Figure 2.

SLP76Y145FKI donor CD8⁺ and CD4⁺ T cells exhibit reduced cytokine production and reduced proliferation

1X10⁶ purified CD8⁺ or CD4⁺ T cells from C57Bl/6 WT or C57Bl/6 SLP76Y145FKI (MHC haplotype b) mice were transplanted into irradiated. BALB/c (MHC haplotype d) mice in separate experiments.

(A and B) At day seven post-allo-HSCT, recipient BALB/c mice transplanted with either CD8⁺ or CD4⁺ T cells were euthanized, and ELISA was performed to determine serum cytokines (IL-33, IL1α, IFN-γ, TNF-α, and IL-17A) from recipient mice.

(C and D) Donor CD4⁺ or CD8⁺ T cells were examined for IFN-γ and TNF-α by intracellular staining after stimulation with anti-CD3/anti-CD28, as determined by flow cytometry.

(E) Donor CD8⁺ or CD4⁺ T cells from several experiments were examined for IFN-γ and TNF-α as above.

(F) Flow cytometry analysis of purified CD4⁺ or CD8⁺WT and SLP76Y145FKI T cells that were mixed at a 1:1 ratio for transplantation into irradiated BALB/c mice. At day seven donor T cells were stimulated with anti-CD3/anti-CD28 and then were gated for expression of H-2K^b, CD45.1, CD 45.2. on WT T cells and SLP76Y145FKI cells and intracellular expression of IFN-γ and TNF-α analyzed by flow cytometry. Combined data from two independent experiments is shown, and the p value for each experiment is shown.

(G) Purified CD8⁺ or CD4⁺ WT or SLP76Y145FKI donor T cells were transplanted into irradiated BALB/c mice. On day seven, donor T cells were analyzed for donor CD8⁺ or CD4⁺ T cell proliferation by examining BrdU incorporation by flow cytometry.

(H) Purified CD8⁺ or CD4⁺ T cells from WT or SLP76Y145FKI mice were mixed at a 1:1 WT:SLP76Y145FKI ratio and transplanted into irradiated BALB/c mice. At day 7, splenic donor T cells were gated for the expression of H-2K^b, CD45.1, and CD45.2 and analyzed for BrdU incorporation. See also Figure S3.