Stimuli leading to Cav-1 expression in murine and human T cells. (A) Expression of Cav-1 in CD4⁺ or CD8⁺ T cells derived from mouse spleens under the indicated conditions as measured by flow cytometry (pooled from 2 experiments, n = 8). The mean fluorescence intensity (MFI) measured for Cav-1 on CD4⁺ or CD8⁺ T cells derived from untreated WT mice was set as “1,” and all values were set in relation to this group. The dotted line indicates the background MFI for Cav-1 that is detected in Cav-1^−/− CD4⁺ or CD8⁺ T cells. WT BM and purified T cells were transplanted as follows: C57BL/6 cells into irradiated BALB/c mice (allogeneic), C57BL/6 cells into irradiated C57BL/6 mice (syngeneic), C57BL/6 cells into non-irradiated BALB/c Rag2^−/−Cγ^−/− mice (allogeneic and lymphopenia), and BALB/c cells into non-irradiated BALB/c Rag2^−/−Cγ^−/− mice (syngeneic and lymphopenia). Untreated WT or Cav-1^−/− mice were used as controls. The analyses of the T cells were performed on day 7 after HCT. *P < .05; **P < .01; ***P < .001. (B) The number of CD3⁺ Cav-1⁺ T cells per HPF is shown within human intestinal biopsies detected by immunohistochemistry. The arrows indicate Cav-1/CD3 double positive cells. Samples from 18 patients were analyzed and different time points after allo-HCT (supplemental Table 1). (C) Expression of Cav-1 in CD4⁺ T cells derived from the PB of healthy controls and patients who underwent allo-HCT without or with acute GVHD. Representative histogram (top) and bar diagram of multiple patients (bottom). The human blood samples were analyzed at multiple time points after allo-HCT (supplemental Table 2). (D) Expression of Cav-1 in in vitro expanded naïve CD4⁺ T cells and naïve Tregs derived from human PB re-stimulated with anti-CD3/28 beads for 3 days. Representative histogram for effector T cells (Teff) and Treg cells (top) and quantification for both Treg and conventional CD4⁺ T cells (n = 3 each) (bottom). The blue lines are the anti–Cav-1 stained cells and the red lines are the isotype-stained cells. The Cav-1 MFI values were normalized in the Teff group (100%). HPF, high power field, n.s., not significant. Ab, antibody; nTreg, naturally occurring Treg.