A potential defensive role of TIM-3 on T lymphocytes in the inflammatory involvement of diabetic kidney disease

Abstract

Objective: The aberrant mobilization and activation of various T lymphocyte subpopulations play a pivotal role in the pathogenesis of diabetic kidney disease (DKD), yet the regulatory mechanisms underlying these processes remain poorly understood. Our study is premised on the hypothesis that the dysregulation of immune checkpoint molecules on T lymphocytes disrupts kidney homeostasis, instigates pathological inflammation, and promotes DKD progression.

Methods: A total of 360 adult patients with DKD were recruited for this study. The expression of immune checkpoint molecules on T lymphocytes was assessed by flow cytometry for peripheral blood and immunofluorescence staining for kidney tissue. Single-cell sequencing (scRNA-seq) data from the kidneys of DKD mouse model were analyzed.

Results: Patients with DKD exhibited a reduction in the proportion of CD3+TIM-3+ T cells in circulation concurrent with the emergence of significant albuminuria and hematuria (p=0.008 and 0.02, respectively). Conversely, the incidence of infection during DKD progression correlated with an elevation of peripheral CD3+TIM-3+ T cells (p=0.01). Both univariate and multivariate logistic regression analysis revealed a significant inverse relationship between the proportion of peripheral CD3+TIM-3+ T cells and severe interstitial mononuclear infiltration (OR: 0.193, 95%CI: 0.040,0.926, p=0.04). Immunofluorescence assays demonstrated an increase of CD3+, TIM-3+ and CD3+TIM-3+ interstitial mononuclear cells in the kidneys of DKD patients as compared to patients diagnosed with minimal change disease (p=0.03, 0.02 and 0.002, respectively). ScRNA-seq analysis revealed decreased gene expression of TIM3 on T lymphocytes in DKD compared to control. And one of TIM-3's main ligands, Galectin-9 on immune cells showed a decreasing trend in gene expression as kidney damage worsened.

Conclusion: Our study underscores the potential protective role of TIM-3 on T lymphocytes in attenuating the progression of DKD and suggests that monitoring circulating CD3+TIM3+ T cells may serve as a viable strategy for identifying DKD patients at heightened risk of disease progression.

Keywords: T cell immunity; TIM-3; diabetic kidney disease; immune checkpoint molecules; inflammation; kidney homeostasis.

Figure 1

Gating strategies to discriminate various T cell subpopulations using multiparametric flow cytometry, with markers indicated on the x and y axes. Lymphocytes were gated based on forward scatter/side scatter dot plot. From the lymphocyte population, T lymphocytes were gated using surface markers CD45 and CD3. Subsequently, CD4+ T cells and CD8+ T cells were gated using their respective surface markers. T cell subpopulations within CD3+ lymphocytes were further gated based on TIM-3, LAG-3, or CTLA-4 expression.

Figure 2

Comparison in proportions of the peripheral T lymphocyte subset [CD3+ (A), CD4+ (B), CD8+ (C), CD4/CD8 (D)] among CD45+ lymphocytes in DKD patients with or without DR, DPN, infection, other GN, and with different degrees of albuminuria or hematuria. **p<0.01. DKD, diabetic kidney disease; DR, diabetic retinopathy; DPN, diabetic peripheral neuropathy; GN, glomerular nephropathy.

Figure 3

Comparison in proportions of the peripheral T lymphocyte subset [CD3+LAG-3+ (A), CD3+TIM-3+ (B), CD3+CD4+CD25+ (C), CD3+CTLA-4+ (D) among CD45+ lymphocytes in DKD patients with or without DR, DPN, infection, other GN, and with different degrees of albuminuria or hematuria. *p<0.05, **p<0.01. DKD, diabetic kidney disease; DR, diabetic retinopathy; DPN, diabetic peripheral neuropathy; GN, glomerular nephropathy.

Figure 4

Analysis of correlation between expression of TIM-3 in peripheral T cells and pathological and clinical parameters. The analyses included (A) global sclerosis, (B) segmental sclerosis, (C) glomerular hematuria, (D) albuminuria, (E) interstitial in filtration, (F) IFTA, (G) Mesangial expansion and (H) Tubular atrophy. *p<0.05.

Figure 5

TIM-3 expression on renal intersitial mononuclear cells in DKD and MCD. (A–D) Representative immunofluorescence staining images (× 40) of kidney CD3+ (green) and TIM-3+ (red) interstitial cells from 2 MCD patients and 2 DKD patients. Double staining is in yellow. Yellow arrow, CD3+TIM-3+; white arrow, CD3+TIM-3-; white triangle, CD3-TIM-3+. (E, F) The numbers of interstitial CD3+cells and the ratio of CD3+/mononuclear cells increased in DKD compared with MCD. (G, H) The numbers of interstitial TIM-3+cells and the ratio of TIM-3+/mononuclear cells increased in DKD compared with MCD. (I, J) The numbers of interstitial CD3+TIM-3+cells and the ratio of CD3+TIM-3+/mononuclear cells increased in DKD compared with MCD. *p<0.05, **p<0.01. DKD, diabetic kidney disease; MCD, minimal change disease.

Figure 6

The single-cell analysis conducted on cells of 12 mouse kidneys from DKD(db/db), advanced DKD (db/db+renin 2d, db/db +renin 2w), and control (db/m) groups to assess the expression of CTLA-4, TIM-3 (Havcr2) and TIM-3’s ligands (Gal-9, Hmgb1). (A) Cells from total 12 mouse kidneys in 4 different groups are projected by uniform manifold approximation and projection (UMAP) plot. (B) Integrated clustering plot of mouse kidney immune cells. (C) Havcr2(TIM-3)+ and CTLA-4 cells in control (db/m), db/db, or db/db +Renin-AAV at 2 days and 2 weeks. (D) Expression of Havcr2(TIM-3) and Ctla4 in T cells across all the groups. (E) Lgals9(Gal-9)+ cells and Hmgb1 in control (db/m), db/db, or db/db +Renin-AAV at 2 days and 2 weeks. (F) Expression of Lgals9(Gal-9) and Hmgb1 in different cells across all the groups. PCT, Proximal Convoluted Tubule; PST, Proximal Straight Tubule; EC, endothelial cells; TAL, thick ascending limb; tAL, one of the TAL subclusters highly expressing Atp10b; MD, macular densa; CNT, connecting tubule; inj.PT, Injuried PT; PC, principal cells; FIB, Fibroblast; PECs, parietal epithelial cells; DCT, distal convoluted tubule; ICA, type A intercalated cells; ICB, type B intercalated cells; Podo, podocyte; DTL, descending thin limbs of Henle’s loop; MΦ, Macrophage; DC, dendritic cell.