American Society of Hematology

Fig. 1.

$Fig. 1. Alloactivated wt (but not TNF−/−) T cells express TNF. TNF is required for maximum GVHD induction, and for target organ damage in thymus, small and large bowel, but not liver. / (A) B6 splenocytes of wt or TNF−/− origin (pooled from 4 animals per group) were coincubated in a primary MLR with irradiated allogeneic (C3HxB6) stimulator cells for 5 days, then harvested, and restimulated for 15 hours with allogeneic or syngeneic irradiated stimulator cells (T-cell depleted, to reduce contamination with cytokine expressing stimulator cells) in the presence of Brefeldin A. All cells (“allo” and “syn”) are initially stimulated with allogeneic stimulators for 5 days for technical reasons (syngeneic “stimulation” does not yield viable cells after 5 days). Only for overnight restimulation on day 5 are responder cells divided into allo- or syngeneic secondary stimulation in the presence of Brefeldin A. This explains the small remaining fraction of IFN-γ–expressing cells in the syngeneic control group. The 2 panels on the right side show a similar analysis of splenocytes that were harvested on day 14 after BMT (from recipients which had received allogeneic TCD-BM and T cells), and subsequently restimulated in vitro as described above. Cells were then stained for intracellular TNF. Populations shown are gated for CD4+, CD44+, and CD62L−. Percentages of TNF-expressing cells are indicated (followed by percentage of isotype-control positive cells in parentheses). One experiment representative of 4 separate experiments is shown. (B,C) Lethally irradiated C3HxB6 mice were given transplants of B6 TCD BM with or without the addition of (B) 2 × 106splenic T cells or (C) 1 × 106 purified splenic CD4+ T cells from B6 wt or TNF−/− mice. Survival from GVHD was monitored and plotted as a Kaplan-Meier curve. Groups consisted of 4 animals for the BM-only control group and 7 to 15 animals for the groups receiving T cells. (D-E) C3HxB6 recipients given transplants as described in (A) were killed on day 28 and organs were harvested. (D) Liver, small bowel, and large bowel were analyzed in a blinded fashion with a semiquantitative GVHD scoring system as described. Higher scores indicate more severe GVHD organ damage. (E) Thymi of the same animals were dispersed and total thymocyte number was recorded.$

Alloactivated wt (but not TNF^−/−) T cells express TNF. TNF is required for maximum GVHD induction, and for target organ damage in thymus, small and large bowel, but not liver.

(A) B6 splenocytes of wt or TNF^−/− origin (pooled from 4 animals per group) were coincubated in a primary MLR with irradiated allogeneic (C3HxB6) stimulator cells for 5 days, then harvested, and restimulated for 15 hours with allogeneic or syngeneic irradiated stimulator cells (T-cell depleted, to reduce contamination with cytokine expressing stimulator cells) in the presence of Brefeldin A. All cells (“allo” and “syn”) are initially stimulated with allogeneic stimulators for 5 days for technical reasons (syngeneic “stimulation” does not yield viable cells after 5 days). Only for overnight restimulation on day 5 are responder cells divided into allo- or syngeneic secondary stimulation in the presence of Brefeldin A. This explains the small remaining fraction of IFN-γ–expressing cells in the syngeneic control group. The 2 panels on the right side show a similar analysis of splenocytes that were harvested on day 14 after BMT (from recipients which had received allogeneic TCD-BM and T cells), and subsequently restimulated in vitro as described above. Cells were then stained for intracellular TNF. Populations shown are gated for CD4⁺, CD44⁺, and CD62L⁻. Percentages of TNF-expressing cells are indicated (followed by percentage of isotype-control positive cells in parentheses). One experiment representative of 4 separate experiments is shown. (B,C) Lethally irradiated C3HxB6 mice were given transplants of B6 TCD BM with or without the addition of (B) 2 × 10⁶splenic T cells or (C) 1 × 10⁶ purified splenic CD4⁺ T cells from B6 wt or TNF^−/− mice. Survival from GVHD was monitored and plotted as a Kaplan-Meier curve. Groups consisted of 4 animals for the BM-only control group and 7 to 15 animals for the groups receiving T cells. (D-E) C3HxB6 recipients given transplants as described in (A) were killed on day 28 and organs were harvested. (D) Liver, small bowel, and large bowel were analyzed in a blinded fashion with a semiquantitative GVHD scoring system as described. Higher scores indicate more severe GVHD organ damage. (E) Thymi of the same animals were dispersed and total thymocyte number was recorded.

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