Kidney tissue hypoxia dictates T cell–mediated injury in murine lupus nephritis

Renal-infiltrating T cells are functionally active and pathogenic

We isolated from nephritic kidneys of 16- to 18-week-old lupus-prone MRL/MpJ-Fas^lpr/J

(MRL/lpr) mice renal-infiltrating T cells, separating them from circulating nonresident cells that potentially lodge in the kidney at time of isolation (fig. S1) (6) and determined their effector capacity. Consistent with published phenotypic analyses of biopsies of human lupus nephritis (7, 8), isolated CD4⁺ T cells expressed interferon-γ (IFN-γ) and CD8⁺ T cells IFN-γ and more granzyme B compared to their counterparts simultaneously isolated from spleens of mice from which kidneys were taken (fig. S2, A and B). Renal T cell populations also had increased T-bet expression, compared to those from spleens (fig. S2C). These phenotypes were replicated in a cohort of 5-month-old lupus-prone B6.Sle1.Yaa male mice (fig. S2, D and F) [lupus in this strain is penetrant in male mice bearing a second Tlr7 locus on the Y chromosome (9)]. Renal-infiltrating T cells expressed programmed cell death–1 (PD-1) (fig. S2G), with PD-1^hi CD4⁺ and CD8⁺ T cells relatively hypofunctional as determined by IFN-γ and granzyme B synthesis, respectively, as previously described (10); yet, most of the renal-infiltrating T cells were PD-1^lo (fig. S2, G to J). These data suggested that renal-infiltrating T cells are transcriptionally and functionally active and distinct from their splenic counterparts.

To confirm pathogenicity of renal-infiltrating T cells, T cell–depleting antibody anti-Thy1.2 was injected every 3 days for 4 weeks into male MRL/lpr mice beginning at age 14 weeks, after onset of renal cell infiltration. T cell depletion effectively reduced peripheral blood T cells by >90% yet had no effect on serum autoantibody titers and glomerular immune complex deposition (fig. S3, A to D), likely secondary to the presence of long-lived autoantibody-secreting plasma cells in lupus mice with established disease (11). In contrast, T cell depletion reduced renal inflammation and prevented features of chronic tissue damage, including glomerulosclerosis, tubular atrophy, and interstitial fibrosis (fig. S3, E to H). These data suggested that renal-infiltrating T cells are functionally active and contribute to tissue damage in lupus nephritis independently of autoantibody production driven by their splenic counterparts.

Adaptation to tissue hypoxia is associated with HIF-1 expression

We next sorted activated (CD44^hi) CD4⁺ and CD8⁺ T cells from nephritic kidneys of 14-week-old MRL/lpr mice, again separating them from circulating cells, and subjected them to RNA sequencing. A total of 128 shared genes were differentially up-regulated (fold change >2; P < 0.05) in both renal-infiltrating CD4⁺ and CD8⁺ T cells compared to splenic cells (data file S1). To determine the transcription factors that promoted expression of these transcripts, we cross-referenced them to a database analyzing perturbations of transcription factors from Enrichr (12). The enriched transcripts in our dataset were preferentially down-regulated upon Nfe2l2 [the gene expressing nuclear factor erythroid 2–related factor 2 (NRF2)] knockout (P = 7.8 × 10⁻¹⁰) and up-regulated by Myc haploinsufficiency (P = 7.8 × 10⁻⁸) ((13–14). The transcriptional regulation of Hif1a (HIF-1α) was determined by referencing TRRUST (Transciptional Regulatory Relationships Unraveled by Sentence-based Text mining database (P = 7.9 × 10⁻³) (15). Because the transcriptional activity of Myc is suppressed upon HIF-1 activation (16, 17) and the redox balance maintained by NRF2 is important for cellular survival in hypoxia (18), we reasoned that the enriched gene sets were a consequence of cellular adaptation to hypoxia. As confirmation, our transcriptome data revealed hypoxia- and HIF-regulated gene signatures in renal-infiltrating CD4⁺ and CD8⁺ T cells (Fig. 1, A and B), compared to those from the spleen. The kidney CD4⁺ T cells were enriched in transcripts up-regulated in a gene set from procollagen-proline dioxygenase [prolyl-4-hydroxylase domain (PHD) proteins]–knockout CD4⁺ T cells (P < 0.001) (4), whereas renal CD8⁺ T cells expressed genes found upon up-regulated HIF-1 activity arising in virally infected mice lacking the Von-Hippel Lindau (VHL) tumor suppressor gene (P < 0.001) (3). Similar hypoxic signatures were found in the transcriptome of renal-infiltrating CD8⁺ T cells (P < 0.001) taken from 23-week-old MRL/lpr mice, with renal-infiltrating CD4⁺ T cells showing a similar trend (P = 0.117) (fig. S4, A and B) (10). These analyses indicated that renal-infiltrating CD4⁺ and CD8⁺ T cells have gene signatures consistent with HIF-1–mediated adaptation to hypoxia, which is known to T cell terminal differentiation and sustains T cell functionality (3).

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To determine whether this transcriptional profile resulted from changes in oxygen tension, pimonidazole (Hypoxyprobe), which binds to proteins and peptides containing thiol groups at oxygen tensions below 10 mmHg (19), was injected into mice before sacrifice. Regions of hypoxia and renal tubular cells were then visualized by anti-pimonidazole staining (fig. S5). By comparison to age-matched C57BL/6 (B6) control mice, in which hypoxia is limited to the renal medulla (fig. S6A), anti-pimonidazole staining revealed extensive hypoxia involving most of the renal cortex of 6-month-old B6.Sle1.Yaa and 5-month-old MRL/lpr as compared to age-matched Fas-intact MRL (MRL^+/+) mice (fig. S6, B to F). Analysis of lymphocytic aggregates revealed that renal-infiltrating CD4⁺ and CD8⁺ T cells were primarily located in regions of hypoxia, with positive pimonidazole staining and nuclear staining of HIF-1α (Fig. 1D). These results were confirmed by flow cytometry, comparing T cells taken simultaneously from kidneys and spleens of MRL/lpr mice (Fig. 1, E to H). Hypoxia and HIF-1α up-regulation were seen in other previously described pathogenic populations, including double-negative T cells (20) and T follicular helper (T_FH)–like cells (fig. S7, A to C) (21). We also examined the expression of HIF-2α in renal T cells, demonstrating that HIF-1α was the dominant form of HIF proteins that accumulated (fig. S8A), with the relative abundance of HIF-1α likely resulting from the differential effect of IFN-γ in regulating its transcript versus that of HIF-2α (22). Predominance of HIF-1α up-regulation in renal-infiltrating T cells was also seen in 6-month-old lupus-prone B6.Sle1.Yaa mice (fig. S8, B and C), suggesting that these changes are common to murine lupus. In summary, T cell adaptation to hypoxia was noted in diseased murine lupus kidneys with up-regulation of HIF-1 expression.

To confirm the causal relationship between T cell infiltration and tissue hypoxia, we analyzed pimonidazole staining in male MRL/lpr mice treated with anti-mouse Thy1.2, with resultant T cell depletion of >90%. This treatment not only prevented extensive tissue damage and fibrosis, as defined by the National Institutes of Health (NIH) activity and chronicity indices (fig. S3, E to H), but also partially reversed hypoxia in the renal cortex (fig. S9, A to C). These findings are consistent with our previous finding that renal-infiltrating T cells are active and that their heightened effector function contributes to tissue damage and hypoxia.

T cell survival adaptation is required in the inflamed hypoxic kidney

We next determined whether renal-infiltrating T cells required modification of survival and apoptotic signals to maintain viability during hypoxia. Although Bcl-2 is overexpressed in tubulointerstitial infiltrates in NZB/W F₁ lupus-prone mice and in human lupus nephritis biopsies (23), we did not find up-regulation of its transcript in renal-infiltrating T cells of diseased MRL/lpr mice.

To explore expression of other prosurvival factors, we determined that of another Bcl-2 family member, BNIP3, transcriptionally controlled by HIF-1 (24). Hypoxia leads to the BH3-only Bcl2 family member (BNIP3)–mediated opening of the mitochondrial permeability transition pore, loss of membrane potential, and increased production of reactive oxygen species, ultimately giving way to hypoxic cell death (25). Yet, certain cells, for example, cancer, robustly express pyruvate dehydrogenase kinase isoform 2 (PDK2) facilitating alternative splicing of exon 3 of Bnip3 (Bnip3Δex3), the product of which stabilizes mitochondrial membrane potential and switches apoptosis to cellular survival by contrast to the full-length protein (26). The up-regulation of PDK2 may be a cellular response to hypoxia, given its transcriptional control by HIF-1 in hematopoietic stem cells (27). To address the involvement of the HIF-1/PDK2/BNIP3 axis in maintaining cellular survival in hypoxia, we measured protein expression in renal-infiltrating CD4⁺ and CD8⁺ T cells, revealing that, compared to their splenic counterparts, those in the kidney expressed more PDK2 (Fig. 2A and fig. S10A). We also found up-regulation of total BNIP3 in renal-infiltrating T cells, as determined by antibody staining (Fig. 2B and fig. S10B), with recognition of an epitope encoded by exon 2 common to both splice variants. However, quantitative polymerase chain reaction (qPCR) showed proportionate up-regulation of the alternatively spliced Bnip3Δex3 transcript in addition to that of full-length Bnip3FL (Fig. 2, C and D), suggesting that the alternative splicing of Bnip3 by PDK2 was involved in maintaining cellular survival in the hypoxic environment.

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To further dissect the T cell response in hypoxia, we demonstrated that splenic CD4⁺ T cells from prediseased (6- to 8-week-old) male MRL/lpr and B6.Sle1.Yaa lupus-prone and 6- to 8-week-old control B6 mice had up-regulation of HIF-1α and PDK2 in hypoxic (1%) conditions (fig. S11, A and B) and along with splenic CD8⁺ T cells, during pseudohypoxia in which the HIF protein was stabilized via addition of the PHD inhibitor dimethyloxalylglycine (DMOG) (fig. S11, C to F). These data suggested that up-regulation of PDK2 in response to HIF-1 expression is a common T cell response to hypoxia.

To confirm the transcriptional control of PDK2 and BNIP3 by HIF-1, chromatin immunoprecipitation (ChIP) and qPCR were performed using CD4⁺ T cells from 6- to 8-week-old B6 mice cultured during DMOG-induced pseudohypoxia. We observed enrichment of HIF-1α binding to the promoter regions of Pdk2 and Bnip3 (Fig. 2E), with loss of protein expression observed after 2 days in hypoxic cultures after knockdown of Hif1a (Fig. 2, F to H). Perturbation of Bnip3Δex3 relative to Bnip3FL was seen in Hif1a and Pdk2 knockdowns as well as with a short hairpin RNA (shRNA) targeting Bnip3Δex3 but with the reverse result upon targeting Bnip3FL (Fig. 2I). Decrease of Bnip3Δex3 expression was associated with decreased T cell survival after 3 days of hypoxia, as shown by increase of apoptotic cells with reduced active mitochondrial membrane potential (MitoTracker Deep Red^lo annexin V⁺) in the transduced cells (Fig. 2J and fig. S12). These data suggested that PDK2 regulation of Bnip3 alternative splicing maintains cell survival in hypoxia, with this pathway transcriptionally controlled by HIF-1 (fig. S13).

Proline metabolism is required for optimal T cell effector function in hypoxia

In addition to the changes in prosurvival factors, pathway analyses of the 128 commonly up-regulated genes in renal-infiltrating CD4⁺ and CD8⁺ T cells revealed involvement of glutathione and amino acid metabolism pathways (table S1). Among the up-regulated genes in renal-infiltrating T cells were genes involved in the Nrf2-regulated glutathione metabolism pathway, including Idh1 [the gene encoding isocitrate dehydrogenase 1 (IDH1)], and glutathione S-transferase genes (Fig. 3A). IDH1 supports redox homeostasis in reductive carboxylation (28). Alpha-ketoglutarate (αKG), a downstream metabolite of glutamine, glutamate, and proline catabolism, is converted to isocitrate through the enzymatic activity of IDH1 while reverting NADPH [reduced form of NADP⁺ (nicotinamide adenine dinucleotide phosphate)] back to NADP⁺ in reductive carboxylation. Confirming our transcriptome analysis, we observed up-regulation of IDH1 in renal-infiltrating T cells from both MRL/lpr and B6.Sle1.Yaa murine lupus models (fig. S14, A and B). Maintenance of redox homeostasis is critical for glycolysis to provide sufficient energy in conditions of mitochondrial dysfunction, such as hypoxia (29). Therefore, up-regulation of amino acid metabolism and the downstream reductive carboxylation may support glycolysis by generating NAD⁺ (nicotinamide adenine dinucleotide) and NADP⁺.

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To further explore amino acid metabolism, we focused on that of proline, essential for meeting cellular energy requirements and survival in hypoxia (30). Metabolomic analysis of cells with mitochondrial dysfunction has reduced proline content, suggesting a prominent role for its metabolism in hypoxia (31). By flow cytometry, we demonstrated up-regulation of proline dehydrogenase (PRODH) in renal CD4⁺ and CD8⁺ T cells from MRL/lpr and B6.Sle1.Yaa mice (Fig. 3B and fig. S14C). Using in vitro hypoxia and pseudohypoxic culture systems, we confirmed that this up-regulation was controlled by hypoxia and HIF-1 in CD4⁺ and CD8⁺ T cells (fig. S15, A to C). HIF-1 is known to globally reprogram cellular metabolism, in part through transcriptional control of Max-interacting protein 1 (MXI1), which ultimately leads to suppression of c-Myc function by interfering with Myc-Max binding (16). Because of the known negative regulation of c-Myc in PRODH expression (32), we determined whether up-regulation of the latter by HIF-1 is through suppression of the negative regulation of c-Myc. The shRNA knockdown of Hif1a, using two different constructs, reduced Mxi1 transcript (Fig. 3C), consistent with transcriptional regulation of Mxi1 by HIF-1 (33). Knocking down either Hif1a or Mxi1 both contributed to down-regulation of PRODH in CD4⁺ T cells after 2 days of hypoxic culture (Fig. 3, D and E), indicating that HIF-1 and MXI1 are both upstream of PRODH regulation. We also found that the expression of the latter was negatively regulated by c-Myc (Fig. 3F), consistent with upstream repression of c-Myc transcriptional activity by HIF-1 and MXI1 (17). These changes during hypoxia were linked to reduced CD4⁺ and CD8⁺ T cell effector capability as evidenced by reduced IFN-γ production in both CD4⁺ and CD8⁺ T cells and reduced granzyme B production by CD8⁺ T cells (fig. 3, G to I).

Both CD4⁺ T and CD8⁺ T cell cells in pseudohypoxic cultures had higher extracellular acidification rate (ECAR) and lower oxygen consumption rate (OCR) (Fig. 3, J to M, and fig. S16, A to D), indicative of increased glycolysis and mitochondrial dysfunction, and consistent with the HIF-1 inhibition of mitochondrial biogenesis and electron transport chain function (17, 34), key features of metabolic changes in hypoxia. The addition of the selective PRODH inhibitor, tetrahydro-2-furoic acid (THFA), reduced ECAR, suggesting that proline metabolism was critical for promotion of glycolysis upon mitochondrial dysfunction. The glycolysis-facilitating effect of proline metabolism is most likely the result of maintenance of NADH (reduced form of NAD⁺)/NAD⁺ balance (Fig. 3, N to O), because progression through glycolysis requires the oxidized form of the cofactor NAD⁺ (fig. S17) (29). Together, these data indicate that HIF-1 controls proline metabolism through suppression of the negative regulation of c-Myc. Up-regulation of proline metabolism serves as a cellular adaptation to maintain a balanced NADH/NAD⁺ ratio to promote glycolysis and is critical for T cell effector functions in a hypoxic microenvironment.

Blockade of hypoxia-induced adaptation eliminates cellular infiltration and reverses tissue injury and hypoxia

Because HIF-1 regulates key adaptations to T cell survival and effector function in hypoxia, it is logical that its blockade might be useful therapeutically to ameliorate tissue damage incurred by infiltrating T cells in lupus nephritis. We accordingly validated hypoxia-regulated transcripts from a published dataset of kidney-infiltrating T cells (10) and then confirmed their phenotypic expression (fig. S18, A to F). Because erythropoietin (EPO)–producing cells are dependent on HIF-2α (35), with the latter also important in stress protection of renal tubular and endothelial cells (36), selective HIF-1 blockade may minimize perturbations in EPO production and stress adaptive renal parenchymal changes. PX-478 was selected for HIF-1α inhibition in vivo, given its role in decreasing both Hif1a transcription and translation of its protein product, alongside its enhanced ubiquitination (37).

To test the efficacy of HIF-1 blockade in vivo, 5-month-old B6.Sle1.Yaa mice, early after onset of proteinuria, were treated with either gavaged phosphate-buffered saline (PBS) or PX-478 (5 mg/kg) every 2 days. After 7 days, renal-infiltrating T cells from animals receiving PX-478 had substantial decreases of HIF-1α, PDK2, and PRODH (fig. S19, A to C). In addition to the perturbation of these adaptive pathways, we observed a reduction in T-bet expression in both CD4⁺ and CD8⁺ T cells in treated mice, whereas there was no difference in the percentage of Foxp3⁺ CD4⁺ T cells (fig. S19, D and E). Along with the reduction of HIF-1α and T-bet, there was reduction of IFN-γ positivity among both CD4⁺ and CD8⁺ T cells after extending therapy for 28 days (fig. S19F). These data are consistent with the role of HIF-1 in favoring T helper 1 (T_H1) cell differentiation and controlling cytokine production (4).

After confirming the efficacy of HIF-1 blockade, an extended 28 days of treatment was performed in proteinuric 5-month-old B6.Sle1.Yaa and 12-week-old male and 10- to 12-week-old female MRL/lpr mice. Four weeks after HIF-1 blockade in male MRL/lpr mice, renal-infiltrating CD4⁺ and CD8⁺ T cells decreased by more than 90% compared to control animals (Fig. 4A). Clinical efficacy was supported by substantial reduction of proteinuria in the treatment group (Fig. 4B). Similar suppression of renal inflammation and lessened proteinuria occurred in treated female MRL/lpr and male B6.Sle1.Yaa mice, compared to controls (fig. S20, A to F). Pathological scores by NIH activity index were also reduced in the PX-478–treated group (Fig. 4C), especially cellular proliferation and glomerular leukocyte and tubulointerstitial infiltration (Fig. 4, D and E, and fig. S21, A to C), the latter around vessels, a feature of nephritis in the MRL/lpr strain. We also observed reduction of vasculitis, a feature of nephritis in the MRL/lpr strain, in treated mice (fig. S21D). Reduced immune complex deposition was noted in the glomeruli of the treated MRL/lpr mice (Fig. 4F and fig. S21E). There was reversal of renal cortical hypoxia in the PX-478–treated mice to nearly the same degree observed in control Fas-intact MRL^+/+ mice (Fig. 4, G to I). Treated mice remained functionally active, with maintenance of hematocrits comparable to control animals (fig. S21F), suggesting lack of off-target effects of PX-478 and that HIF-2–regulated erythropoiesis was unaffected. Extended treatment for 12 weeks with PX-478 was well tolerated in MRL/lpr mice, with preservation of renal function (fig. S21, G and H) and improvement in survival (Fig. 4J).

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In addition to eliminating renal-infiltrating T cells through blockade of hypoxia adaptation, we found reduced splenic size and anti-dsDNA (double-stranded DNA) antibody production in MRL/lpr and B6.Sle1.Yaa mice treated with PX-478 for 4 weeks (fig. S22, A to F), coincident with the reduction in immune complex deposition in glomeruli, indicating suppression of systemic autoimmunity. These findings were likely the result of perturbation of plasma cell survival and interruption of ongoing T cell–B cell interactions and consequent extrafollicular (38) or germinal center (GC) B cell maturation. Consistent with the finding that hypoxia is required for bone marrow plasma cell survival (39), we found reduction of plasma cells in 16-week-old MRL/lpr mice treated with PX-478 for 4 weeks (fig. S22, G and H). The reduction in autoantibody titers, which was more pronounced in the PX-478–treated B6.Sle1.Yaa than treated MRL/lpr mice, was perhaps a consequence of less reduction in PSGL-1^lo PD-1^hi CXCR4^hi extrafollicular helper T cells, which drive B cell maturation in the latter strain, compared to PD-1^hi CXCR5^hi T_FH cells which are B cell drivers in B6.Sle1.Yaa mice and were reduced upon treatment (fig. S22, I and J). There was also abolition of GC formation in PX-478–treated B6.Sle1.Yaa mice (fig. S22K), likely related to GC hypoxia and the reliance of HIF-1 in regulating antibody class switch (40, 41).

One of the limitations of pharmacological blockade is its generalized systemic effect. To determine the role of T cell–specific HIF-1α in cellular adaptation with promotion of tissue damage, we generated Cd4cre.Hif1a^fl/fl.B6.Sle1.Yaa mice, finding that they did not have apparent T cell developmental defects, similar to nonautoimmune Cd4cre.Hif1a^fl/fl mice (3), nor did we observe any effect in development of systemic autoimmunity as measured by spleen size and anti-dsDNA production, compared to Hif1a^fl/fl.B6.Sle1.Yaa mice (fig. S23, A to C). The lack of effect in autoantibody production was likely due to the functional redundancy of HIF-1α and HIF-2α in T_FH cells leading to generation of antibody-producing cells (42). Consistent with this finding, GC sizes were similar between Cd4cre.Hif1a^fl/fl.B6.Sle1.Yaa and Hif1a^fl/fl.B6.Sle1.Yaa controls (fig. S23, D to F). However, CD4⁺ and CD8⁺ T cells isolated from Cd4cre.Hif1a^fl/fl.B6.Sle1.Yaa mice were less glycolytic and had higher oxidative phosphorylation (fig. S23, G to J) than controls, consistent with the known role of HIF-1 in promoting glycolysis and suppressing mitochondrial respiration (17).

Upon analysis of kidneys of Cd4cre.Hif1a^fl/fl.B6.Sle1.Yaa mice, we observed reduction in renal-infiltrating T cells compared to Hif1a^fl/fl.B6.Sle1.Yaa controls, analogous to pharmacological blockade (Fig. 5A). Although T cell selective knockout of Hif1a had no effect on proteinuria (Fig. 5B), there was reduction in total pathological scores, with complete ablation of pathological findings as quantified by NIH activity index in 7 of 16 mutant Cd4cre.Hif1a^fl/fl. B6.Sle1.Yaa mice (Fig. 5C). Mutant mice had similar degrees of glomerular pathology and immune complex deposition as control animals, but with reduction of tubulointerstitial inflammation (Fig. 5, D to F, and fig. S23, K to M). Because systemic autoimmunity was unaffected in T cell–specific mutant mice, their glomerular pathology was likely the result of inflammation triggered by immune complex deposition (fig. 23 N). In contrast, blocking tissue adaptation to hypoxia largely eliminated T cells in the hypoxic tubulointerstitial compartment, which, when damaged in autoimmunity, is most correlated with poor renal function by contrast to glomerulopathy (43). We also demonstrated reduced hypoxia in the renal cortex of the genetically modified mutant mice (Fig. 5, G to I), in agreement with a role of infiltrating T cells in initiation of tissue damage and resultant hypoxia. Yet, depletion of T cell tubulointerstitial infiltration did not completely reverse renal cortical hypoxia, suggesting that immune complex deposition and subsequent glomerular pathology can cause tissue hypoxia. The reduction of T cell infiltration in the tissue also corresponded with increased survival of mutant Cd4cre.Hif1a^fl/fl B6.Sle1.Yaa mice compared to controls (Fig. 5J), further indicating the relevance of pathogenic T cell in dictating organ damage and outcome. In summary, T cell–selective HIF-1α depletion is effective in suppressing renal inflammation, implicating adaptation to hypoxia and induction of a HIF-1 transcriptional program as a feedback loop promoting tissue damage independently of humoral autoimmunity. These data also provide evidence that targeting environmental factors that regulate adaptive T cell survival and function is therapeutically beneficial in mice.

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Pathways promoting tissue adaptation and effector function are up-regulated in human lupus nephritis

We next determined whether HIF-1–driven transcriptional pathways that are up-regulated in murine lupus nephritis were similarly enhanced in renal biopsy specimens from patients with SLE. As in mice, lupus nephritis in humans is characterized by not only glomerular immune complex deposition and regions of dense lymphocytic aggregates, predominantly in tubulointerstitial regions, but also periglomerular and perivascular (21, 43). We first analyzed single-cell RNA-sequencing data from the Accelerating Medicine Partnership (AMP) SLE project (44). Renal-infiltrating T cells from patients with SLE expressed Hif1a transcripts, and other HIF-1–regulated genes identified in our work, including BNIP3 and PDK2 (fig. S24, A to C). The low PRODH signal was likely the result of its relatively low expression among T cell transcriptomes (fig. S24D). Meanwhile, IDH1, which confers redox balance, was also expressed in renal-infiltrating T cells as in our murine lupus models (fig. S24E).

To confirm expression of these pathways, we performed immunohistochemistry staining of lupus nephritis biopsy samples. A key enzyme for reductive carboxylation, IDH1, and HIF-1–regulated proteins PDK2 and PRODH, were expressed in areas of dense T cell infiltration (Fig. 6, A to C). By contrast, these markers were not present in T cell–dense areas (T cell zones) of inflamed human tonsillar sections (Fig. 6, D to F), although they were observed in tonsillar GCs, consistent with its hypoxia (40). The generalized diffuse staining of these markers in regions of dense lymphocytic infiltration in lupus nephritis biopsy samples was different from that of T cells in inflamed human tonsils (Fig. 6, G to I). We further confirmed the expression of these hypoxia-adaptive markers in T cells in biopsy samples using confocal microscopy (Fig. 6, J to L), whereas these markers were only stained a portion of renal tubular cells from noninflamed biopsies, with less fluorescence intensity compared to the inflamed region (fig. S25). The comparison between human lymphoid tissue (tonsil) and nephritis samples was compatible with our findings that up-regulation of hypoxia-adaptive pathways exists in renal-infiltrating T cells in murine lupus compared to splenic T cells and support the idea that pharmacologic HIF-1α blockade should be considered as a therapeutic option in treatment of lupus nephritis and, more generally, in autoimmune tissue damage.

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