Journal: iScience
Article Title: Single cell profiling of hematopoietic stem cell transplant recipients reveals TGF-β1 and IL-2 confer immunoregulatory functions to NK cells
doi: 10.1016/j.isci.2024.111416
Figure Lengend Snippet: CD56 bright NK cells expanded in IL-2 and TGF-β1 exhibit regulatory functions in vitro (A) Method of NK cell isolation, PBMCs were stained with FITC conjugated antibodies for the following lineage markers: CD3, CD4, CD8α, CD14, CD15, CD19, CD20, TCRαβ, TCRδγ, CD33, CD34, CD203c, FCεr1α, CD79α, and CD138. NK were sorted as live CD56 bright CD16 − or CD56 dim CD16 + , and CCR6 − and CRTH2 − to exclude ILC2s and ILC3s. (B) Representative gating strategy to sort CD56 bright CD16 − NK cells and CD56 dim CD16 + NK cells from healthy PBMCs. (C) Fold expansion of NK cells after 19 days of in vitro culture in IL-2, IL-15, and IL-18 cytokines, or IL-2, TGF-β1, and anti-IFN-γ referred to as NK conv and NK TGF-β , respectively. (D) Representative and summary cytokine expression of CD56 bright NK conv and NK TGF- β at day 20 post-expansion, stimulated with phorbol 12-mystrate 13-acetate (PMA)/ionomycin for a duration of 6 h ( n = 7, 3). (E) Active TGF-β1 within supernatants of NK cells plated at 1 M/mL at D20 ( n = 14, 16). (F and G) Naive autologous CD4 + T cells were labeled with cell proliferation dye (CPD) for a short term 4-day culture with either NK conv ( n = 4) or NK TGF-β ( n = 6) with anti-CD3 and anti-CD28 antibodies. Suppression was measured by percent divided of the live CD3 + CD4 + CD56 − CD8 − in comparison to naive CD4 + T cells activated with anti-CD3 and anti-CD28 antibodies. Dashed line indicates 0 percent suppression. (H) Summary of CPD + HELIX-NP + and Annexin-V + K562 cells. Helix-NP + Annexin-V + indicates apoptotic cells ( n = 5, 6). (I) Expression of FOXP3 and CD25 within co-cultures with NK cells ( n = 3–6). (J) Following 20 days co-culture of CD4 + T cells with IL-2 and TGF-β1-treated CD56 bright NK cells, CD25 + CD127 low/− T cells were sorted then co-cultured with CPD stained allogeneic naive T cells for 3 days ( n = 3, 4). Induced Tregs (iTregs) were generated using standard induction protocols of IL-2 and TGF-β1 ( n = 3, 4). Suppression as measured by percent divided of CD4 + CD3 + CPD + T cells. Asterisk indicates a significant difference, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Significance was determined by Kruskal-Wallis test (C–E and G), (F) Mann-Whitney U test, and (I) Signed Wilcoxon-rank test. Data are represented as mean ± SEM (D and I) or median (E, F, H, and I) are shown.
Article Snippet: APC anti-human CD25 (clone BC96) , eBioscience , Cat#17-0259-42; RRID: AB_1582219.
Techniques: In Vitro, Cell Isolation, Staining, Expressing, Labeling, Comparison, Co-Culture Assay, Cell Culture, Generated, MANN-WHITNEY
