoring showing increased colitis in the hematopoietic NOD2-deficient chimera. Error bars indicate SEM.