The Immune Microenvironment of Transplant Glomerulitis

Abstract

Introduction: Transplant glomerulitis is a morphological lesion seen in kidney allograft rejection that is associated with poor outcomes; however, little is known about how immune cells infiltrate and organize specifically within glomeruli.

Methods: We used Co-Detection by Indexing (CODEX) multifluorescent imaging to measure 52 protein markers in a retrospective cohort of 41 human allograft nephrectomies (ANs) and evaluated the immunological landscape of transplant glomerulitis.

Results: Characterization of 18 cell types identified diverse immune cells within inflamed glomeruli, with unique phenotypes and compositions compared with the extraglomerular microenvironment. Immunological phenotypes were conserved across glomeruli within individuals and associated with the general state of injury, with M1 macrophages and effector CD8 T cells associated with mild inflammation. Distance-based spatial analysis further revealed a profibrotic community composed of M2 macrophages, memory CD8 T cells and exhausted CD8 T cells surrounding endothelial cell hubs. These interaction networks were associated with regions of adverse glomerular remodeling, expression of profibrotic proteins, and were more prevalent in individuals with C4d-positive rejection.

Conclusion: These results implicate distinct cell-cell interactions as hallmarks of alloimmune injury and chronic remodeling during transplant glomerulitis and may give rise to new tools for histological risk assessment of clinical rejection syndromes.

Keywords: cell-cell communication; kidney rejection; single-cell; transplant glomerulitis.

Graphical abstract

Figure 1

Experimental overview. (a) Donor characteristics of allograft nephrectomy samples used in the study. (b) Hematoxylin and Eosin (H&E) stain demonstrating the histological spectrum of transplant glomerulitis lesions identified within allograft nephrectomy samples and a normal glomerulus from a tumor nephrectomy. Scale bar is 50 μm. (c) Schematic outline of the nephrectomy tissue microarray, CODEX fluorescence and single-cell segmentation images. AN, allograft nephrectomy; AR, acute rejection; ATN, acute tubular necrosis; CN, control nephrectomy; CR, chronic rejection; CODEX, Co-Detection by Indexing; DD, deceased donor; DN, diabetic nephropathy; ESRD, end-stage renal disease; GN, glomerulonephritis; HA, hyperacute rejection; HTN, hypertension; LRD, living related donor; LURD, living unrelated donor; PKD, polycystic kidney disease.

Figure 2

A single-cell atlas of the alloreactive kidney microenvironment. (a) UMAP representation of the single-cell CODEX dataset colored by identity of major cell types and (b) higher resolution sub-clustering for immune cells. (c) Protein expression for marker features across all major cellular populations. Colors indicate the average protein expression for all cells within a cluster, and dot size indicates the percentage of cells within each cluster expressing the protein. (d) Composition of cell types across all control and allograft nephrectomy samples. Control nephrectomies are shown in shades of red, allograft nephrectomies in shades of blue or purple. CODEX, Co-Detection by Indexing; MPO, myeloperoxidase; HLA, human leukocyte antigen; IFNγ, interferon gamma; UMAP, uniform manifold approximation and projection.

Figure 3

The cellular diversity of transplant glomerulitis. (a) Periodic acid-Schiff (PAS) stain and CODEX fluorescent images for representative control and allograft nephrectomy tissue cores showing CD68+ macrophages, CD4 and CD8 T cells within segmented glomeruli. CD31 is shown to identify glomerular endothelial cells. Scale bar is 100 μm. (b) Distribution of broad glomerular cell populations from the 39 nephrectomy samples that contained glomeruli. Nonimmune cell populations are colored gray. (c) Ranking of 234 individual glomeruli from 39 nephrectomy samples containing glomeruli according to inflammatory score (log2 ratio of immune-to-nonimmune cells). Red bars indicate glomeruli from control nephrectomies (CN) with few immune cells, blue bars are glomeruli from allograft nephrectomy (AN) samples. (d) Cell count normalized by glomerular surface area for n =234 individual glomeruli ranked by inflammatory score. Myeloid cells, CD8 T cells, CD4 T cells, and B cells are colored separately and shown on the same axis. CODEX, Co-Detection by Indexing.

Figure 4

CD8 T cells and macrophages promote endothelial cell injury in inflamed glomeruli. (a) Single-cell resolution CODEX fluorescent images demonstrating canonical protein markers for infiltrating glomerular CD8 T cell and macrophage subsets. Scale bar is 10 μm. (b and c) Composition of CD8 T cell (b) and macrophage (c) subsets within all glomeruli grouped according to inflammatory scores for low (bottom 20th percentile, n = 50), intermediate (n = 137) and high (top 20th percentile, n = 47) ranking. (d and e) Inverse relationship between endothelial cell PD-L1 protein expression and its distance to CD8 Teff (d) or M1 macrophages (e) within inflamed glomeruli. P values represent comparisons to a null distribution of uniform endothelial cell PD-L1 expression using a linear mixed effects model. CODEX, Co-Detection by Indexing.

Figure 5

Spatial organization of the glomerular alloimmune microenvironment. (a) Conceptual overview of compartmentalized spatial analysis to identify glomerular endothelial-centered communities (EC). For each endothelial cell, 10 nearest neighboring cells are identified and clustered into communities. (b) Distribution of the distance between index endothelial cells and their nearest cell neighbor (k1, light gray) or their 10th nearest cell neighbor (k10, dark gray). (c) Heatmap showing the cellular composition of 7 distinct endothelial-centered communities. Z-scores represent scaling across rows. Left panel shows the abundance (percent total) of each community summarized across glomeruli for all control and allograft nephrectomy cores. (d and e) Representative CODEX fluorescence images showing the spatial organization of (d) communities within glomeruli from control and (e) allograft nephrectomy samples. Endothelial cells are colored by community phenotype. Scale bar is 20 μm. (f) Distribution of EC composition across each nephrectomy tissue core. (g) Frequency of each EC within individual glomeruli from all tissue cores grouped by low (n = 50), intermediate (n = 128), and high (n = 36) glomerular inflammatory score ranking. Each dot represents a single glomerulus, 1-way analysis of variance. CODEX, Co-Detection by Indexing.EC, endothelial-centered communities.

Figure 6

Macrophages regulate glomerular remodeling within tissue regulatory niches. (a) Representative CODEX fluorescence images of 2 allograft nephrectomy cores demonstrating EC_Remodeling within sclerotic glomeruli. Outer images show endothelial cells colored by EC membership; inner images show corresponding fluorescent markers. Scale bar is 40 μm. (b) Number of M1 macrophages (top) and M2 macrophages (bottom) per 10-cell community across all ECs. (c and d) Normalized fluorescence intensity of collagen IV (c) and αSMA (d) protein expression in structural cells from glomeruli containing EC_Remodeling compared with glomeruli without EC_Remodeling communities. n = 289 cells from EC_Remodeling positive glomeruli, n = 3105 cells from EC_Remodeling negative glomeruli, Wilcoxon rank-sum test. (e) Experimental overview for exploring endothelial-mesangial-immune cell interactions. (f) Distributions of collagen IV (left) and αSMA (right) normalized protein expression in structural cells from ECs containing macrophages (blue), immune cells with no macrophages (red), or no immune cells (gray). Wilcoxon rank-sum test comparing immune-containing distributions to the non-immune controls. CODEX, Co-Detection by Indexing; EC, endothelial-centered communities.

Figure 7

Association of profibrotic cell-cell interactions with C4d tissue staining. (a) Cell counts normalized by glomerular surface area for indicated broad cell types within allograft nephrectomy samples stratified by C4d positive versus negative staining evaluated separately for each donor. Each dot represents one individual glomerulus, n = 90 from C4d positive individuals and n = 93 from C4d negative individuals. Wilcoxon rank-sum test. (b) Percent abundance of EC_Remodeling within individual glomeruli from tissue cores grouped by C4d tissue staining. n = 86 from C4d negative individuals, n = 77 from C4d positive individuals, Wilcoxon rank-sum test.EC, endothelial-centered communities.