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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2008 Sep 2;84(6):1400–1409. doi: 10.1189/jlb.0907651

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

Dolores B Ascon *,1, Miguel Ascon *, Shailesh Satpute *, Sergio Lopez-Briones *, Lorraine Racusen *, Robert B Colvin †, Mark J Soloski *, Hamid Rabb *,1
PMCID: PMC2614602  PMID: 18765477

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 CD44CD62L 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-γ and TNF-α 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.

Keywords: DN T cells, NK cells, cytokines, ischemia, rodents

INTRODUCTION

The immune system is organized into primary and secondary lymphoid organs, where lymphocytes develop and mature to fight pathogens. Organs such as liver, small intestine, lung, and skin also harbor resident lymphocyte populations of the innate (NK and NKT cells) and adaptive (T and B lymphocytes) immune system [1,2,3,4,5,6]. In these organs, a high proportion of resident T lymphocytes is of unconventional phenotypes such as CD3+CD4+CD8+ double-positive, CD3+CD4−CD8− double negative (DN), CD3 intermediate cells (phenotype CD3intIL-2Rβ+), TCRγδ, and CD8αα. Resident lymphocytes in nonimmune organs mediate multiple, specialized, local functions including tolerogenic responses, immune homeostasis, maintaining a barrier to invading pathogens, release of cytokines, antigen recognition, disposal of infected and malignant cells, and alteration of the development of inflammatory diseases [7,8,9,10,11]. The mammalian kidney is not conventionally considered an organ that harbors resident lymphocyte populations. However, using electron microscopy, lymphocytes, dendritic cells, and macrophages have been observed in the peritubular interstitium of rat kidney [12]. The kidney of the teleost fish is an organ that supports hematopoiesis, indicating precedence for the kidney to harbor immune cells [13]. Additionally, we recently found lymphocytes in the kidneys of healthy, exanguinated mice; however, blood contamination was not excluded by perfusion, and rigorous characterization was not performed [14].

Unlike the liver, the lymphocyte populations in normal mice kidneys have not been well characterized or implicated in disease. It is unclear if the kidney harbors lymphocytes of unconventional phenotypes or the activation state of classic CD4 and CD8 T lymphocytes. There is a suggestion that the kidney of rodents contains lymphocytes of an unconventional phenotype including CD3int T cells, NK, and NKT cells [14, 15]. CD3int T cells and NK cells are resident populations of the liver, intestine, and lung, where they play a role in the local immune response and in disease pathogenesis [1, 6,7,8, 16]. Lymphocytes mediated renal ischemia reperfusion injury (IRI), as well as IRI of other organs such as the liver, lung, heart, and brain [17,18,19,20]. Despite several studies demonstrating a role for lymphocytes in renal IRI, there have been no rigorous studies to date seeking to determine whether kidney injury is orchestrated by infiltrating lymphocytes into an affected organ or by lymphocytes already present. The increased number of CD3int T cells found in ischemic kidneys of mice suggests an important role of unconventional cells in renal IRI [15]. There is also evidence that resident liver NK and NKT cells are important mediators in ischemic injury [16]. In liver ischemia, it has been suggested that resident lymphocytes alter the inflammatory response by producing proinflammatory cytokines and chemokines to recruit additional lymphocytes and neutrophils [11].

Given the body of evidence demonstrating that nonlymphoid organs harbor several lymphocyte populations of an unconventional phenotype, which are important mediators in disease pathogenesis, we hypothesized that unconventional and activated lymphocytes could also be found in normal mice kidneys. Unlike previous studies, we extensively perfused normal mice prior to isolation of kidney mononuclear cells (KMNC) for flow cytometry and immunohistochemistry staining. Here, we report that lymphocytes found in normal mice kidneys have clear differences from spleen and blood lymphocytes and were not contaminants. Kidney lymphocytes had many similarities, as well as differences, with liver and lung lymphocytes. In addition, we observed a significant increased percentage of CD4+ and CD8+ T cells expressing IFN-γ and TNF-α after 24 h of renal IRI; however, no changes were observed in DN T cells. The data suggest the important role of lymphocytes harbored in the kidney in the pathogenesis process of renal IRI.

MATERIALS AND METHODS

Mice

Male C57BL/6J wild-type mice, 5–8 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained under specific pathogen-free conditions. All experiments were performed in accordance with the Animal Care and Use Committee guidelines.

Human samples

Kidney graft biopsies of healthy, live donors were obtained after perfusion of the kidneys and prior to implantation in the recipient (n=6; average age, 37 years; range, 28–48 years old). The kidney tissue was analyzed by immunohistochemistry staining. Institutional Review Board approval was obtained.

Mouse renal ischemia model

An established model of renal ischemia and reperfusion in mice was used. Briefly, mice were anesthetized with an i.p. injection of sodium pentobarbital (75 mg/kg). Following abdominal incisions, renal pedicles were bluntly dissected, and a microvascular clamp (Roboz Surgical Instrument, Gaithersburg, MD, USA) was placed on each renal pedicle for 30 min. During the procedure, animals were kept well hydrated with warm saline and at a constant temperature (37°C). After 30-min ischemia, the clamps were removed, and the wounds were sutured. Then, the animals were allowed to recover with free access to food and water. Sham-operated mice underwent the same surgical procedure without clamping of the renal pedicle.

Assessment of renal function

Blood samples were obtained at 0, 3, and 24 h after renal IRI. Serum creatinine levels (mg/dL) were measured to monitor renal function using a commercial creatinine kit and an autoanalyzer (Roche Diagnostics Corp., Indianapolis, IN, USA).

Immunohistochemistry

Staining was performed on formalin-fixed kidney tissue. Kidney sections (4 μm) were immersed in 3% hydrogen peroxide methanol for 5 min to block endogenous peroxidase. For antigen retrieval, slides were pressure-cooked in antigen-decloaker solution for 3 min (AEC Biocare Medical, Walnut Creek, CA, USA). After treatment with normal goat serum (1/100) and two drops of avidin D (100 mg/ml PBS), CD3, CD19, and B220 mAb were added to the sections at a 1/200 dilution. Sections were then incubated overnight at 4°C and then rinsed in PBS and treated with biotin (10 μg/L PBS) to block the biotin-binding sites. After three washes in PBS, the slides were incubated with a biotin-conjugated goat anti-rabbit IgG secondary antibody for 35 min at room temperature. Sections were once again washed and were then incubated for 45 min with streptavidin peroxidase. Kidney sections were exposed with AEC chromogen (Romulin AEC Biocare) to visualize the immunocomplex and counterstained with hematoxylin. All kidney sections were examined by a pathologist and a nephrologist in a blinded manner.

Isolation of lymphocytes

Before harvesting the kidneys and other organs, mice were perfused with 30 ml PBS at 37°C using a perfusion pump. Kidney and liver lymphocytes were isolated using a technique described previously with few modifications [14]. Briefly, organs were disrupted mechanically using a Stomacher 80 Biomaster (Sewart, UK), and then cells were suspended in 36% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ, USA), gently overlaid onto 72% Percoll, and centrifuged at 1000 g for 30 min at room temperature. The lung was digested with collagenase for 30 min at 37°C, and then KMNC were isolated using the Percoll technique as described above. Spleen and blood lymphocytes were isolated using standard techniques.

Cell count

The absolute number of kidney lymphocytes was determined using the TruCount tubes following the instructions from the manufacturer (BD Biosciences, San Jose, CA, USA). The absolute number of cells was expressed as number of cells per two kidneys (cell/organ).

Antibodies

The fluorochrome-conjugated mAb to mouse antigens used for flow cytometry analysis were: anti-CD16/CD32 (2.4G2), anti-CD3 allophycocyanin (APC; 145-2C11), anti-TCRαβ (H57-597), anti-IL-2Rβ (TM-B1), anti-CD4 PerCP (RM4-5), anti-CD8b FITC (53-5.8), anti-CD19 PE (1D3), anti-B220 PE (RA3-6B2), anti-CD69 PE (H1.2F3), anti-CD25 PE (3C7), anti-NK1.1 PE (PK136), anti-CD44 FITC (IM7), and anti-CD62 ligand (anti-CD62L) PE (MEL-14; BD PharMingen, San Diego, CA, USA).

Flow cytometry analysis

Lymphocytes were preincubated with anti-CD16/CD32 FcR for 10 min to minimize nonspecific antibody binding. Cells were then incubated with various combinations of mAb for 25 min at 4°C, washed twice with FACS buffer, and fixed with 1% paraformaldehyde. Four-color immunofluorescence staining was analyzed using a FACSCalibur instrument (BD Biosciences). The lymphocytes were gated using forward- and side-scatter to exclude debris and dead cells, and then 10,000 events were acquired in each assay for analysis.

Analysis of variable β chain (Vβ) TCR clones by flow cytometry

Kidney and spleen lymphocytes of three normal mice were pooled and stained with anti-CD8 PE, anti-CD4 PerCp, anti-CD3 APC, and anti-TCR Vβ FITC mAb: anti-2 (B20.6), anti-3 (KJ25), anti-4 (KT4), anti-5.1 and -5.2 (MR9-4), anti-6 (RR4-7), anti-7 (TR310), anti-8.3 (1B3.3), anti-8.1 and -8.2 (MR5-2), anti-9 (MR10-2), anti-10b (B21.5), anti-11 (RR3-15), anti-12 (MR11-1), anti-13 (MR12-3), anti-14 (14-2), and anti-17a (KJ23; BD PharMingen). Flow cytometry analysis was performed as described above.

Identification of NKT cells with a specific tetramer

To identify CD1d-restricted NKT cells, we labeled kidney cells with anti-CD4 PerCp, anti-TCRαβ APC, and CD1d tetramer-Alexa 647 loaded with PBS57 (1:500), an analog of α-galactosyl ceramide (α-GalCer), which yields results identical with α-GalCer (National Institutes of Health Tetramer Facility, www.research.yerkes.emory.edu/tetramer_core). Unloaded CD1d tetramer-Alexa 647 was used as a control.

Identification of CD4+CD25+forkhead box p3+ (Foxp3+) T regulatory cells (Tregs) from normal mouse kidney

The CD4+CD25+Foxp3+ Tregs from the kidney were determined using a mouse Treg staining kit (eBioscences, San Diego, CA, USA). In brief, surface molecules CD4 and CD25 were stained, and then cells were fixed and permeabilized with special buffer for 2 h at 4°C in the dark. Next, the anti-mouse/rat Foxp3 (FJK-16 s) mAb or isotype control in permeabilization buffer was added and incubated at 4°C for at least 30 min in the dark. Cells were washed in permeabilization buffer and resuspended in appropriate volume flow cytometry staining buffer and analyzed by flow cytometry.

Intracellular cytokines staining by flow cytometry

Freshly isolated KMNC from perfused mice were stimulated with 5 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of monensin. The samples were incubated for 5 h at 37°C in a 5% CO2 humidified atmosphere incubator. Surface staining of stimulated cells was performed with mAb anti-CD8 FITC, anti-CD4 PerCp, and anti-CD3 APC for 25 min at 4°C. Cells were then permeabilized with perm/wash solution for 20 min and stained with PE-conjugated mAb anti-IFN-γ and anti-TNF-α or the appropriate isotype-matched control antibodies and analyzed by flow cytometry.

Statistical analysis

Data were expressed as means ± sd or sem using Sigma Stat 3.0. (Richmond, CA, USA). Statistical comparisons between two groups were performed by the Student’s t-test. Comparisons between multiple groups were performed by a one-way ANOVA test followed by the Student-Newman-Keuls test where appropriate. Statistical significance was determined as P < 0.05.

RESULTS

Lymphocytes in normal murine and human kidneys detected by immunohistochemistry

We first determined by immunohistochemistry if normal mouse kidney contains lymphocytes after extensive perfusion. Whole mouse was perfused with 30 ml intracardiac PBS. The kidney was then stained by immunohistochemistry and analyzed by a renal pathologist and nephrologist. We found T cells [n=3; 8±5.3 cells/10 high-powered fields (HPF; Fig. 1A)] and rare B cells (0.1±0.1 cells) among the endothelium, interstitium, and peritubular capillaries in kidneys of normal mice. We also examined kidney biopsies of healthy human donors prior to implantation and after perfusion by immunohistochemistry staining. Human kidney tissue showed T lymphocytes localized in the interstitium and peritubular capillaries (n=6; 11±4 cells/10 HPF) and between intraepithelial cells (0.5±0.1 cells; Fig. 1B). Few B cells were detected in the interstitium (1±0.2 cells). These data demonstrate that normal murine and human kidneys have T and rare B lymphocytes.

Fig. 1.

Fig. 1.

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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.

Phenotypic characterization of lymphocytes isolated from normal murine kidney

We then isolated lymphocytes from mouse kidney for phenotypic characterization by flow cytometry. We compared the percentage of kidney lymphocytes with those isolated from liver, lung, spleen, and blood to determine the differences and similarities. The total number of KMNC isolated from both kidneys was 1.4 × 106 cells ± 2.8 × 105 cells (n=6). Multiparametric flow cytometry analysis of KMNC showed CD3+ T cells (mean±sd; 31%±5%; range between 23% and 36%), CD19+ B cells (25%±6%; 18–36%), and NK cells (phenotype CD3−NK1.1+; 8%±1%; 7–10%). The percentage of NK cells was increased significantly in the kidney, liver, and lung (*, P<0.001) when compared with those in the spleen and blood (Fig. 2A). Unexpectedly, we observed that 50% of intra-renal T lymphocytes were CD3int cells of phenotype CD3intIL-2Rβ+ (16%±2%; 14–17%; Fig. 2B).

Fig. 2.

Fig. 2.

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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 CD3intIL-2Rβ+ phenotype. The arrow indicates the population of CD3int 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).

Conventional and unconventional T cell subsets in normal mouse kidney

We found a significant increased percentage of unconventional TCRαβ+CD4−CD8− DN T cells in kidney (23%±4%; 18–32%; P<0.001), liver (22%±3%; 16–26%; P<0.001), and lung (14%±5%; 11–17%; P<0.001) when compared with spleen (5%±1%; 4.4–6.1%) and blood (3%±1%; 2.5–4.4%) lymphocytes (Fig. 3A). We found that 11% of kidney DN T cells expressed the phenotype B220+TCRαβ+CD4−CD8− (Fig. 3B). Interestingly, we observed that the expression of CD69 on DN T cells increased significantly in kidneys of 8-week-old mice (81%±4%; P=0.001) when compared with kidneys of 5-week-old mice (47%±7%; Fig. 3C). We also found that normal mice kidneys had a similar percentage of the classic CD4+ (55%±4%; range between 51% and 61%) and CD8+ (21%±4%; 17–30%) T cell subsets as liver lymphocytes. Among the T cells isolated from normal mice kidneys, 95% expressed TCRαβ and 5% TCRγδ. Thus, T lymphocytes in a normal mouse kidney are of conventional and unconventional phenotype.

Fig. 3.

Fig. 3.

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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).

Activated, effector/memory NKT and Foxp3 T cells in normal mice kidneys

It is well documented that in the liver, intestine, and lung, resident CD4 and CD8 T cells have phenotypes distinct from those found in lymphoid organs [6, 21, 22]. By flow cytometry, we analyzed the physiologic state of classic CD8+ and CD4+ T cells with the expression of the early activation marker CD69. In perfused mice, kidney CD8+ (6%±2%) and CD4+ (28%±6%) T cells and liver CD8+ (2.2%±2%) and CD4+ (24%±11%) T cells showed higher expression of CD69 when compared with lung, spleen, and blood lymphocytes (Fig. 4A). We observed a similar percentage of CD8+CD69+ (8%±1%) and CD4+CD69+ (30%±1%) T cells in exanguinated mice (Fig. 4, B and C) when compared with perfused mice. We analyzed the homing receptors CD44 and CD62L on the classic CD4+ and CD8+ T cells to distinguish them from naïve (CD44loCD62hi) or effector/memory phenotype (CD44hiCD62Llo/neg). In perfused mice, an increased percentage of effector/memory CD4+CD44highCD62Lneg T cells (57%±7%) in the kidney when compared with lung, spleen, and blood lymphocytes was observed (Fig. 5). The CD8+ T lymphocytes in the kidney also expressed the effector/memory phenotype (49%±4%). We also found a greater percentage of NKT cells in the kidney (phenotype CD4+NK1.1+; 7%±1.7%; 0.9–1.5%; P=0.002) compared with lung, spleen, and blood (Fig. 6A). To investigate if CD4+NK1.1+ cells observed in perfused mouse kidney were CD1d-restricted NKT cells, we stained kidney lymphocytes from perfused and exanguinated mice with a CD1d tetramer. We found a similar percentage of CD4+CD1d+ NKT cells in perfused (10%±2%) and exanguinated (12%±2%) mice kidneys (Fig. 6B). Finally, we determined the percentage of CD4+ T cells expressing the IL-2R CD25 and the forkhead transcription factor Foxp3 of these cells. A small population of CD4+CD25+ Tregs in perfused (1.97%±0.2%) and exanguinated (2.2%±0.15%) mice was observed. We then analyzed Foxp3+ expression in gated CD4+CD25+ T cells (R2; Fig. 7A). No significant differences existed in the percentage of CD4+CD25+Foxp3+ in perfused (45%±2%) and exanguinated (51%±2%) mouse kidney (Fig. 7B). Thus, renal CD4+ and CD8+ T cell subsets had a preactivated phenotype, expressed effector/memory phenotype, and a population of T cells with a regulatory phenotype (CD4+CD25+Foxp3+).

Fig. 4.

Fig. 4.

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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.

Fig. 5.

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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 (CD44highCD62Lneg) 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.

Fig. 6.

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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.

Fig. 7.

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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).

Profile of TCR Vβ chains expressed by kidney and spleen T cell subsets

To further characterize the phenotype of kidney lymphocytes, we compared the kidney and spleen CD4+, CD8+, and DN TCR Vβ profile. In a normal mouse kidney, an increased percentage of CD4+ T cells expressing Vβ2, Vβ7, and Vβ8.1/8.2 chains compared with spleen lymphocytes was observed. We found an increased percentage of kidney DN T cells expressing Vβ2, Vβ3, Vβ5.5/5.2, Vβ7, Vβ8.3, Vβ8.1/8.2, and Vβ13 chains when compared with spleen lymphocytes. Among the CD8+ T cells, we observed an increased percentage of Vβ8.3 and Vβ9 clones compared with spleen (Fig. 8). These data provide additional evidence that kidney lymphocytes are of a distinct phenotype when compared with splenic lymphocytes.

Fig. 8.

Fig. 8.

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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.

Decreased percentage of DN T cells and increased levels of IFN-γ/TNF-α in CD4+ and IFN-γ CD8+ T cells after renal IRI

Given that we found abundant DN T lymphocyte cells in kidneys of normal mice, we further investigated these cells after 30 min of ischemic injury. After 24 h of renal IRI, serum creatinine increased significantly in the IRI mice (n=6; 1.9±0.2 mg/dL) as compared with normal (0.4±0.03 mg/dL) and sham-operated mice (n=8; 0.4±0.1 mg/dL; *, P<0.001; Fig. 9A). Additionally, H&E staining of kidney tissue showed extensive tubular damage and proteinaceous casts in IRI kidneys. After 3 h of renal IRI, a similar number of DN T cells in sham-operated and IRI mice were observed after extensive perfusion of mice. However, a significant decrease in the number of DN T cells in IRI mice (0.83×104±0.66×103 cells) when compared with normal (1.6×104±1.4×103 cells; *, P=0.010) and sham-operated mice (1.1×104±1.1×103 cells) was observed 24 h after renal IRI (Fig. 9B).

Fig. 9.

Fig. 9.

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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.

In a previous study, we have found a significant increased production of intracellular cytokines IFN-γ and TNF-α by CD3+ T cells isolated from kidneys of IRI mice [14]. However, it was unknown what subpopulation of T cells was producing these proinflammatory cytokines. Here, freshly isolated KMNC from normal, sham-operated, and IRI mice were examined for intracellular cytokine production by CD4+, CD8+, and DN T cells after 24 h of renal IRI. Flow cytometry analysis of KMNC from IRI mice revealed significant increased production of IFN-γ by CD4+ (mean±sem; 18%±3%; *, P<0.002) and CD8+ (19%±5%; *, P<0.018) T cells when compared with normal (CD4+: 6.6%±1%; CD8+: 7.1%±3%) and sham-operated mice (CD4+: 3.7%±1%; CD8+: 4.8%±1%; Fig. 9C). A significantly increased production of TNF-α was observed after ischemia by CD4+ T cells (19%±4%; *, P<0.007) and not by CD8+ T cells (4.9%±2%) when compared with normal (CD4+: 5%±1%; CD8+: 2.2%±0.3%) and sham-operated mice (CD4+: 5%±1%; CD8+: 3%±1%; Fig. 9D). In contrast, DN T cells did not show significant increases of intracellular cytokines after renal IRI.

DISCUSSION

In the present study, we found that normal mice kidneys harbor diverse populations of lymphocytes with conventional (CD4+ and CD8+ T cells) and unconventional (CD3int and DN T cells) phenotypes. These populations are similar to liver cells and significantly different from blood and spleen lymphocytes. Although in normal mice, these populations are likely part of the natural immunity raised by natural antigen contact in the kidney, the results suggest that these populations are also involved in pathogenic processes in the kidney, such as those observed after renal IRI stimulation.

A high proportion of kidney T lymphocytes was of the CD3int phenotype, which is characterized by the constitutive expression of the IL-2R β-chain and intermediate levels of the TCR-CD3 complex (Fig. 2B). CD3int T cells are of extrathymic origin, as they have been found in athymic nu/nu mice and contain a high frequency of self-reactive clones, which are thought to participate in autoimmunity responses [23, 24]. In contrast, a low proportion of CD19+ B cells in the kidney when compared with other organs was observed (Fig. 2A). Similar to resident lymphocytes of the liver, the increased percentage of kidney NK cells suggests that kidneys are important sites of innate immune responses. In the liver, these cells are abundant and likely play an important role in local immune responses as well as in the maintenance of tolerance [1, 7, 8]. The data found in mice corroborate our pilot data in humans, where T and B lymphocytes, in extensively perfused, normal kidneys, prior to transplantation, were identified (Fig. 1B). Further study of kidney lymphocytes could alter our current approach to immune-mediated kidney diseases and challenge the conventional paradigm that lymphocyte-mediated kidney diseases are solely a result of infiltrating lymphocytes.

In a comparative study with other organs that harbor specific lymphocyte populations, we determinate the proportion of conventional (CD4 and CD8 T cells) and unconventional (DN T cells) phenotypes from the CD3+ population (Fig. 3A). We found that normal mice kidneys harbored approximately 11% of DN T cells expressing the B220 B cell marker (Fig. 3B), which are also abundant in the liver. Also, an activated phenotype of DN T cells was observed (Fig. 3C). It has been reported that DN T cell subpopulations B220+TCRαβ+CD4−CD8− and NK1.1+TCRαβ+CD4−CD8− DN Tregs play important immunoregulatory functions in nonlymphoid organs. In lymphoproliferative diseases, DN T cells expressing B220 accumulate progressively as a result of the down-regulation of TCRαβ CD4 or CD8 coreceptors [25,26,27,28]. These results suggest that unconventional lymphocytes isolated from normal mouse kidneys could be resident kidney lymphocytes, similar to resident lymphocytes of liver and lung [4, 8]. We also found that kidney CD4 and CD8 T lymphocytes have a distinct phenotype compared with blood and spleen lymphocytes. These kidney cells were markedly more activated CD69+ (Fig. 4) and expressed effector/memory phenotype CD44+CD62L (Fig. 5), suggesting that normal mice kidneys are a site of antigen encounter. These results are in agreement with those observed in the liver and intestine, where resident CD4+ and CD8+ T lymphocytes also show an activated phenotype in a steady-state condition [3, 9]. Phenotypic activation on T lymphocytes in healthy, nonlymphoid organs is believed to be from the rapid response to the antigens that are arriving continuously from the portal vein and gut to these organs [3, 7]. Human kidneys receive a large proportion of total blood flow relative to their size (20% of cardiac output); thus, kidney lymphocytes could be responding to blood-borne antigens, similar to what happens in the liver and intestine. In addition, in normal, perfused, and exanguinated kidneys, we observed small populations of CD1d-restricted NKT (Fig. 6) and CD4+CD25+Foxp3+ T cells (Fig. 7). Recently, it has been reported that NKT cells are involved in the pathogenesis of the kidney after 24 h of ischemia [29], and increased populations of Foxp3 Tregs have been observed in long-term, allogenic transplants [30]. These results suggest that the kidney could modulate the immune responses during pathogenic processes after kidney injury.

To further define the origin, selection, and diversity of kidney T cells, we have determined Vβ gene expression profiles in DN and single-positive TCRαβ cells of normal mice. The analysis of the TCR Vβ repertoire of CD4+, CD8+, and DN T cell subsets shows a distinct population of reactive clones between kidney and spleen lymphocytes, principally among the DN T lymphocytes (Fig. 8). These reactive clones could be raised as a consequence of the natural exposure of antigens in kidney. Similar differences were found in the expression of TCR Vβ chains among hepatic and splenic lymphocytes in DBA/2 mice [31].

Finally, we reported previously an increased percentage of CD3+ T cells expressing IFN-γ and TNF-α after 24 h of renal IRI [14]. However, the production of these cytokines by subpopulations CD4, CD8, or DN (CD3+CD4−CD8−) T cells was not determined. In this study, we found that after IRI, there was a significant increased production of IFN-γ and TNF-α by CD4+ T cells (Fig. 9, C and D) and IFN-γ by CD8+ T cells (Fig. 9C). In contrast, no cytokine changes were observed in DN T cells. These data demonstrate that CD4+ and CD8+ T cells from the perfused kidney become activated and generate a response to ischemic injury by producing inflammatory mediators. Production of cytokines by liver resident lymphocytes has been suggested to play an important role in local immune responses and shaping the composition of lymphocyte populations [7, 32,33,34]. More recently, it has been suggested that resident lymphocytes modulated the immune response to ischemia through the production of cytokines, which serve to recruit peripheral lymphocytes and neutrophils [11]. In renal IRI, the up-regulation of cytokines and chemokines, including IL-1, IL-2, IL-6, IL-8, IL-10, IFN-γ, TNF-α, keratinocyte-derived chemokine, MIP-2, and GM-CSF, has been identified in whole kidney tissue [35, 36]. However, it has been reported that B220+ DN T cells, whether generated in vitro or isolated from lpr (Fas-deficient) and gld (FasL-deficient) mice, are capable of suppressing T cell proliferation and cytokine production [28]. Two hypotheses exist to explain how lymphocytes in nonimmune organs arise: the local generation/differentiation of lymphocytes and the selective recruitment of lymphocytes from the periphery [37, 38]. From our results, it seems that the first hypothesis could explain how the lymphocytes are harboring in normal kidneys and after ischemia. However, further studies will be necessary to understand in detail the real mechanism involved.

In conclusion, these data demonstrate for the first time that a healthy mouse kidney harbors resident lymphocytes of the innate and adaptive immune system. The kidney resident lymphocytes have different phenotypes than blood, spleen, and lung lymphocytes but have many similarities to liver resident lymphocytes. During renal IRI, CD4+ and CD8+ T cells have up-regulated proinflammatory function in a consistent mode with kidney pathogenesis. Resident kidney lymphocytes could play an important role in normal kidney immunology as well as participate in immunologic diseases and are therefore potential targets for therapeutic interventions.

Acknowledgments

This study was supported by National Institutes of Health grants R01 DK54770 (H. R.), 3R01DK054770-05S1 (D. B. A.), 3R01DK054770-06A1 (M. A.), SCCOR HL073944 (H. R.), RO1 AI42287 (M. J. S.), and R21 AI063133 (M. J. S.). We thank Dr. Abdel Hamad for advice with DN T cells. We also thank Dr. Hye Ryoun Jang for help in kidney surgery.

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