Figure 2
Effect of MyD88 deficiency in TCD-BM on the expansion and infiltration of donor-type T cells into the intestine. Lethally irradiated F1 recipients were given 5 × 106 WT or MyD88KO TCD-BM cells plus 1 × 106 purified WT T cells isolated from congenic B6 donors (CD45.1+; n=5, each group, allo WT and allo KO). (a) The expansion of donor T cells (CD45.1+) and non-T cell (CD45.1−) compartments in the spleen, mesenteric lymph node (MLN) and small intestines (SI) were assessed by staining of the leukocytes for expressions of CD45.1, CD4, and CD8 on day 13 post transplantation. (b) The frequencies CD45.1+ cells (donor-type T cells) and CD45.1− cells (non-T-cell compartment) in the leukocytes isolated from different organs shown in a and their absolute numbers are plotted. (c) The frequencies and numbers of donor-type (CD45.1+) CD4+ and CD8+ T cells are plotted. Data are presented as means±s.e.m. and are representative of duplicate experiments. (d) Infiltration of CD45.1+ cells in the SI during GVHD. SI were harvested from F1 recipient mice (n=5) on day 13 and sectioned for staining with anti-CD45.1 Ab as described in “Methods”. Magnification: × 100 (left) and × 200 (right). (e) Lethally irradiated B6 (syn WT and syn KO) or F1 (allo WT and allo KO) recipients underwent transplantation from WT B6.Ly-5a (CD45.1) donor T cells plus either WT or MyD88KO TCD-BM cells. Splenocytes from each group of mice were isolated and analyzed on days 5 and 7 (n=5 per group). CFSE-labeled T cells before the transplantation were used as CFSE-positive controls. Expression of CCR9 was examined by gating the donor T-cell-derived CD45.1+CD4+ or CD45.1+ CD8+ cells. Data from one of five independent experiments are shown. FACS data of syngeneic controls are shown. Data are presented as means±s.e.m. (f) Quantitative analysis of CCL25 protein production was determined by enzyme-linked immunosorbent assay on days 5 and 7. CCL25 expressions of SI were compared between the WT and MyD88KO recipients (n=5 per group). Data from one of two replicate experiments that yielded similar results are shown. Data are presented as means±s.e.m. (g, h) After irradiation, F1 recipients underwent transplantation with WT B6.Ly-5a (CD45.1) donor T cells plus either WT or MyD88KO TCD-BM cells. Splenocytes were isolated and analyzed on day 4. Cells are gated on either CD45.1+CD4+ or CD45.1+CD8+ cells, and the percentages of BrdU- and Annexin V-positive cells (g), and Bax- and Caspase 9-positive cells (h) were determined (n=6 per group). Data in g and h from one of two replicate experiments that yielded similar results are shown. Data are presented as means±s.e.m. BMT, bone marrow transplantation; CFSE, carboxyfluorescein succinimidyl ester; GVHD, graft-vs.-host disease; KO, knockout; TCD-BM, T-cell depleted bone marrow; WT, wild type.
