Renal tubular epithelial cell-derived IL-7 drives expansion and protective effects of double-negative T cells in acute kidney injury

Abstract

Previous studies have demonstrated that double-negative (DN) αβ T cells play an important role in immune responses, particularly during ischemic acute kidney injury (AKI). Here, we investigate the role of γ-chain cytokines in driving DN T cell proliferation in both steady state and AKI, focusing on IL-2, IL-7 and IL-15. All three cytokines promoted significant in vitro DN T cell proliferation, with IL-7 inducing the most robust expansion. Co-culture experiments revealed that renal tubular epithelial cells (RTECs) are a source of IL-7 that is essential for DN T cell proliferation, as IL-7 blockade significantly reduced their expansion. In vivo administration of IL-7 complexes in wild-type mice markedly increased DN T cell numbers in the kidney, highlighting the physiological importance of IL-7 in DN T cell homeostasis. IL-7 selectively expanded the PD-1+ DN T cell subset which are regulatory and noncytotoxic NK1.1+ DN T cells. During AKI, kidney DN T cells expanded in wild-type, but not IL-7 receptor knockout mice, with IL-7 deficiency linked to worsened renal outcomes. Furthermore, primary human RTECs activated peripheral human DN T cells in vitro as indicated by CD69 upregulation, suggesting that our mice data were relevant to humans. These results establish IL-7 as pivotal for DN T cell expansion and underscore RTECs role in homeostasis of DN T cell.

Introduction

Double-negative (DN) T cells, a subset of αβ T cells lacking the CD4 and CD8 coreceptors, have emerged as an intriguing component of the immune system with roles in regulating autoimmunity, inflammation, and tissue homeostasis (Martina, et al., 2016). These cells are enriched in various tissues and appear to exert protective effects in the context of organ-specific stress and injury(Sadasivam, et al., 2019). In the normal kidney, DN T cells are actively proliferating and then respond to ischemic acute kidney injury (AKI), potentially limiting damage and promoting repair. The factors driving their expansion and function in this setting are not well-defined.

Previous data implicate direct cell contact with renal tubular epithelial cells (RTECs) and soluble mediators in driving spontaneous proliferation of kidney DN T cells (Sadasivam, et al., 2023). Integrins were mediators of direct cell-cell contact in the aforementioned study, but soluble mediators were not studied. Common gamma-chain (γc) cytokines, which include IL-2, IL-7, IL-15, and IL-21, are critical regulators of T cell development, homeostasis, and function (Rochman, et al., 2009). These cytokines share the gamma chain (γc) subunit and are essential for lymphocyte proliferation, survival, and differentiation, each playing distinct and sometime overlapping roles (Rochman, et al., 2009). Among these, IL-7 is particularly vital for T cell development and maintenance, contributing to immune homeostasis under both resting and stress conditions (Jacobs, et al., 2010). Produced in various tissues, IL-7 sustains T cell subsets and promotes immune resilience in challenging environments. IL-7 has been widely studied in relation to CD4+ and CD8+ T cells, but its role in DN T cell biology, particularly in tissue-specific immune responses, remains poorly understood (Bradley, et al., 2005). Recent evidence suggests that IL-7 may play a role in local immune regulation by supporting the survival and activity of T cells in inflamed or injured tissues, including the kidney (Bikker, et al., 2012).

In this study, we investigate the role of γc cytokines in regulating DN T cell proliferation and survival particularly within the kidney using in vitro and in vivo models. We examined the role of RTECs as a local IL-7 source and assessed the impact of IL-7 stimulation on DN T cell expansion and function during AKI. Our results establish IL-7 as pivotal for DN T cell expansion and underscore RTECs role in DN T cell effects in kidney especially under conditions of acute injury.

Material and Methods

Mice

C57BL/6J (wild-type) and B6.129S7-Il7r^tm1Imx/J (IL-7 receptor knockout) mice were purchased from Jackson Laboratory. All mice were maintained under specific pathogen-free conditions at the Johns Hopkins University Animal Facility. For the study, age-matched mice, aged 8 to 10 weeks, were used unless specified otherwise. All animal procedures were conducted in accordance with protocols approved by the Johns Hopkins University Animal Care and Use Committee.

Human kidney samples

Human kidney tissues were obtained from the non-tumor sections of kidneys removed during nephrectomy procedures for renal cell carcinoma, with participants recruited by Johns Hopkins University. The collection of these samples followed the principles outlined in the Declaration of Helsinki and received approval from the Johns Hopkins Medicine Institutional Review Board (IRB number CR00031498). All participants provided written informed consent prior to participation, and all data were anonymized.

Antibodies

Antibodies used in this study were purchased from BD Biosciences, BioLegend, and eBioscience for flow cytometry analysis. The monoclonal antibodies (mAbs) targeting mouse antigens included: CD45-APC-Cy7 (30-F11), αβTCR-Pacific Blue (H57–597), CD8α-PerCP (53–6.7), CD4-APC (RM4–5), Annexin V-PE, NK1.1-FITC (PK136), and PD-1-BV421 (J43). For human antigens, the mAbs included: CD45-APC-Cy7 (HI30), αβTCR-FITC (IP26), CD8α-PE-Cy7 (HIT8a), CD4-APC (OKT4), HLA-ABC (W6/32), and HLA-DR/DP/DQ (WR18). Recombinant mouse IL-2, IL-15, and IL-7 were obtained from Prospec (NJ, USA). Mouse CD1d tetramers were sourced from the NIH Tetramer Core Facility (USA). The CellTrace^™ CFSE Cell Proliferation Kit was purchased from Thermo Scientific (USA).

Isolation of lymphocytes from kidneys and lymph nodes

Mononuclear cells were isolated from kidneys following established protocols (Martina, et al., 2014, Sadasivam, et al., 2019). In brief, kidneys were minced and incubated in a 5 mg/mL collagenase D solution (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 minutes. The digested tissue was mechanically disrupted using 70-μm strainers (BD Biosciences) to create a single-cell suspension, which was then filtered through 40-μm strainers. The cell suspension was centrifuged at 400 × g for 10 minutes at 4°C to pellet the cells. The resulting pellet was resuspended and subjected to Percoll gradient centrifugation to isolate the mononuclear cells. To determine the absolute number of viable lymphocytes in each sample, a trypan blue exclusion assay was performed using a hemocytometer and a light microscope. The number of each lymphocyte subset was determined by multiplying the total cell count per organ by the percentage of that specific subset, as assessed by flow cytometry (FACS). Lymphocytes from peripheral lymph nodes were isolated using similar procedures. Isolated lymphocytes were subsequently used for staining or co-culture experiments with renal tubular epithelial cells (RTECs) for further analysis.

Isolation of renal tubular epithelium (RTEC) and generation of monolayers

Renal tubular epithelial cells (RTECs) were isolated from kidneys using a modified method as described previously (Rabb, et al., 2000). In brief, kidneys were minced and incubated with 5 mg/mL collagenase D (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 minutes. The tissue was mechanically disrupted using 70-μm strainers (BD Biosciences) and then filtered through 40-μm strainers to generate a single-cell suspension. This suspension was plated onto culture plates and incubated for 2 hours to allow glomeruli to adhere. After this incubation, the non-adherent tubular cells were collected and seeded onto collagen-coated culture plates. The cells were maintained in a humidified incubator at 37°C with 5% CO2 for 48 hours, during which time the tubules were left undisturbed. The culture medium was refreshed every 2 days. After 7 days, confluent monolayers of RTECs were evaluated for morphology and purity. These cells were then used for co-culture experiments with lymphocytes.

Flow cytometric analysis and gating strategy

Flow cytometric analysis was performed following standard procedures (Ahmed, et al., 2019). Cells were first stained in FACS buffer (PBS, 2% FBS, 0.1% sodium azide) and incubated with an anti-CD16/CD32 antibody for 10 minutes to block non-specific binding. Following this, cells were incubated with a mixture of fluorochrome-conjugated monoclonal antibodies (mAbs) for 30 minutes at 4°C. After washing, cells were resuspended in FACS buffer for analysis. Fluorescence minus one (FMO) controls were included for each marker to establish accurate gating thresholds. FMO controls consisted of cells stained with all antibodies in the panel except the fluorochrome-conjugated antibody corresponding to the specific marker being analyzed. These controls were used to account for background fluorescence and spillover from other fluorochromes, enabling precise identification of positive populations for all markers included in the analysis. Data acquisition was carried out using an Attune^™ NxT Acoustic Focusing Cytometer, equipped with three laser lines (405 nm, 488 nm, and 637 nm). Flow cytometry data were analyzed using FlowJo V10 software (Treestar Software, USA).

CFSE cell proliferation assay

T cell proliferation was assessed using a standard CFSE dye dilution assay, as previously described (Ahmed, et al., 2019, Sadasivam, et al., 2023). Briefly, lymphocytes were isolated, stained with 5 μM CFSE for 3 minutes at room temperature, washed, and resuspended in RPMI medium. The cells were then counted and stimulated with appropriate antigens or mitogens. After a specific incubation period (e.g., 3–5 days), cells were harvested and stained with fluorescently labeled antibodies to identify specific populations (e.g., CD4+, CD8+ T cells). Flow cytometry was used to analyze CFSE dilution, as the dye is distributed equally between daughter cells during division, leading to a progressive decrease in fluorescence intensity that reflects the number of cell divisions.

Annexin V apoptosis assay

Apoptosis was assessed using the PE-Annexin V Apoptosis Kit (BD Pharmingen, USA) following the manufacturer’s instructions. Cells were first stained for surface markers for 30 minutes at 4°C, washed twice with FACS buffer, and resuspended in Annexin V binding buffer. PE-Annexin V was then added, and the cells were incubated in the dark at room temperature for 15 minutes. Samples were acquired without washing using a flow cytometer and analyzed with FlowJo software. Apoptotic cells were identified as Annexin V-positive.

In Vivo Analysis of IL-7 Effects Using IL-7/Anti-IL-7 mAb Complex Treatment

IL-7/anti-IL-7 mAb complexes (IL-7 CMPLX) were prepared by incubating 1.5 μg of recombinant mouse IL-7 (ProSpec, NJ, USA) with 7.5 μg of anti-IL-7 mAb (M25) in PBS for 18 hours at 4°C to allow stable complex formation. These complexes were then administered to WT mice via intraperitoneal (i.p.) injection. Five days post-injection, mice were euthanized, and lymphocytes were isolated from the kidneys. The cells were stained with fluorescently labeled antibodies to identify specific populations, such as CD4+ and CD8+ T cells or B cells. Stained cells were subsequently analyzed by flow cytometry using FlowJo software to evaluate changes in cell numbers, activation markers, and other relevant parameters.

Assessing the role of IL-7 in ischemic acute kidney injury in mice

An established bilateral renal ischemia-reperfusion (IR) model in mice was used as previously described (Kurzhagen, et al., 2023, Lee, et al., 2020, Martina, et al., 2016). Mice were anesthetized via intraperitoneal injection of ketamine (130 mg/kg) and xylazine (7 mg/kg). A midline abdominal incision was performed to expose the renal pedicles, which were carefully dissected and clamped with microvascular clamps (Roboz Surgical Instrument, Gaithersburg, MD) for 30 minutes. Throughout the procedure, the animals were maintained at a constant body temperature (37°C) and hydrated with warm saline. After 30 minutes of ischemia, the clamps were removed to allow reperfusion, and the incision was sutured. Mice were then allowed to recover with unrestricted access to food and water. Kidney function was assessed by serum creatinine (SCr) levels and glomerular filtration rate (GFR). SCr levels were measured by Cobas Mira Plus automated analyzer system (Roche) with creatinine reagent (Pointe Scientific Inc) at baseline and 24 hours post-ischemia. GFR was measured by transcutaneous fluorescein isothiocyanate (FITC)-sinistrin (inulin analogue) with a fluorometer device (MediBeacon, St. Louis, MO) at baseline and 24 hours post-ischemia. Lymphocytes were isolated from the kidneys and lymphoid organs 24 hours post-injury, stained, and analyzed by flow cytometry (LSRII) using FlowJo software.

Statistical analysis

Data are presented as mean ± SEM, with n representing the number of animals per group, and were collected from at least three independent experiments. Unpaired t-tests were used for comparing repeated measures within the same group. For comparisons between multiple groups, one-way or two-way ANOVA was applied, followed by Tukey’s or Sidak’s multiple comparison tests, as appropriate. All statistical analyses were conducted using GraphPad Prism 6 software, and statistical significance was defined as P < 0.05.

Results

Exogenous IL-7 is sufficient to expand DN T Cells in vitro and in vivo

To investigate the contributions of γ-chain cytokines to DN T cell proliferation, we used an in vitro culture system. Lymphocytes isolated from peripheral lymph nodes of B6 mice were cultured with or without IL-2, IL-15, or IL-7. After five days, cultured lymphocytes were analyzed by flow cytometry for DN T cell frequency, and absolute cell numbers, proliferation, and apoptosis. Our findings show that all three cytokines significantly expanded DN T cells in the absence of direct TCR stimulation. Both the percentage and absolute numbers of DN T cells increased substantially with cytokine stimulation compared to controls (Fig. 1A). However, IL-7 was the most potent inducer, yielding a twofold increase in DN T cell expansion compared to IL-2 or IL-15. DN T cells cultured with IL-7 also exhibited significantly enhanced proliferation and survival compared to those cultured alone (Fig. 1B). To further investigate IL-7 secretion, we confirmed its production by renal tubular epithelial cells (RTEC) in coculture using RT-PCR and ELISA. We first measured the mRNA expression of IL-7 in RTEC cultured either alone or in the presence of sorted DN T cells, CD4, or CD8 T cells. The results revealed a significant increase in IL-7 mRNA expression in RTEC co-cultured with DN T cells, with expression levels approximately 4 times higher than those in RTEC cultured alone. Also, IL-7 mRNA expression was significantly lower in RTEC co-cultured with CD4 or CD8 T cells compared to RTEC with DN T cells. We also analyzed the supernatants collected after 5 days of coculture for IL-7 levels using ELISA. The ELISA results revealed that the supernatants from the RTEC-DN T cell coculture contained significantly higher levels of IL-7 compared to those from RTEC cocultured with CD4 or CD8 T cells (Fig. 1C). These findings confirm that RTEC secrete IL-7 in response to interaction with DN T cells. To assess physiological relevance in vivo, we administered IL-7 complex—a long-acting IL-7 form—to wild-type mice, which resulted in a significant increase in DN T cell frequency and number within the kidneys (Fig. 1D). These results indicate that γ-chain cytokines and IL-7 in particular effective in driving DN T cell proliferation both on vitro and in vivo.

Figure 1: Exogenous IL-7 expands DN T cells In vitro and In vivo.

Lymphocytes from lymph nodes were isolated and cultured in the presence or absence of recombinant γ-chain cytokines (IL-2, IL-15, and/or IL-7) for 5 days. Cells were then harvested and analyzed as described.

(A) Representative dot plots depict the percentages of CD4, CD8, and DN T cells after 5 days of culture. Numbers within quadrants indicate percentages. Graphs show the cumulative frequency and absolute numbers of DN T cells.

(B) Representative histograms illustrate proliferation, measured by CFSE dilution, and apoptosis, measured using annexin V staining, in gated DN T cell subsets from different culture groups. Graphs display cumulative data for both assays.

(C) Graph shows relative IL-7 mRNA expression in renal tubular epithelial cells (RTEC) co-cultured with DN, CD4, or CD8 T cells. The graph also depicts IL-7 protein levels in the culture supernatant of these co-cultures.

(D) Representative dot plots show the percentages of CD4, CD8, and DN T cells in the kidney and lymph nodes of B6 wild-type (WT) mice 5 days after receiving IL-7 complex (IL-7 CMPLX) transfer. Numbers within quadrants indicate percentages. Graphs show cumulative frequency and absolute numbers of DN T cells. Data points (mean ± SEM) represent at least three independent experiments (n = 3 mice per group). *P < 0.05, **P<0.01, ****P < 0.0001, ^nsnon-significant.

RTECs-derived IL-7 is necessary for proliferation of DN T cells

RTECs are known to produce IL-7, particularly under conditions of stress or inflammation. This localized IL-7 production may contribute to immune responses within the kidney by promoting the survival and activity of T cell subsets, especially during injury or infection (Hsieh, et al., 2012). Based on the potent expansion induced by exogenous IL-7, we next examined whether renal tubular epithelial cells (RTECs) produce IL-7 to support DN T cell expansion and proliferation. Using co-culture experiments where lymphocytes from lymph nodes were added to primary murine renal tubular epithelial cells (mRTECs) monolayers and incubated at 37°C. We assessed DN T cell proliferation in the cocultures in the presence or absence of an IL-7-blocking antibody. The presence of the blocking antibody significantly reduced DN T cell frequency, absolute cell numbers, and proliferation rate (Fig. 2A–B), demonstrating that IL-7 from RTECs is critical for DN T cell expansion. These findings underscore the importance of RTEC-derived IL-7 for DN T cell activation and expansion.

Figure 2: RTECs induced proliferation of DN T cells is IL-7 dependent.

Blockade of IL-7 decreases the percentage and absolute cell number of peripheral DN T cells in RTEC co-culture.

(A) Dot plots show representative percentages of CD4, CD8, and DN T cells in co-culture in the presence and absence of anti-IL-7 blocking antibody. Numbers in quadrants indicate percentages. Graphs show cumulative frequency and absolute numbers of DN T cells.

(B) Representative plots show CFSE dilution of gated DN T cell subsets from co-culture. Graph shows cumulative frequency. Data points (mean ± SEM) are from at least three independent experiments (n=3 mice). *P<0.05, **P<0.01, ****P<0.0001.

IL-7 selectively expands PD-1+ subset of kidney DN T cells

DN T cells are typically classified based on their expression of surface markers, especially PD-1 and NK1.1 (Sadasivam, et al., 2019). PD-1⁺ DN T Cells expresses the programmed cell death protein 1 (PD-1), which is an immune checkpoint molecule that when engaged with its ligands (PD-L1 and PD-L2) on other cells, it delivers inhibitory signals to the T cell, dampening its activation and effector functions (find a reference). Consequently, PD-1⁺ DN T cells play roles in regulating immune responses and preventing excessive inflammation or autoimmunity. They have been observed to accumulate in certain tissues during inflammatory responses or stress, suggesting a regulatory function that could protect tissues from immune-mediated damage. NK1.1⁺ DN T cells express NK1.1, a marker commonly associated with natural killer (NK) cells, and share features with both T and NK cells, making them part of the “NKT-like” population (Neyt, et al., 2016). NK1.1⁺ DN T cells can be cytotoxic and may contribute to the immune response by directly targeting infected or stressed cells. They tend to have more pro-inflammatory functions compared to PD-1⁺ DN T cells. These subsets likely have distinct roles, with PD-1⁺ DN T cells leaning towards immune regulation and NK1.1⁺ DN T cells contributing to immune surveillance and cytotoxicity. We further explored IL-7’s selective effects on DN T cell subsets. We cultured lymph node lymphocytes with or without IL-7 and analyzed the expansion of PD-1+ and NK1.1+ DN T cells. IL-7 selectively expanded PD-1+ DN T cells, increasing both their percentage and absolute cell numbers, whereas NK1.1+ DN T cells showed no significant change (Fig. 3A). To validate this in vivo, we administered IL-7 complex to wild-type mice. Consistent with in vitro results, IL-7 injection led to an increase in kidney PD-1+ DN T cells, with minimal changes in peripheral PD-1+ DN T cell populations (Fig. 3B). These observations suggest that IL-7 selectively promotes the proliferation of kidney-resident PD-1+ DN T cells.

Figure 3: IL-7 Selectively expands the PD-1+ subset of kidney DN T cells.

Lymphocytes were isolated from lymph nodes and cultured with or without recombinant IL-7 (rIL-7) for 5 days. After culture, cells were harvested and analyzed to determine the percentage and absolute numbers of PD-1⁺ and NK1.1⁺ DN T cells.

(A) Representative dot plots display the percentages and absolute numbers of PD-1⁺ and NK1.1⁺ DN T cells in vitro. Numbers within the quadrants indicate percentages. Graphs show cumulative data.

(B) Representative dot plots show the percentages and absolute numbers of PD-1⁺ and NK1.1⁺ DN T cells from the kidney and lymph nodes 5 days after mice received i.p. IL-7 complex. Percentages are indicated within the quadrants. Graphs display cumulative results. Data points (mean ± SEM) represent at least three independent experiments (n = 3 mice). *P < 0.05, **P < 0.01, ^nsnon-significant.

DN T cells rely on IL-7 for expansion during ischemic AKI

To assess IL-7’s role in DN T cell expansion within a disease context, we analyzed DN T cell responses to ischemic acute kidney injury (AKI). Wild-type (WT) and IL-7 receptor knockout (IL-7r KO) mice were subjected to bilateral renal ischemia-reperfusion injury (IRI), and DN T cell expansion was measured in the kidney. Renal injury was confirmed by elevated serum creatinine levels. IRI induced significant DN T cell expansion and upregulation of IL-7r in WT mice. By contrast, response of DN T cells was markedly impaired in IL-7r KO mice (Fig. 4A). IL-7 receptor expression on kidney DN T cells was also upregulated following IRI in WT mice (Fig. 4B). Furthermore, IL-7r KO mice displayed more severe ischemic injury, with elevated serum creatinine levels and reduced glomerular filtration rate (GFR) compared to WT mice (Fig. 4C). These findings highlight the requirement for IL-7 in DN T cell expansion and protection during ischemic AKI.

Figure 4: IL-7 promotes expansion of kidney DN T cells during ischemic AKI.

WT and IL-7r KO mice were subjected to ischemia-reperfusion injury (IRI), and serum creatinine levels were measured 24 hours later to confirm kidney injury. Lymphocytes were subsequently isolated from the kidneys of WT, IL-7r KO, WT-IRI, and IL-7r KO-IRI mice, and the frequencies and absolute numbers of T cell subsets were analyzed by flow cytometry.

(A) Representative dot plots depict the percentages of CD4, CD8, and DN T cells in the kidneys. Numbers within the quadrants indicate percentages. Graphs display cumulative data.

(B) Representative histograms show IL-7r expression on different T cell subsets from kidney tissue. Graphs depict the percentage of IL-7r-positive T cells in the kidneys of the indicated mouse groups.

(C) Graphs present serum creatinine (SCr) levels and glomerular filtration rate (GFR) in WT and IL-7r KO mice before and after IRI. Data points (mean ± SEM) are derived from at least three independent experiments (n = 3 mice). *P < 0.05.

Human renal tubular epithelial cells (hRTECs) activate peripheral blood DN T Cells

To assess the translational relevance of our findings, we investigated the ability of primary human renal tubular epithelial cells (hRTECs) to promote the expansion of DN T cells. Primary hRTECs were isolated from patients who underwent nephrectomy for localized renal cell carcinoma and cultured as monolayers. These hRTEC monolayers were then co-cultured with human peripheral blood mononuclear cells (PBMCs) for 5 days at 37°C. Subsequently, T cells were harvested, and various subsets were analyzed by flow cytometry for DN T cell expansion and activation. Notably, RTEC did not induce expansion of any subset including DN T cells (Fig. 5A). However, RTEC clearly resulted in activation of DN T cells was evidenced by a significant upregulation of CD69 expression (Fig. 5B). Furthermore, the activation of DN T cells by hRTECs was found to be MHC-independent, as blocking HLA class I or II molecules did not affect the response (Fig. 5C). These results, combined with our previous findings in murine models, collectively demonstrate that RTECs possess the capacity to support growth and activation of DN T cells in humans.

Figure 5: Human renal tubular epithelial Cells (hRTECs) activate peripheral DN T cells.

Primary hRTECs were isolated from human kidney tissues obtained from patients who underwent nephrectomy, grew as monolayers and peripheral blood mononuclear cells (PBMCs) were added and incubated at 37 ⁰C for 5 days. Lymphocytes were harvested percentage and absolute numbers and analyzed for activation as indicated by CD69 upregulation and proliferation using CFSE assay.

(A) Representative dot plots show the percentages of CD4, CD8, and DN T cells after 5 days of co-culture. Numbers in quadrants indicate the percentage of each T cell subset. Graphs show cumulative data.

(B) Representative histograms show expression of CD69 by CD4, CD8, and DN T cells after 5 days of co-culture with hRTECs. The graph shows cumulative frequency of CD69+ T cells.

(C) Representative histograms show expression of CD69 by gated DN T cells in the co-culture with or without the HLA MHC class I or class II blockade. The graphs show cumulative frequency of CD69+ DN T cells. Data points (mean ± SEM) are from at least three independent experiments (n = 3). *P<0.05, **P<0.01, ***P<0.001, ^nsnon-significant.

Discussion

Our study demonstrates the important role for IL-7 in supporting the expansion and survival of double-negative (DN) T cells, particularly in kidney tissue. We found that IL-7, among other γ-chain cytokines tested, was the most effective at promoting DN T cell proliferation in vitro and in vivo. Specifically, IL-7 significantly increased the numbers of DN T cells, selectively enhancing the expansion of PD-1+ DN T cell subsets. Furthermore, RTECs were identified as a primary source of IL-7, essential for driving DN T cell responses in the kidney. In ischemic acute kidney injury (AKI), the absence of IL-7 signaling in IL-7 receptor knockout mice resulted in impaired DN T cell expansion and worsened renal outcomes, underscoring the cytokine’s protective role in kidney injury. Furthermore, primary human RTECs activated human peripheral blood DN T cells in vitro, suggesting that our mouse studies were relevant in humans. These results establish IL-7 as pivotal for DN T cell expansion and underscore RTECs role in supporting DN T cells in kidney health and injury.

Our findings align with previous reports on IL-7’s involvement in T cell homeostasis and tissue-specific immune responses (Jacobs, et al., 2010). Recently, we have identified two distinct populations of DN T cells based on their expression of PD-1 and NK1.1 found in mouse and human kidneys (Sadasivam, et al., 2019). Furthermore, the PD-1+ DN T cells was found to be the major responder to external stimuli caused by other conventional T cells or by ischemic AKI. IL-7 has been shown to sustain other T cell subsets under stress conditions, and our results suggest that DN T cells share this dependency on IL-7, especially during renal stress such as AKI. The selective expansion of PD-1+ DN T cells observed here also mirrors reports of tissue-specific immune responses in which IL-7 regulates not only cell numbers but also subset-specific immune functions. The expression of PD-1 on expanded DN T cells may indicate an immunoregulatory role, aligning with studies that link PD-1+ T cells to protective responses in inflamed tissues.

Our study has a number of limitations. First, while we demonstrated that RTECs are a source of IL-7, further investigation is needed to clarify whether other kidney-resident cells may also contribute to IL-7 production. Additionally, our findings were based on murine in vitro and in vivo models and in vitro human cell cultures; we do not know if these mechanisms are occurring in human AKI and other kidney diseases. Future research should explore the interactions between IL-7 and other cytokines in regulating DN T cell activity, as well as the potential therapeutic implications of modulating IL-7 signaling in kidney diseases.

Overall, our study sheds light on IL-7 as a crucial driver of DN T cell expansion and highlights RTECs’ role in maintaining kidney immune homeostasis, opening new avenues for therapeutic strategies targeting IL-7 in renal injury.

Data availability statement.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Abdel Rahim A. Hamad (ahamad@jhmi.edu).