Figure 5. Dysregulated cytokine production by DOCK8-deficient CD4⁺ T cells is greatly improved following HSCT.

Naive and memory CD4⁺ T cells were sort-purified from the peripheral blood of healthy donors (n = 7–25), untransplanted DOCK8-deficient patients (n = 2–7), and DOCK8-deficient patients following HSCT (DOCK8 pHSCT) (n = 6–18). The cells were labeled with CFSE and then cultured under Th0 conditions (TAE beads; naive and memory), or Th1- (+IL-12), Th2- (+ IL-4), or Th17-polarizing (IL-1β, IL-6, IL-21, IL-23, TGF-β, prostaglandin E2) conditions (naive only) for 5 days. (A and B) Cells and culture supernatants were harvested to assess proliferation of (CFSE dilution) and cytokine secretion of (A) Th1 cytokines (IFN-γ/TNF-α), Th2 cytokines (IL-4/IL-5/IL-13), or Th17 cytokines (IL-17A/IL-17F) by memory CD4⁺ T cells, and of (B) of Th1 cytokines (IFN-γ/TNF-α), Th2 cytokines (IL-5/IL-13), or Th17 cytokines (IL-17A/IL-17F) naive CD4⁺ T cells. (C and D) Cells were restimulated with PMA/ionomycin before permeabilization and intracellular staining to determine proportions of cells expressing Th1 (IFN-γ, TNF-α) and Th2 (IL-4, IL-13) cytokines. Data are presented as (C) the ratio of cells expressing Th2 versus Th1 cytokines and (D) the combined percentage of cells from individual donors and patients expressing Th1 (i.e., %IFN-γ⁺/TNF-α⁺/IFN-γ⁺TNF-α⁺ cells) versus Th2 (i.e., %IL-4⁺/IL-13⁺/IL-4⁺IL-13⁺ cells) cytokines. (E and F) Intracellular expression of IL-21 by memory CD4⁺ T cells (E) and naive CD4⁺ T cells cultured under Th0- or Th1-polarizing conditions (F) cells was measured. Graphs show mean ± SEM. Statistical performed with Prism using unpaired t test with Welch’s correction; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.