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.