Fig 2: HIOs promote proliferation and maturation of systemic human ILCPs(A) Gating strategy for PBMC-derived ILCPs, pregated on live, single, CD45+ cells (FMOs, blue and magenta).(B) Schematic of HIO + ILCP co-cultures, indicating the presence of mesenchymal cells and CD45+ ILCPs.(C) Representative plot overlays of EpCAM+ IECs, double-negative mesenchymal cells (M, magenta), and CD45+ ILCs (blue) after 14-day co-cultures, highlighting Matrigel debris in gray, and quantified in (D) as count of EpCAM-, CD45+, LINn- ILCs and (E) fold change expansion of ILCs after 14-day co-culture relative to the number of ILCPs seeded on day 1 (N = 3–15 across seven experiments).(F) Representative image of CD45+ ILCPs co-cultured with mesenchyme-depleted, EpCAM+ HIOs (scale bar: 25 µm). Error bars represent SEM; p values are from unpaired Student’s t tests.(G) Count of Live, EpCAM-CD45+LIN-ROR?t+ ILCs after 14-day co-culture with SD-HIOs or epithelial-depleted HIO-STROs expressing markers CCR6, NKp44, and/or T-bet (ILCPs from N = 3 PBMC donors).(H) Relative group 1 (Live, CD45+Lin-CRTH2-c-kit- [CD127+CD161+ in primary gut ILC only] and [EpCAM-ROR?t-GATA3- in SD-HIO and HIO-STRO only]), group 2 (Live, CD45+Lin-CRTH2+c-kit+/-, [CD127+CD161+/- in primary gut ILC only] and [EpCAM-ROR?t-GATA3+ in SD-HIO and HIO-STRO only]), group 3 (Live, CD45+Lin-CRTH2-c-kit+, NKp44+/- and [CD127+ CD161+ in primary gut ILC only] and [EpCAM-ROR?t+GATA3- in SD-HIO and HIO-STRO only]), and other LIN- ILCs (e.g., undifferentiated precursors; Live, CD45+, Lin-, CRTH2-, c-kit- and [CD127+ CD161+ in primary gut ILC only] and [EpCAM-, ROR?t-, GATA3- in SD-HIO and HIO-STRO only]) in unstimulated, primary human intestine (N = 13, adapted from Krämer et al., 2017), SD-HIOs (N = 13 from six experiments), and HIO-STROs (N = 7 from three experiments).

Fig 3: Proliferating CD4+CD45RO+ T Cells Infiltrate Preferentially the High Active Lesion, whereas CD8+CD45RO+ T Cells Are More Globally Expanded, Related to Figures 5 and 6 and Tables S4 and S5(A) Correlation of the number of shared unique productive TCRVß sequences in cloneset samples 1 (M) and 2 (N) with the number of unique productive TCRVß sequences in cloneset sample 1 (M) multiplied by the number of unique productive TCRVß sequences in cloneset sample 2 (N), in order to normalize the number of shared TCRVß sequences for the cloneset size (Zvyagin et al., 2014). Correlations were performed for overall TCRVß sequences and TCRVß sequences that were identified in the peripheral CD4+CD45RO+ or CD8+CD45RO+ T cell compartment (Spearman’s rank correlation test).(B) Frequencies of shared unique productive TCRVß sequences in the AP compartment and the brain lesions of MS patient 1 (experiment B). Colors indicate, which TCRVß sequences were also identified in the peripheral CD4+CD45RO+ (green) or CD8+CD45RO+ (blue) T cell compartment. Circles correspond to brain-matching TCRVß sequences which are found in both CFSEhi and CFSEdim cells, and triangles correspond to brain-matching TCRVß sequences only found in CFSEdim cells. The position of the symbols reflects the corresponding frequency of the TCRVß sequence inside the AP compartment or the brain lesion.(C) Brain-homing CD4+ TCCs from the AP compartment of MS patient 1 were analyzed for the expression of the brain-homing receptors CXCR3 and CCR6. Prior to analysis, gates were set on live cells.

Fig 4: Gut-matured ILC2 upregulates ST2 on transfer to HLO culture(A) Representative image of SD-HIOs and SD-HLOs showing E-cadherin+ (E-CAD) epithelium, CD45+ ILCs, and nuclei (Hoechst) after 14-day co-culture (scale bars: 50 µm).(B) Count of EpCAM-, CD45+, LIN- ILCs after 14-day co-culture with SD-HIOs or SD-HLOs, with corresponding count of EpCAM-, CD45- mesenchyme.(C) Representative flow plots of ROR?t, CCR6, IL-22, IL-17A, IFN-?, and NKp44 after 14-day co-culture with SD-HIOs or SD-HLOs after 4-h PMA/Ionomycin stimulation (pregated population in gray, representative of N = 3).(D) IL-5 and IL-13 expression in putative GATA3+ ILC2s after 14-day co-culture and 4-h PMA/Ionomycin stimulation (FMOs, magenta and cyan).(E) Expression of CD25 and ST2 in putative GATA3+ ILC2s after 14-day co-culture with SD-HIOs or SD-HLOs.(F and G) Relative frequency of CD25+ and ST2+ putative ILC2s (F) and GeoMFI of ST2 in the same population with corresponding histogram overlay of ST2 (PE) after 14-day co-culture with SD-HIOs or SD-HLOs, followed SD-HIO-to-HIO, SD-HIO-to-HLO, or SD-HIO-to-HLO with 50 ng/mL hIL-33 neutralizing antibody (G). All experiments were performed with ILCPs from N = 3 donors; unpaired two-tailed Student’s t tests were used; and error bars represent SEM.

Fig 5: AP Involves Classical and Non-classical Th1 Cells with Increased Proinflammatory Responses in MS(A) AP (% CFSEdim) and cytokine secretion profile for each donor (n = 32 HDs; n = 32 REM) after 7-day culture. Subject values are organized according to their AP strength. The graphs under the heatmap represent the mean cytokine secretion in supernatants (mean ± SEM; Mann-Whitney U test).(B) Mean cytokine response following AP in the presence of blocking HLA-DR, CD4, or isotype controls (n = 5 REM).(C) Intracellular IFN-? and phosphorylation of STAT1 in CFSEhi and CFSEdim (AP) T cells. Representative example out of 5 REM.(D) Proportion of CD4 and CD8 subsets in AP IFN-?+ T cells (mean ± SEM; n = 5 REM; Mann-Whitney U test).(E) RNA sequencing data of sorted CFSEhi and CFSEdim (AP) CD4+ T cells (n = 5 REM) for Th1, Th2, and Th17 gene sets, each defined by 17 subset-specific genes. MKI67 served as control transcript for proliferation. The differential expression shows only significantly upregulated genes (log2 >1.0; false discovery rate [FDR] <0.01) in AP CD4+ T cells and is expressed by the z-score based on the reads per kilobase million (RPKM) values. Th gene sets were tested for significance (Fisher test).(F) Distribution of classical Th1 (CXCR3+CCR6–), non-classical Th1 (CXCR3+CCR6+), and Th17 (CXCR3–CCR6+) cells in the CFSEhi and CFSEdim compartment, exemplarily shown for AP, MLR, and PHA in one donor and as mean for AP in HDs (n = 9) and REM (n = 13).See also Figure S2 and Table S1.