Fig 2: Tumor ILC3s, especially NKp44+ ILC3s are negatively correlated with pathological stage. Distribution of ILCs and ILC subtypes by FACS. MNCs from tumor, proximal, and distal regions of 58 patients with colon cancer were prepared. (A) The gating used to define ILC subtypes: MNCs were stained for Lin cocktail (CD3, CD14, CD19, and CD20), CD94, CD34, CD1a, TCRα/β, TCRγ/δ, CD45, CD127, CRTH2, and CD117. Total ILCs were identified as Lin− CD94− CD34− CD1a− TCRα/β− TCRγ/δ− CD45+ CD127+, ILC1s were identified as Lin− CD94− CD34− CD1a− TCRα/β− TCRγ/δ− CD45+ CD127+ CRTH2− CD117−, ILC2s were identified as Lin− CD94− CD34− CD1a− TCRα/β− TCRγ/δ− CD45+ CD127+ CRTH2+, and ILC3s were identified as Lin− CD94− CD34− CD1a− TCRα/β− TCRγ/δ− CD45+ CD127+ CRTH2− CD117+. ILC3s were further divided into NKp44+/− ILC3s. (B) ILC levels among the CD45+ cells in the indicated tissues. (C) Percentage of ILC1s, ILC2s, and ILC3s among CD45+ cells and total ILCs in the indicated tissues. (D) Percentage of NKp44+/− ILC3s among CD45+ cells in the indicated tissues. (E, F) Correlation between the percentage of ILC3s or NKp44+/− ILC3s in tumor (T) versus distal (D) tissue and the pathological stage of cancer. The distal tissue was considered normal tissue and was used for normalization to the background, here T/D. (G) Percentage of ILC3s among CD45+ cells in colon glandular cancer and mucous carcinoma tissue. In (B–D, G), each symbol represents the indicated tissue from one patient (circle, tumor; square, proximal region; triangle, distal region). In (A, E, F), each dot represents one patient. A paired t-test and Spearman test were used for statistical comparison. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Isolation and in vitro expansion of human Tregs. (A) Schematic overview of the cell expansion procedure. Tregs are stimulated in vitro for six days with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and then transferred to 6-well plates 24 hours prior to nucleofection in the absence of TCR stimulation. Read-out of the KO was performed 3 to 7 days after nucleofection. (B) Representative FOXP3 expression before and after FACS sorting CD4+CD25+CD127- cells. (C) Cell numbers (left panel) and proliferation rate (right panel) of in vitro expanded Tregs (n = 16 independent donors). Data is depicted as mean ± SD. (D) FOXP3 expression before stimulation and after seven days in culture of one representative donor (left panel) and 16 independent donors (right panel). (E) Cell viability after seven days in culture (n = 16 independent donors). Error bars represent mean ± SD. (F) Suppressive capacity of Tregs after seven days of in vitro culture, measured as the ability of Tregs to suppress CD4+ T cell (Tresp) proliferation. FACS data of one representative donor (left panel) and cumulative data of 3 independent donors (right panel) are shown. (G, H) DNA methylation pattern of the Treg-Specific Demethylation Region (TSDR) assessed in 7 day-in vitro expanded CD4+CD25-CD127+ Tconv and CD4+CD25+CD127- Treg cell subsets. (G) Average methylation rate for 9 CpG sites of the TSDR. Each dot denotes a donor (n =3 different donors matched for Tconvs and Tregs). (H) In the heatmap plot, each box represents the percentage of methylation of a single CpG residue for each sample (n= 3 matched donors for Tconvs and Tregs). Bar colors designate: yellow: 0% methylation; green: 50% methylation; blue: 100% methylation. Error bars represent mean ± SD. Significance was calculated using one-way ANOVA (C) or two-tailed paired t test (F, G). **p ≤ 0.01, ***p ≤ 0. 001 and ****p ≤ 0.0001.

Fig 4: Knock-out of IL2RA in human Tregs impairs suppressive capacity and IL-2-mediated signaling. Human Tregs were cultured in vitro for 7 days prior gene editing (following protocol described in Figure 1A). (A) FOXP3 and CD25 expression were analyzed in mock and IL2RA-KO Tregs that were harvested 4 days after RNP nucleofection. FOXP3 plots are pre-gated on living cells and CD25 plots are pre-gated on living FOXP3+ cells. Data are from one representative experiment. (B) CD25 expression was studied in mock and IL2RA-KO Tregs 4 to 7 days after nucleofection. n = 11 independent donors. Error bars represent mean ± SD. (C) RNP-transfected cells were FACS-sorted for CD25 expression and as living (PI-) CD4+CD127- cells. Top panel displays pre-sorted cells and bottom panels represent re-analysis post sorting. (D) FOXP3 expression after CD25-sorting in mock (n = 3), CD25+ (n = 6) and IL2RA-KO (n = 6) Tregs. Error bars represent mean ± SD. (E) STAT5 phosphorylation was studied in CRISPR-edited Tregs. FACS plot of one representative experiment (left panel) and combined data of three independent donors (right panel). Error bars represent mean ± SD. MFI is normalized over IL-2-stimulated mock. (F) Suppressive capacity of IL2RA-KO Tregs was measured by their ability to suppress T cell proliferation in vitro (ratio 1:2). Left panels show FACS plots of one representative donor. Cells are pre-gated for viability (all columns) and CD4 or CD8 expression (middle and right column, respectively). Right panels show CD4+ and CD8+ T cell proliferation, displayed by dilution of the cell proliferation dye CFSE. Proliferation is normalized over T cell proliferation in the absence of Tregs (n = 3-6 independent donors). Error bars represent mean ± SD. Significance was calculated by two-tailed paired t test (B), Kruskal-Wallis test (D, F) or one-way ANOVA (E). *p ≤ 0.05, **p ≤ 0.01. ****p ≤ 0.0001.

Fig 5: Identification and distribution of group 2 innate lymphoid cells in healthy donors and NSCLC patients. (A) The representative gating strategy of human ILCs (CD45+Lin (CD3, CD14, CD16, CD19, CD20, CD56)-CD127+). ILC1s: CD45+Lin-CD127+CRTH2-CD117-; ILC2s: CD45+Lin-CD127+CRTH2+; ILC3s: CD45+Lin-CD127+CRTH2-CD117+. (B) The proportions of total ILCs among CD45+ cells from HD-PB (PBMCs from healthy donors, n=20), LU-PB (PBMCs from NSCLC patients, n=20), and LU-CA (tumor single cell suspension from NSCLC patients, n=20). (C) The representative results of ILC subset distribution in HD-PB, LU-PB, and LU-CA samples assessed via flow cytometry. (D) The proportions of ILC2s among total ILCs in HD-PB, LU-PB, and LU-CA (for each group, n=20). (E) The proportions of ILC2s among total ILCs of LU-CA at different stages (for each group, n=20). NS, not significant; ****P<0.0001. In (B, D), P values were calculated by one-way ANOVA and post hoc comparisons were performed via Tukey’s test. In (E), P values were obtained in a non-paired two-tailed student’s t-test.