Dedicator of cytokinesis 8 (DOCK8) mutation impairs the differentiation of helper T cells by regulating the glycolytic pathway of CD4+ T cells

Abstract

Dedicator of cytokinesis 8 (DOCK8) deficiency is a primary immunodeficiency disease caused by mutations in exon 45 of the DOCK8 gene. The clinical signs primarily consist of increased serum IgE levels, eczema, repeated skin infections, allergies, and upper respiratory tract infections. Using CRISPR/Cas9 technology, we generated a DOCK8 exon 45 mutation in mice, mirroring the mutation found in patients. The results indicated that DOCK8 mutation impairs peripheral T cell homeostasis, disrupts regulatory T cells (Tregs) development, increases ICOS expression in Tregs within peripheral lymph nodes (pLn), and promotes Th17 cell differentiation within the spleen and pLn. Upon virus infection, DOCK8 mutation CD4⁺ T cells have a Th2 effector fate. RNA-bulk sequencing data revealed alternations in the mTOR pathway of DOCK8 mutant CD4⁺ T cells. We observed that DOCK8 mutation upregulates the glycolysis levels in CD4⁺ T cells, which is related to the Akt/mTOR/S6/HIF-1α pathway. In summary, our research elucidates that DOCK8 regulates the differentiation of helper T cells by modulating the glycolytic pathway in CD4⁺ T cells, thereby advancing the comprehension and offering potential treatment of diseases in DOCK8-deficient patients.

Keywords: DOCK8; T cell; differentiation; glycolytic.

FIGURE 1

DOCK8 mutation increases serum IgE levels and decreases dedicator of cytokinesis 8 (DOCK8) protein in T cells. (A,B) The DNA of DOCK8 in wild type (WT) and DOCK8 mutant mice was amplified by PCR and sanger sequencing (Mut = DOCK8 mutant mice). (C) The level of IgE in serum was analyzed by enzyme linked immunosorbent assay (ELISA) (n = 6). (D) Western blotting of DOCK8 in CD4⁺ T cells. (E) The size of the peripheral lymph nodes (pLn) was shown (n = 9). (F) H and E staining results of tissues (scale bar = 100 µm).

FIGURE 2

DOCK8 mutation cripples peripheral T cell homeostasis without affecting thymus T cell development. (A–E) Flow cytometry analysis of T cells in the thymus, spleen, peripheral lymph nodes (pLn), and mesenteric lymph nodes (mLn). Representative plots of flow cytometry (A). Quantification of the percentages (B,C) and cell numbers (D,E) of CD4⁺ T cells and CD8⁺ T cells (SP4 ,  CD4 single positive; SP8, CD8 single positive; DP, double positive; DN, double negative; n = 6). (F–K) Flow cytometry analysis of naive and activated T cells in the spleen and pLn from WT and DOCK8 mutant mice. Representative plots of flow cytometry (F,G). Quantification of the percentages of naive and activated CD8⁺ T cells (n = 6) (H,I). Quantification of the percentages of naive and activated CD4⁺ T cells (n = 6) (J,K). (L) Flow cytometry analysis of apoptotic CD4⁺ T cells in the spleen. Quantification of the percentages of late apoptotic cells (Q2) and early apoptotic cells (Q3, n = 3). (M) Proliferation of CD4⁺ T cells in the spleen after stimulation. Quantification of the division index by Flowjo software (n = 3).

FIGURE 3

DOCK8 mutation disturbs the development and ICOS expression of Treg cells in the peripheral lymph nodes (pLn). (A–K) Flow cytometry analysis of CD4⁺CD25⁺ Treg cells and CD4⁺Foxp3⁺ Treg cells in the spleen, pLn, and mesenteric lymph nodes (mLn). Representative plots of flow cytometry (A,D). The percentages and cell numbers of CD4⁺CD25⁺ Treg cells (B,C) and CD4⁺Foxp3⁺ Treg cells (E,F) were quantified (n = 6). The MFI of Foxp3 (G) was quantified in CD4⁺Foxp3⁺ Treg cells (n = 6). The MFI of ICOS (H), PD‐1 (I), CTLA‐4 (J), and NRP1 (K) was quantified in CD4⁺CD25⁺ Treg cells (n = 6).

FIGURE 4

DOCK8 mutation promotes CD4⁺ T cells differentiation to Th17 cells. (A) Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of remarkably different genes in CD4⁺ T cells (p < 0.05). (B) Gene enrichment analysis (GSEA) showing enrichment of the Th17 cell differentiation pathway in CD4⁺ T cells (p < 0.05). (C–J) Flow cytometry analysis of cytokine expression of CD4⁺ and CD8⁺ T cells in the spleen, peripheral lymph nodes (pLn), and mesenteric lymph nodes (mLn). Representative plots of flow cytometry (C,E,F). The percentages of IL‐17 (D), IFN‐γ (G,H), IL‐4 (I), and IL‐2 (J) were quantified in CD4⁺ and CD8⁺ T cells (n = 6). (K) The mRNA of IL‐17F, IL1R1, SOX4, and RORC was examined by RT‐PCR (n = 3).

FIGURE 5

DOCK8 mutation CD4⁺ T cells are biased to Th2 effector fate after lymphocytic choriomeningitis virus (LCMV) infection. (A–C) WT and DOCK8 mutant mice were injected intraperitoneally with LCMV. One week later, flow cytometry analysis of cytokine expression was done in the spleen. Representative plots of flow cytometry (A). The percentages of CD4⁺ and CD8⁺ T cells were examined in the spleen (n = 4) (B). The percentages of IL‐2, IL‐4, IL‐17, and IFN‐γ were analyzed in CD4⁺ T cells (n = 4) (C). (D) The level of IgE in serum was quantified by ELISA after LCMV infection (n = 4).

FIGURE 6

DOCK8 mutation upregulates the glycolysis level of CD4⁺ T cells associated with Akt/mTOR/S6/HIF‐1α pathway. (A) Gene enrichment analysis (GSEA) showing enrichment of the mTOR signaling pathway in CD4⁺ T cells (p < 0.05). (B) Western blotting of pAkt, Akt, pmTOR, mTOR, pS6, S6, PKM2, and HIF‐1α in CD4⁺ T cells. (C–F) The mean fluorescence intensity (MFI) of PK Mito (C,D) and dihydroethidium (DHE) (E,F) were analyzed by confocal, respectively. (G) Extracellular acidification rate (ECAR) detection of CD4⁺ T cells (n = 3). (H) Oxygen consumption rate (OCR) detection of CD4⁺ T cells (n = 3).