Fig 2: HO-1, ROS, and RNS Levels in Myeloid Cells Isolated from Severely Diseased Regions of Human Tuberculous Lung Tissue(A) The sequential flow cytometry gating strategy for identification of monocytes, macrophages, neutrophils, and T cells from freshly isolated CD45+ cells within the distinct pathological regions of human TB lung. Cells were identified by the following cell-surface markers: monocytes (CD14+HLA-DR+CD11clowCD66b−CD3−), macrophages (HLA-DR+CD11c+CD206+CD11b+CD86+CD16−CD66b−CD3−), neutrophils (CD11b+CD66b+CD16+CD14−CD3−), and T cells (CD3+CD14 −CD11b−).(B) Representative histogram of HO-1 expression in neutrophils.(C and D) Percentage of HO-1+ neutrophils (C) and MFI of HO-1 in neutrophils (D).(E) Representative histogram of HO-1 expression in macrophages.(F and G) Percentage of HO-1+ macrophages (F) and MFI of HO-1 in macrophages (G).(H) Representative histogram of HO-1 expression in monocytes.(I and J) Percentage of HO-1+ monocytes (I) and MFI of HO-1 in monocytes (J).(K and L) Percentage of ROS-positive neutrophils, macrophages, and monocytes (K) and their respective MFI of ROS (L).(M and N) Percentage of RNS-positive neutrophils, macrophages, and monocytes (M) and their respective MFI within isolated lung immune cells (N).n = 9–12 individual patients per group (each dot represents an individual patient). Statistical testing was performed using the unpaired Student’s t test. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Black curve in (B), (E), and (H) represents isotype control (Iso).

Fig 3: Immune cell and inflammation response in KidVD mice following selective podocyte injury. Flow cytometry analysis of immune cell populations in the kidney at days 0, 2, and 7 following dimerizer delivery to KidVD+POD mice and their POD littermates. (a) Total CD45+ cells/g kidney tissue; (b) total CD3e+ cells; (c) CD4+ T cells % of CD3e+; (d) CD8+ T cells % of CD3e+ (e) mRNA expression levels for the murine genes encoding IL‐6 (Il6), IL‐1 beta (Il1b), NGAL (Ngal), and TNFα (Tnfa) in the kidney at day 2 following injury normalized to POD at the same time; note the different y‐axis for Tnfa. n = 4 POD, 5 KidVD+POD. (f) Cytokine mRNA expression profile in the kidney at day 7 following injury normalized to POD at the same time. n = 4 POD, 4 KidVD+POD. (g) mRNA expression for fibrosis of the murine genes encoding α‐SMA (Acta2), Type I collagen (Col1a1), Type IV collagen (Col4a1), and fibronectin (Fn) in the kidney at day 7 following injury normalized to POD at the same time. n = 4 POD, 4 KidVD+POD. (h) Picrosirius (PS) red staining on day 7 kidney sections of POD and KidVD+POD mice. Scale bars = 500 µm. (i) Quantified percentage of cortex images positive for intense PS red staining at day 7. n = 5 POD, 6 KidVD+POD. Open circles indicate POD and closed circles represent KidVD+POD mice. All data are represented as ±SD. Statistical comparisons were made using two‐way ANOVA with Tukey's correction, *p < 0.05 compares POD to KidVD genotype effect at same time point, #p < 0.05 effect from baseline or over time for the same genotype. ANOVA, analysis of variance; DAPI, 4′,6‐diamidino‐2‐phenylindole; SD, standard deviation

Fig 4: Impact of expanded renal lymphatics on IRI outcomes. (a) Immunofluorescence for lymphatics in the renal cortex labeled for LYVE‐1 (red) and DAPI (blue) 1 and 7 days post‐injury. Scale bars = 100 µm. (b) Count of LYVE‐1+ lumen/artery on tissue sections of the cortex. All the images were taken at 10× magnification and, for quantification, five different fields/section were imaged. (c) Serum creatinine (SCr); and (d) transdermal glomerulation filtration rate (GFR) measured 1 and 7 days following IRI in KidVD mice and their WT littermates. n = 7–10 WT and KidVD. (e) mRNA expression levels for the murine genes encoding IL‐6 (Il6), IL‐1 beta (Il1b), NGAL (Ngal), and TNFα (Tnfa). n = 4 WT, 5 KidVD. Flow cytometry analysis of immune cell populations in the kidney 1 and 7 days following IRI in KidVD mice and their WT littermates include (f) total CD45+ cells/g kidney tissue; (g) CD3e+ % of CD45+ cells; (h) CD4+ T cells % of CD3e+; (i) CD8+ T cells % of CD3e+; n = 3–5 WT, 3–5 KidVD. (j) mRNA expression levels for the murine genes encoding smooth muscle alpha‐actin (Acta2), type I collagen (Col1a1), type IV collagen (Col4a1), and fibronectin (Fn) in the kidney at day 7 following injury normalized to POD at the same time. n = 4 WT, 5 KidVD. (k) Picrosirius (PS) red staining on day 7 post‐IRI kidney sections of WT and KidVD mice. Bars = 500 µm. (l) Quantified percentage of cortex images positive for intense PS red staining at day 7. n = 9 WT, 12 KidVD. Open circles indicate WT and closed circles represent KidVD mice. All data are represented as ±SD. Statistical comparisons were made using two‐way ANOVA with Tukey's correction, *p < 0.05 compares POD to KidVD genotype effect at same time point, #p < 0.05 effect over time for the same genotype. ANOVA, analysis of variance; DAPI, 4′,6‐diamidino‐2‐phenylindole; IRI, ischemia‐reperfusion injury; SD, standard deviation; WT, wild‐type

Fig 5: CD22KO GC B cells display poor proliferation and cell cycle phase progression, as well as increased apoptotic cell death(A and B) Representative flow cytometric histograms of BrdU+ cells in WT and CD22KO naive and GC B cells (A) and quantification of percentage BrdU+ cells in WT and CD22KO B cells (B). Pooled data from 2 independent experiments; n = 12 mice.(C and D) Representative flow cytometric histograms of BrdU+ cells in WT and CMAHON naive and GC B cells (C) and quantification of BrdU+ cells in WT and CMAHON B cells (D). Pooled data from 2 independent experiments; n = 8 mice.(E) Gating strategy for the flow cytometry-based analysis of cell-cycle phase progression.(F and G) Quantification of WT and CD22KO GC B cells (F) and WT CMAHON GC B cells (G) in different cell cycle phases. n = 6 or 7 mice/group.(H) Quantification of percentage apoptotic naive, DZ GC, and LZ GC B cells from WT, CD22KO, and CMAHON mice, at day 14 PI. n = 6–8 mice/group.(I) Model for glycan remodeling on GC B cells. In naive B cells (upper left), CD22 forms nanoclusters with CD45 that sequester CD22 away from the BCR. Downregulation of the CMAH in the GC (upper right) results in a switch from Neu5Gc to Neu5Ac, leading to downregulation of CD22 ligands. This glycan remodeling is predicted to increase CD22-BCR association and enhance trans interaction with glycan ligands on CD4+ TFH cells. Constitutive CMAH expression (lower left) or CD22 deficiency (lower right) in GC B cells does not enable CD22 to negatively regulate the BCR, leading to a defective GC response.Data plots are presented as mean ± SEM. Statistical analysis was performed using paired Student’s t test (B, D, F, and G), and Dunnett’s multiple-comparison post one-way ANOVA (H).