Normal mouse kidneys contain activated and CD3+CD4- CD8- double-negative T lymphocytes with a distinct TCR repertoire

Abstract

Healthy liver, intestine, lung, and skin harbor resident lymphocytes with conventional and unconventional phenotypes. Lymphocytes also have been detected in healthy mice kidneys; however, these cells have not been well studied and have been largely overlooked. To better characterize the intra-renal lymphocytes, we extensively perfused C57BL/6J mice with PBS and then isolated mononuclear cells for flow cytometry analysis. We observed T cells, B cells, and NK cells in normal mice kidneys after extensive perfusion. Approximately 50% of kidney T lymphocytes expressed intermediate levels of CD3 (CD3int T cells). Similar to liver and lung, a high percentage of unconventional CD3+CD4(-)CD8(-) double-negative T cells was observed in normal mice kidneys, from which 11% expressed B220 antigen. Unlike the spleen and blood, the classic CD4+ and CD8+ T lymphocytes in the kidney had a high proportion of activated CD69+ and effector/memory CD44- CD62L ligand phenotypes. Also, a small percentage of CD4+CD25+forkhead box p3+ and NKT cells was observed in perfused and exanguinated kidneys. In addition, a distinct TCR repertoire was found on intra-renal conventional and unconventional T cells compared with those from the spleen. Finally, after 24 h of renal ischemia reperfusion injury (IRI), increased production of cytokines IFN-gamma and TNF-alpha by CD4+ and CD8+ T cells, isolated from perfused kidneys, was observed. These data suggest that some of these cells harbored in the kidney could be implicated in the immune response of the IRI pathogenic process.

Fig. 1.

Immunohistochemistry staining of mice and human kidney sections. (A) Normal mouse kidney showing CD3⁺ T lymphocytes after extensive perfusion with 30 ml PBS at 37°C over 2 min. The arrows indicate T cells between kidney intraepithelial cells (mean±sd; n=3). (B) Normal human kidney donor biopsy showing CD3⁺ T lymphocytes localized in the intraepithelial cells of the cortex (n=6). Original magnification, ×200.

Fig. 2.

Lymphocyte populations in the kidney, liver, lung, spleen, and blood of normal mice. (A) Bar graph shows the percentage of CD3⁺ T cells, CD19⁺ B cells, and NK1.1⁺ cells in the kidney compared with other organs. A statistically significant increased percentage of NK cells (phenotype CD3⁻NK1.1⁺) in the kidney, liver, and lung (*, P<0.001) when compared with spleen and blood. (B) Approximately 50% of T cells isolated from normal mice kidneys show the CD3^intIL-2Rβ⁺ phenotype. The arrow indicates the population of CD3^int T cells. Results are expressed as percentage of positive cells from the gated lymphocyte area of 10,000 events collected. Results represent the mean of three independent experiments (mean±sd; n=6).

Fig. 3.

Percentage of DN, CD4⁺, and CD8⁺ T cells and DN T cells expressing B220 and CD69 antigens in healthy mice kidneys. (A) In perfused mice, kidney, liver, and lung show a significant increased percentage of DN T cells (P<0.001) compared with spleen and blood. The arrow indicates the population of DN T cells. (B) The bar graph shows that 11% of DN T cells express the phenotype B220⁺TCRαβ⁺CD4⁻CD8⁻. (C) Expression of the CD69 antigen on DN T lymphocytes isolated from kidneys of mice from different ages. In 8-week-old mice, there is a significantly increased percentage of DN T cells expressing CD69 antigen when compared with 5-week-old mice (P=0.001). Data were obtained from the gated lymphocyte and CD3⁺ T cell areas of 10,000 events collected. Data are expressed as the mean percentage of positive cells ± sd of six independent experiments (n=16).

Fig. 4.

CD4⁺ and CD8⁺ T cells are activated in kidneys of perfused and exanguinated, normal mice. (A) Perfused mice, freshly isolated mononuclear cells, were four-color-stained with mAb anti-CD8 FITC, anti-CD69 PE, anti-CD4 PerCp, and anti-CD3 APC and analyzed by flow cytometry. The kidney and liver show increased expression of the early activation marker CD69 on CD8⁺ and CD4⁺ T cells when compared with lung, spleen, and blood lymphocytes. (B) Dot plots show the percentage of CD8⁺CD69⁺ and CD4⁺CD69⁺ T cells in kidneys of exanguinated mice. Percentages are expressed from the gated lymphocyte and CD3⁺ T cell areas. Dot plots are representative examples and show the percentage of positive cells of three independent experiments (mean±sd; n=8). (C) The bar graph shows similar percentages of CD8⁺CD69⁺ and CD4⁺CD69⁺ T cells in kidneys of perfused and exanguinated normal mice.

Fig. 5.

CD4⁺ T lymphocytes expressing effector/memory phenotype. Mononuclear cells were stained with mAb anti-CD44 FITC, anti-CD62L PE, anti-CD4 PerCp, anti-CD8 PerCp, and anti-CD3 APC and analyzed by flow cytometry. A high percentage of kidney and liver CD4⁺ T cells expresses effector/memory phenotype (CD44^highCD62L^neg) when compared with lung, spleen, and blood lymphocytes. Percentages are expressed from the gates: lymphocytes and CD3⁺ and CD4⁺ T cell areas. Dot plots are representative examples of three independent experiments (n=8).

Fig. 6.

Percentage of NKT cells in the kidney of perfused and exanguinated, normal mice. (A) Perfused mice. Dot plot graphs show the percentage of CD4⁺NK1.1⁺ cells in the kidney compared with those of the liver, lung, spleen, and blood. The liver and kidney show a significantly increased percentage of NKT cells compared with the lung, spleen, and blood (P=0.002). (B) Dot plots show a similar percentage of CD4⁺CD1d⁺ NKT cells in kidneys of perfused and exanguinated mice. Results were obtained from the gated lymphocyte and CD3⁺ T cell areas of 10,000 events collected. Results are representative of three independent experiments (mean±sd; n=6).

Fig. 7.

Percentage of CD4⁺CD25⁺ T cells and CD4⁺CD25⁺Foxp3⁺ Tregs in normal kidneys of perfused and exanguinated mice. KMNC were surface-stained with mAb anti-CD4 FITC and anti-CD25 APC, and then cells were fixed and permeabilized for intracellular Foxp3-PE staining followed by flow cytometry analysis. (A) Percentage of CD4⁺CD25⁺ T cells (gate R2) from the gated lymphocyte area of perfused and exanguinated mice. (B) Data show similar percentages of CD4⁺CD25⁺Foxp3⁺ Tregs in kidneys of perfused and exanguinated mice. Percentages of CD4⁺CD25⁺Foxp3⁺ Tregs were obtained from the gated CD4⁺CD25⁺ T cell areas. Dot plots are representative examples of two independent experiments (mean±sd; n=8).

Fig. 8.

TCR Vβ profile of kidneys and spleen, CD4⁺, CD8⁺, and DN T cell subsets. Mononuclear cells obtained from kidneys and spleens of three mice were stained with mAb anti-TCR Vβ FITC, anti-CD8 PE, anti-CD4 PerCp, and anti-CD3 APC for flow cytometry analysis. Bar grafts show the percentage of TCR Vβ-positive CD4⁺ T cells from the R1 gate, CD3⁺CD4⁻CD8⁻ DN T cells from the R2 gate, and CD8⁺ T cells from the R3 gate.

Fig. 9.

Number of DN T cells and increased percentage of CD4⁺ and CD8⁺ T cells expressing IFN-γ and TNF-α after renal IRI. After extensive perfusion of mice, cells were isolated from kidneys of normal, sham-operated (laparotomy) and IRI (laparotomy and 30-min ischemia) mice. (A) A significant increase in serum creatinine 24 h after renal IRI. *, P < 0.001. (B) The absolute number of TCRαβ⁺CD4⁻CD8⁻ DN T cells was obtained from the gated lymphocyte and TCRαβ⁺ T cell areas of 10,000 events collected. Data are expressed as means ± sem of three independent experiments (n=10). *, P < 0.01. (C) Intracellular cytokine staining in kidney CD4⁺, CD8⁺, and DN T cells 24 h after renal IRI. KMNC were stained with mAb anti-CD8 FITC, anti-CD4 PerCp, anti-CD3 APC, and anti-IFN-γ PE and analyzed by flow cytometry. The bar graph shows the percentage of CD4⁺, CD8⁺, and DN⁺ T cells expressing IFN-γ-positive cells. Compared with normal and sham-operated mice, IRI mice showed a significant increased percentage of intracellular, IFN-γ-producing CD4⁺ (P<0.002) and CD8⁺ T cells (P<0.018). (D) Data show a significant increased percentage of CD4⁺ T cells expressing intracellular TNF-α in IRI mice (P<0.007) when compared with normal and sham-operated mice. Data were obtained from lymphocyte and CD3⁺ T cell-gated areas from two independent experiments (means±sem; n=4). *, P < 0.05.