Figure 7. Lethal GVHD of C57BL/6 recipients induced by BALB/c CD4+ CD25− effector T cells can be suppressed by co-injected C57BL/6 CCR9+ DCs.

(a) C57BL/6 mice received 2 × 450 rads of total body irradiation, 2 × 10⁶ BALB/c T-cell depleted bone marrow cells and 0.5–1 × 10⁶ BALB/c splenic CD4+CD25− T cells. Three cohorts of mice received either coinjected sorted CCR9⁺ pDCs, CCR9+ pDCs or no pDCs at all (no DC control) at 0.2–0.5 × 10⁶ DCs/mouse from pooled peripheral lymph nodes of Flt3L-treated B6 mice. Combined data from two independent experiments are shown, with a total of 9–10 animals per treatment group. The two independent experiments showed similar results with roughly 50% mortality in the absence of protection by CCR9⁺ pDCs. *, P = 0.027, **, P = 0.010 using the logrank test comparing the survival of allogeneic bone marrow and effector T cell treated mice receiving CCR9⁺ pDCs versus no pDCs or CCR9⁺ pDCs versus CCR9− pDCs respectively. (b) Intracellular cytokine staining of splenocytes and peripheral lymph nodes from irradiated C57BL/6 (Thy1.2⁺) mice three weeks after i.v. transfer of 2 × 10⁶ BALB/c (Thy1.2⁺) T-cell depleted bone marrow cells, 0.5–1 × 10⁶ splenic Thy1.1⁺ effector CD4⁺CD25⁻ T cells from congenic BALB/c.Thy1.1 mice and either no pDCs or sorted CCR9⁺ or CCR9⁻ pDCs from Flt3L-treated B6 (Thy1.2⁺) mice. Cells were gated on CD4⁺ Thy1.1⁺ effector T cells and each bar represents the percentage of gated cells producing intracellular IL-17 and IFN-γ from 2–3 pooled mice. Data presented are from one of two experiments with similar results. (c) Analysis of the same groups of mice in B for the expression of intracellular foxp3 and surface CD25 from unstimulated splenocytes and MLN cells. Cells were gated on CD4⁺ Thy1.1⁺ effector T cells and the gates were set based on the isotype controls for the anti-Foxp3 and anti-CD25 mAbs to include <1% in the positive stained gates. Representative FACS plots from two experiments.