Interactions of the Immune System with Human Kidney Organoids

Abstract

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

Keywords: immunity; implantation; kidney; organoids; stem cells.

FIGURE 1

Immune cells infiltrate kidney organoids in vitro. (A) Schematic drawing of the organoid-PBMC co-culture. (B) Brightfield image of an organoid (cultured without immune cells) harvested at day 25 of differentiation. Scale bar indicates 1 mm. (C) Expression of the distal tubular marker ECAD, the proximal tubular marker Villin and the glomerular marker WT1 in kidney organoid sections at day 25 of differentiation (without immune cells). Scale bars represent 100 µm. (D) mRNA expression of CD45 in organoid-PBMC co-culture compared to only organoid. Data is expressed as ratio with GAPDH x 1000 with SE (n = 12). (E) Immunohistochemistry for CD45 depicting infiltration of CD45⁺ leukocytes in glomerular (left) and stromal structures (right). Asterisk marks a tubular structure. Scale bar represents 100 µm. (F) Immunohistochemistry for CD3, CD4 and CD8 in consecutive slides showing the distribution of CD4⁺ and CD8⁺ T-cells (asterisk marks a glomerular structure). Scale bar represents 100 µm. (G) Immunohistochemistry for CD20, revealing a rare presence of B-cells. Scale bar represents 100 µm. (H) Immunohistochemistry for CD68 showing presence of macrophages in the interstitial space. CD163 staining shows the presence of regulatory macrophages. Scale bar represents 100 µm. (I) Comparison of the number of infiltrated cells for various immune cell subsets through quantification of immunohistochemical staining by image analysis.

FIGURE 2

Weak activation status of immune cells infiltrated in in vitro organoids. (A) Gene expression analysis of inflammatory cytokines. Data is expressed as ratio with GAPDH x 1000 with SE (n = 12). (B) Double immunofluorescence staining for CD45 (green) and Ki-67 (red) showing absence of proliferating leukocytes. Scale bar represents 100 µm. (C) Immunohistochemistry for differentiation markers WT1, Villin-1 and ECAD showing maintenance of differentiation in the organoids exposed to immune cells. Scale bar represents 100 µm. (D) Immunohistochemistry for collagen type 1 in organoids cultured with and without immune cells. Scale bar represents 100 µm. (E) Collagen 1 type 1 (Col1A1) quantification in immunohistochemical staining in organoids cultured with and without immune cells.

FIGURE 3

Immune cells infiltrate vascularized implanted kidney organoids. (A) Timeline of in vivo organoid implantation model and adoptive transfer of PBMC. (B) Immunohistochemical staining of CD4 and CD8 in immune cell-infiltrated whole organoids. Scale bar represents 2 mm. (C) Immunohistochemistry for CD4 and CD8 in consecutive slides in implanted organoids, showing infiltration of immune cells in all compartments of the organoids. Arrows indicate CD4 and CD8 staining in nephron structures. Scale bars represent 100 µm. (D) Proportion of CD4⁺ and CD8⁺ T-cells in organoids determined by flow cytometry (n = 18 organoids from 11 mice in 4 individual experiments). (E) Representative Periodic acid-Schiff (PAS) staining of implanted organoids retrieved from animals without immune cell administration (organoid) and from animals that received immune cells (organoid + immune cells). Scale bars represent 100 µm.

FIGURE 4

Composition and activation status of infiltrated T-cells in implanted organoids. (A) Proportion of activated CD25⁺CD4⁺ T-cells and CD25⁺CD8⁺ T-cells in blood, spleen, and organoids of animals that received immune cells determined by flow cytometry (n = 6 or 7 mice from 2 individual experiments). The proportion of activated CD25⁺CD4⁺ T-cells and CD25⁺CD8⁺ T-cells in spleens of animals that received immune cells only (no organoid) is shown as control (n = 2 from 2 individual experiments). (B) mRNA gene expression analysis of inflammatory cytokines, indicating the initiation of an immune response in organoids with immune cells. Data is expressed as ratio with GAPDH x 1000 with SE (n = 12). (C) Immunohistochemistry for Granzyme B⁺ cells, revealing Granzyme B⁺ cells surrounding tubular structures and in the interstitial space. Scale bar represents 100 µm. (D) Double immunofluorescence staining for CD45 (green) and Ki-67 (red) showing presence of proliferating leukocytes (red arrows). Scale bar represents 100 µm. (E) Triple immunofluorescence staining for CD3 (white), CD25 (green) and PD-1 (yellow) showing that many of the infiltrated CD3⁺ T-cells display CD25 expression and many show co-staining with PD-1. Panels from left to right, top to bottom: nuclear Dapi staining; CD25 staining; CD3⁻CD25-PD-1 triple staining; PD-1 staining; CD3 staining. Scale bar represents 100 µm. (F) Immunohistochemistry for PD-L1 in immune cell-infiltrated organoids (left panel) showing expression in epithelial cells (arrows) but not in the stromal areas (asterisks). Organoids without immune cells show no PD-L1 expression (right panel). Scale bar represents 100 µm. (G) Immunohistochemistry for FOXP3 demonstrates the presence of T-cells with a regulatory phenotype. Scale bar represents 100 µm. (H) Proportion of tissue resident CD4⁺ T-cells and CD8⁺ T-cells in the blood, spleen, and organoids of animals that received immune cells determined by flow cytometry (n = 8 mice from 2 individual experiments). The proportion of tissue resident CD4⁺ T-cells and CD8⁺ T-cells in spleens of animals that received immune cells only (no organoid) is shown as control (n = 4 mice from 2 individual experiments).

FIGURE 5

Single cell transcriptomic analysis of implanted kidney organoids. (A) UMAP presentation of single cell transcriptomic analysis of organoid cells with and without PBMC by sample and by cell clusters identified on basis of highly variable genes. (B) UMAP representation of infiltrated immune cells. Diverse T-cell populations in the organoid are identified by Celltypist. (C) Dot plot visualization of T-cell subsets, displaying key markers for the diverse subsets indicated on the X-axis. The size of the dots reflects the frequency of T-cells in each subset that expresses the gene indicated on the X-axis, and the color of the dots reflects the level of expression. (D) Frequency distribution of T-cell subsets in four immune cell-infiltrated organoids.

FIGURE 6

Effect of immune cell infiltration on implanted organoids. (A) Ligand-receptor interactions in immune cell-infiltrated organoids. Left: Aggregated cell-cell communication network between all human cells from the immune cell-infiltrated organoids. Circle size denotes number of cells in each group; width of lines denotes total interaction strength between cell groups. Right: Scatter plot showing the dominant senders (ligands) and receivers (receptors). Dot size denotes total number of inferred links. Axis measure the total outgoing or incoming communication strength associated with each cell cluster. (B) Differentially expressed (DE) pathways between nephron cells, stromal cells and endothelial cells from control organoids vs. organoids infiltrated with immune cells, demonstrating upregulation of immune response pathways and metabolic pathways upon immune cell infiltration. Dot size indicates number of differentially expressed genes within the pathways. (C) Dot plot visualization comparing the expression of HLA genes in all cell types of kidney organoids with and without immune cells. Each dot represents the averaged relative gene expression of 4 organoids.

FIGURE 7

Effect of immune cell infiltration on gene expression of nephron structures and stromal cells. (A) Single cell mRNA expression analysis of tubular structural and functional markers VIL1, CDH1, LRP2 and CUBN, the glomerular markers NPHS1, PODXL and WT1, the DNA damage marker H2AFX and the stress response markers E2F4 and MTOR in nephron cells from organoids with and without infiltrated immune cells. (B) Single cell mRNA expression analysis of stromal markers PDGFRA and PDGFRB, the DNA damage/injury markers H2AFX and BAX and the immune response marker CCL2 in stromal cells from organoids with and without infiltrated immune cells.