Fig 2: Application of reporter cell line assay in multiplex detection of anti-HLA antibodies in serum samples from allo-sensitized patients. (A) K528-derived fluorescence-barcoded reporter cell lines were transduced to express HLA class I molecules along with human β2M or class II molecules along with human CD74. (A) The resultant cell lines were pooled, and the surface expression of HLA molecules on individual cell lines were validated with a panel of reference monoclonal antibodies. Example gating strategy is shown in Figure S3 . Demultiplexed reporter cell lines with MFI values above three-fold of the average MFI value of internal control cell lines (K528-FP0000 and K528-FP1111) were scored as positive and the histograms were highlighted in orange. (B) Multiplex detection of anti-HLA antibodies with 10 serum samples from allo-sensitized patients. W6/32 (hIgG1) was used as positive control, and hIgG1κ and hIgG1λ ( Figure S4 ) as negative controls in staining. Positive readouts that are also positive in clinical HLA SAB readouts were considered as true positive (TP) and indicated with green arrows. Negative readouts that are positive in bead readouts were considered as false negative (FN) and indicated with blue arrows. Positive readouts that were bead not tested (BNT) in SAB assay are indicated with gray arrows. MFI values are listed in Table S1 . (C) Clinical HLA SAB MFI readouts. The color codes are in accordance with those in B), with TPs in both assays highlighted in green, and TPs in SAB assay while FNs in reporter cell assay in blue. HLA alleles are listed in abbreviations as indicated in parentheses in (A). For class II molecule lines, bulk cell line after antibiotic selection were used.

Fig 3: Disease stage-dependent gene expression pattern in myeloid cells during experimental CNV Trajectory analysis reveals alterations in gene expression pattern over time and disease state (d0, d3, d7). Above: Trajectory heatmap depicting loss of homeostatic microglial gene expression signatures (P2ry12, Tmem119, Csf1r, SiglecH) and induction of genes involved in cytoskeleton modifications (Actb, Tuba1c), cell cycle progression (Cdk1, Ptma), and antigen presentation (Cd74, H2-Aa). Highlighted molecules (asterisks) were confirmed by immunohistochemistry shown in (B–D). Below: Trajectory plot showing the trajectory followed by myeloid cells upon CNV progress.Above: P2ry12 expression is shown by trajectory analysis and represented in a t-SNE plot. Expression is high in homeostatic microglia (see Figs 1G and H and 7H). Below: Typical immunohistochemistry for P2RY12 (green) in unlesioned and lesioned areas. Arrows indicate lost P2RY12 expression. Asterisks mark microglial cells that maintained P2RY12 expression. Iba1 immunohistochemistry (red) to show microglia. Pictures are representative of two mice. Scale bars represent 25 µm.Above: Tmem119 expression visualized by trajectory analysis and represented in a t-SNE plot. High signals in homeostatic microglia (see Fig 1G and H). Below: TMEM119 immunofluorescence (green) by Iba1+ (red) microglia inside lesioned and non-lesioned areas in the retina and RPE. Arrows indicate microglia with lost TMEM119 expression whereas asterisks mark microglial cells that maintained TMEM119 expression. Picture is representative of two mice. Scale bars represent 25 µm.Above: Ptma expression presented by a t-SNE plot and its trajectory analysis over the disease course. Ptma expression correlates with proliferation of rMGs at d7 during CNV in Cx3cr1CreERT2:Rosa26-tdTomato mice. Below: Representative immunofluorescence of proliferating EdU+ (white) Iba1+ (red, Tomato) microglia at sites of CNV on d7 (arrow) next to an EdU-Tomato+Iba1- cell (asterisks) after EdU treatment from d0 to d6. Arrowhead points toward an intravascular myeloid EdU-Tomato-Iba1+ cell. Typical pictures from three mice obtained from one independent experiment are shown. Scale bar represents 25 µm.

Fig 4: Single-cell RNA-seq from all cells in the healthy murine eye t-SNE representation of individual cells from all eye compartments measured by scRNA-seq. Each dot represents an individual cell. Color code indicates the respective cell types.Unbiased cluster analysis of cell subpopulations found in the retina, ciliary body, and cornea.Stacked bar plot (Marimekko chart) representation of macrophage subsets to a given cluster.Heatmap of the 30 most regulated genes per cluster (adjusted P-value < 0.05 based on the negative binomial distribution). The scale bar represents the color-coded z-scores. Genes presented in t-SNE plots in (E) are marked by asterisks. t-SNE plot of typical genes for microglia subsets (P2ry12, Tmem119, Hexb), macrophage cluster (Cd74, H2-Aa, Apoe), Rods/Cone cluster (Rho, Prph2, Pdc), and epithelial cells cluster (Emp1, Gsto1, Krt5).

Fig 5: Inhibition of TGFβ signaling increases surface expression of CD74 in microglia. (A) Scheme of experimental workflow for flow cytometry, created with BioRender.com. (B) Gating strategy to detect CD74 surface expression in F4/80+ microglia. Relative fluorescence intensities (RFI) of CD74 in microglia (out of F4/80+ cells) were analyzed after 1 (C), 3 (D), 5 (E), and 7 days (F). (C–F) Quantifications and statistical analyzes of relative fluorescence intensities (RFI) of CD74 after indicated treatments and time points. Data are given as means ± SEM for at least three independent experiments. p-values derived from one-way ANOVA followed by Tukey’s multiple comparison tests are shown as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.