Fig 1: Changes in BM T cell function in children with B-ALL and AML at diagnosis.BMMNCs from B-ALL (n = 13), AML (n = 17), or HD (n = 5) were cultured alone or with PMA/ionomycin in the presence of GolgiStop. After 4 hours of culture, cells were stained with dead cell exclusion dye as well as antibodies to detect surface CD3, CD4, CD8, PD-1, TIGIT, intracellular IFN-?, and IL-2 and analyzed using flow cytometry. (A) Proportion of CD8+ and CD4+ T cells secreting IFN-? and IL-2. (B) IFN-? secretion by cells expressing PD-1 and/or TIGIT. Figure shows a representative plot from patient with AML. All graphs show mean ± SEM. *P < 0.05 by Mann-Whitney U test with Bonferroni’s correction for multiple comparisons.
Fig 2: Changes in BM T cell transcription factors in children with B-ALL and AML at diagnosis.BMMNCs from B-ALL (n = 13), AML (n = 17), or HD (n = 5) were cultured alone or with PMA/ionomycin in the presence of GolgiStop. After 4 hours of culture, cells were stained with dead cell exclusion dye as well as antibodies to detect surface CD3, CD4, CD8, PD-1, TIGIT, intracellular IFN-?, and IL-2 and analyzed using flow cytometry. (A) Expression of TCF1, T-bet, GATA3, and EOMES transcription factors in memory CD4+ (left) and CD8+ (right) T cells in BMMNCs from HD and patients with B-ALL. (B) Expression of TCF1, T-bet, GATA3, and EOMES transcription factors in memory CD4+ (left) and CD8+ (right) T cells in BMMNCs from HD and patients with AML. (C) Tregs (CD3+CD4+CD25+CD127–FOXP3hi) as percentage of total T cells in BM from HD (n = 11) and patients with B-ALL (n = 36) and AML (n = 28). (D) Heatmaps showing characteristics of the TCF1hi and TCF1– CD8+ memory T cells from HD, B-ALL, and AML. All graphs show mean ± SEM. **P < 0.01, ***P < 0.001 by Mann-Whitney U test with Bonferroni’s correction for multiple comparisons.
Fig 3: Differences in BM T cells in children with B-ALL and AML at diagnosis.BM mononuclear cells (BMMNCs) from patients with B-ALL (n = 36), AML (n = 28), and healthy donors (n = 11; n = 5 for 4-1BB) were characterized using single cell mass cytometry. (A) Figure shows percent naive (CCR7+CD45RO–), central memory (TCM; CCR7+CD45RO+), effector memory (TEM; CCR7–CD45RO+), and terminal effector (TERM Eff; CCR7–CD45RO–) CD8+ T cells in B-ALL and HD BM. (B) Percent naive, central memory, effector memory, and terminal effector CD8+ T cells in AML and HD BM. (C) Expression of CD69 on memory CD8+ T cells from B-ALL, AML, and HD marrow. (D) CD8+ T cells expressing 4-1BB in B-ALL, AML, and HD BM. (E) Figure shows expression of inhibitory immune checkpoints PD-1, TIGIT, and LAG3 on CD4+ and CD8+ T cells in B-ALL and HD BMMNCs. (F) Expression of inhibitory immune checkpoints PD-1, TIGIT, and LAG3 in CD4+ and CD8+ T cells from AML and HD BM. All graphs show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Mann-Whitney U test.
Fig 4: Expression of surface markers of pTFH and pTFR cells. (A–D) Quantification of the percentage of cells expressing CCR5, TIGIT, PD1, or ICOS on pTFR and pTFH cells, respectively. HIV- (n = 8, mean ± SEM). HIV+ (in red, n = 6, mean ± SEM) (Mann–Whitney U test). (A,B) In both pTFH and pTFR cells, the percentage of TIGIT-expressing cells was similar in HIV-infected and non-infected individuals. In HIV-infected and non-infected individuals, the expression of TIGIT was higher in pTFR than in pTFH cells. (C,D). In both pTFH and pTFR cells, the percentage of cells expressing PD1/ICOS was similar in HIV-infected and non-infected participants of the MACS. (ns: p > 0.05, *: p = 0.05, **: p = 0.01).
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