Fig 2: IAV infection alters cell surface expression of ligands for NK cell receptors. A549 cells were infected with FM-MA, PR8, or CA/07 at an MOI of 1. At 17 h, cells were fixed and immunostained to determine cell surface levels of NK cell ligands; cells were subsequently permeabilized for immunostaining of intracellular IAV proteins. (A) Flow cytometry analysis of cells immunostained with a pan-HLA-A/B/C antibody, or antibodies specific for class I HLA proteins HLA-B, HLA-C, or HLA-E or isotype antibody controls. (B) Flow cytometry analysis for cells immunostained with antibodies to detect NK cell-activating ligands MICA/B and CD155/PVR, or inhibitory ligand CD113/NECTIN3, or isotype antibody controls. Representative histograms (top) show results of a single experiment; the vertical lines represent the expression level of the target in uninfected cells. (Bottom) Mean fluorescence intensity (MFI) relative to uninfected cells. Each data point represents an independent experiment. Means ± SD are shown. *, P < 0.05; *2, P < 0.01; *3 P < 0.001. (C) A549 cells were mock infected or infected with PR8 for 16 h or CA/07 for 15 h at an MOI of 1, fixed, permeabilized, and stained with polyclonal antibodies specific for IAV proteins (green). DNA was stained with Hoechst-33342 (blue). Scale bar represents 20 µm.

Fig 3: Identification of a panel of surface markers that can further purify MICB+PVR+ MKPs.(A) Twenty index sorts of A1ATD1 MKs. Rank indicates the first occurrence of a marker in a depth-first search of the decision trees making up the bagging model; genes with consistently low rank are discriminative for a given replicate. (B) Determining cutoffs for MKP markers based on all index sorts of A1ATD1 MKs. Outliers were excluded, and the 20 datasets were normalized and combined. The resulting dataset was then divided into 60 bins based on PVR expression and the colonies and noncolonies counted in each bin. This allowed the calculation of (1 - proportion colony formation per bin) (y axis). Cells were then ordered with decreasing PVR expression, and the proportion of cells with higher than this value of PVR is plotted on the x axis. The dataset was then subsetted on this expression level of PVR (top 20%), and the analysis was repeated for MICB, CD51, CD168, and VEGFR1. Areas with maximum colony formation are shown with shaded boxes. (C) Final sort strategy for MKPs. Cells are first purified as MICBhiPVRhi, then as CD51hiCD168hi, and lastly as VEGFR1lo.

Fig 4: Identifying MKP surface markers by scRNA-seq.(A) UMAP of SMART-Seq scRNA-seq of a D44 A1ATD1 culture. Insert: Density clustering decision plot. (B) Normalized putative marker expression per cluster in (A). (C) Flow cytometry analysis of MICB, PVR, CD41, and CD42 expression during FFDK and QOLG3 MK differentiation. (D) qPCR analysis of MICB and PVR transcript levels during A1ATD1 MK differentiation. (E) Index sort schematic. (F) Index sort results: A1ATD1 MKs. Expression in cells that did not (N) and did form a colony (Y). CD41 (n = 7), CD42 (n = 7), MICB (n = 6), and PVR (n = 8). (G) Sorting scheme. (H to J) Colony counts (H), bright-field microscopy (I), and flow cytometry analysis (J) on D12 after D75 A1ATD1 MKs were sorted for MICB or PVR. (K) Sorting scheme. (L and M) Colony counts (L) and flow cytometry analysis (M) on D11 or D13 after D33 or D35 A1ATD1 MKs were sorted into CFU assays. (N) qPCR analysis of sorted D23 A1ATD1 MKs. Replicates are shown as points. For flow cytometry, viable cells were selected as DAPI negative and thresholds set on a corresponding isotype control for each sample. All colony counts were averaged per experiment (n = 2) and normalized to plated cell number and Live cell condition.

Fig 5: TIGIT+ B cells suppress the proliferation of CCR6+ Tfh cells.(A–C) Sorted CD4+CD45RA–CXCR5+ Tfh cells and CD4+CD45RA–CXCR5– non-Tfh cells were stimulated with anti-CD3/anti-CD28 antibodies (each 1 µg/mL) for 3 days. Representative flow data for CD155 expression (A, left) and the proportion of CD155+ cells (A, right) (n = 10) are shown. (B) PVR mRNA expression was measured relative to B2M by qPCR (n = 7). (C) Proportion of CD112+ cells (n = 10). (D) Experimental workflow for coculture assays with sorted CD20+CD27+ memory B cells and CD4+CD45RA–CXCR5+ Tfh cells. (E) Representative flow data for Tfh cell proliferation. (F) Proportion of proliferated CellTrace Violet+ (CTV+) cells. (G) IL-17 and IFN-? expression in the supernatants of coculture assays was evaluated by ELISA. (H) Correlation between CD4+CD45RA–CXCR5+CCR6+ Tfh cells (percentage of CD4+CD45RA–CXCR5+ Tfh cells) and TIGIT+ cells (percentage of CD20+CD27+ memory B cells). Data for healthy donors are indicated by blue dots (n = 15) and by red dots for patients with MS (n = 16). Linear regression is shown with a 95% CI (pink area). (I) Proportion of CCR6+ Tfh cells between healthy donors (n = 18) and patients with MS (n = 17). Data are presented as the mean ± SEM and were evaluated by 2-tailed, unpaired Student’s t test (A–C and I) or Wilcoxon matched-pairs, signed-rank test (F and G).