Fig 2: Increased longevity of CD73+ memory T cells(A) NT5E, BCL2, IL7R, and KIT expression quantified by RT-PCR. Data are shown as 2(-delta Ct) *10-5.(B) CD73+ and CD73- memory CD4 T cells, respectively, were cultured in the absence of cytokines. Cells were stained with Annexin V and 7-AAD. Results are shown as representative contour plots (left) and summary data (right).(C and D) CD127 (IL-7R) expression on CD73+ versus CD73- cells of CD4 (C) and CD8 (D) memory T cell subsets. Representative histograms (left) and summary data (right).(E) Cells were cultured in the presence or absence of IL-7 (10 ng/mL) for indicated times and stained with Annexin V and 7-AAD. Contour plots are representative of four experiments.Data were compared by two-tailed paired t test. ***p < 0.001, ****p < 0.0001.See also Figure S3.

Fig 3: Immune cell populations in colonic tissue from HSCR and control patients and in vitro T-cell conversion assay. (A) Flow cytometric analysis of mononuclear cells isolated from different aganglionic and ganglionic colonic segments of HSCR and control patients. Frequencies of total cells are shown. IL17+ Th17 T cells were gated in viable CD3+ CD4+ lymphocytes restimulated with PMA/ionomycin. Treg cells were defined as CD127- Foxp3+ cells in unstimulated viable CD3+ CD4+ lymphocytes. Fiber-low (n = 30); fiber-high (n = 13); AG DC (n = 10); G DC (n = 10); control DC (n = 5). CD14+ MF were gated in viable HLA+ CD64+ cells. Fiber-low (n = 26); fiber-high (n = 13); AG DC (n = 10); muscle (n = 10); G DC (n = 10); control DC (n = 5). Exact P values indicated in Table 10. (B) Frequencies of NCR+ (Nkp44+) and NCR- (Nkp44-) ILC3 (CD117+) subsets. Fiber-low (n = 17); fiber-high (n = 9); AG DC (n = 6); G DC (n = 6); control DC (n = 3). Figure 15 shows flow cytometric gating strategy. Scatter plots with bar show means ± standard error of the mean. Significance was determined by multiple comparison using one-way analysis of variance (*P = .05; **P = .01, ***P = .001, ****P = .0001); exact P values indicated in Table 10. (C) Quantitative RT-PCR analysis of full-thickness colonic tissue or isolated muscle tissue from different aganglionic and ganglionic colonic segments of HSCR and control patients (CX3CR1, circles and CCR2, squares). Significance was determined by using Wilcoxon matched-pairs signed-rank test. Fiber-low (n = 31); fiber-high (n = 13); AG DC (n = 12); G DC (n = 11); control DC (n = 5). (D and E) Representative brightfield microscopic images (original magnification, ×10) of M2 (D) and M1 (E) after 10 days of differentiation. Scale bar: 20 µm. (F and G) Flow cytometric analysis of blood-derived M1 and M2 macrophages from adult healthy donors. Histograms show CD14 (F) and CX3CR1 (G) expression in M1 (green) and M2 (blue) macrophages as well as in unstained controls (filled histograms). Representative analysis out of 3 independent experiments is shown. (H) Th17 and Treg conversion assay using blood-derived M1 and M2 macrophages. Frequencies of Th17 and Treg cells were determined, and Th17/Treg ratio was calculated. One out of 3 independent experiments is shown. Significance was determined using unpaired t test. Figure 15 indicates FACS gating strategy.

Fig 4: Characterization of Lin- lymphoid cells among human PBMCs A CD127+ and CD127- cells from Lin-CD56- population after gating on lymphoid, singlet, live, CD45+ cells of PBMCs. Lineage (Lin) markers include antibodies against: CD3, CD4, TCRaß, TCR?d, CD19, CD20, CD22, CD34, FceRIa, CD11c, CD303, CD123, CD1a, and CD14.B Heatmap of differentially expressed genes by RNA-Seq, sorted Lin-CD56-CD16-CD127+ versus Lin-CD56-CD16-CD127- cells, from PBMCs of five donors (log2 fold change > 1, Padj < 0.01 determined by DESeq2, the postsort profiles are shown in Appendix Fig S1).C Normalized counts of Lin-CD56-CD127+ (red) and Lin-CD56-CD127- (blue) cell-related genes from (B) by DESeq2 (n = 5).D, E Reactome analysis based on enriched transcripts of Lin-CD56-CD127+ cells (D) or Lin-CD56-CD127- cells (E).F Gating of Lin-CD56hi NK, Lin-CD56dim NK, Lin-CD56- NK cells, and Lin-CD56-CD16-CD127+ILCs.G The indicated populations in (F) were detected with isotype controls or antibodies against TBX21, CRTH2, and ROR?T. Data information: All data were generated using blood from healthy HIV-1-negative donors.

Fig 5: Cytokine profile and immune cell status in colon of HSCR patients. (A) Luminex analysis of full-thickness colonic tissue isolated from different aganglionic (AG) and ganglionic (G) colonic segments of HSCR and control patients. IL23 was determined by enzyme-linked immunosorbent assay. Fiber-low (n = 30); fiber-high (n = 13); AG DC (n = 12); G DC (n = 12); control DC (n = 5). IL23 protein expression was not detectable in 5 fiber-low, 3 fiber-high, 3 aganglionic DC, 5 ganglionic DC, and 3 control DC samples. (B–E) FACS analysis of mononuclear cells isolated from different aganglionic and ganglionic colonic segments of HSCR and control patients. Frequencies of IL17+ Th17 T cells (B), CD127-Foxp3+ Treg cells (C). Fiber-low (n = 30); fiber-high (n = 13); AG DC (n = 10); G DC (n = 10); control DC (n = 5). (D) Ratio Th17/Treg cells. (E) Frequencies of CD11c+ and CD14+ MF. Fiber-low (n = 26); fiber-high (n = 13); AG DC (n = 10); muscle (n = 10); G DC (n = 10); control DC (n = 5). (F) Immunofluorescence of CD64+ MF from a colonic muscle region (left), a mucosal region from a fiber-low patient (center), and a mucosal region from a fiber-high patient (right). Scale bar: 10 µm and 30 µm (muscle). Images from 3 fiber-high and 3 fiber-low patients were analyzed for MF shape. A total of 100–200 CD64+ cells per patient were counted, and the percentage of bipolar cells was calculated (bar graph). Scatter plots (with and without bars) show means ± standard error of the mean. Significance was determined using one-way analysis of variance multiple comparison analysis (A–F) and an unpaired nonparametric Mann-Whitney test (*P = .05; **P = .01; ***P = .001). Table 10 shows P values. Figure 15 shows the FACS gating strategy.