Blockade of tumor necrosis factor superfamily members CD30 and OX40 abrogates disease activity in murine immune-mediated glomerulonephritis
Authors: Katharina Artinger, Alexander H. Kirsch, Agnes A. Mooslechner, Daniel J. Cooper, Ida Aringer, Max Schuller, Corinna Schabhüttl, Konstantin A. Klötzer, Kerstin Schweighofer, Philipp Eller, Hideo Yagita, Anna L. Illert, Alexander R. Rosenkranz, Peter J. Lane, Kathrin Eller
Journal: Kidney International (2021)

Co-stimulation is a prerequisite for pathogenic activity in T cell-mediated diseases and has been demonstrated to achieve tolerance in organ-specific autoimmunity as a therapeutic target. Here, we evaluated the involvement of the tumor necrosis factor family members CD30 and OX40 in immune-complex mediated kidney disease. In vitro stimulation and proliferation studies were performed with CD4+ cells from wild type and CD30/OX40 double knock-out (CD30OX40-/-) mice. In vivo studies were performed by induction of nephrotoxic serum nephritis in wild type, CD30OX40- /- , CD30-/-, OX40-/-, reconstituted Rag1-/- and C57Bl/6J mice treated with αCD30L αOX40L antibodies. CD30, OX40 and their ligands were upregulated on various leukocytes in nephrotoxic serum nephritis. CD30OX40-/- mice, but not CD30-/- or OX40-/- mice were protected from nephrotoxic serum nephritis. Similar protection was found in Rag1-/- mice injected with CD4+ T cells from CD30OX40-/- mice compared to Rag1-/- mice injected with CD4+ T cells from wild type mice. Furthermore, CD4+ T cells deficient in CD30OX40-/- displayed decreased expression of CCR6 in vivo. CD30OX40-/- cells were fully capable of differentiating into disease mediating T helper cell subsets, but showed significantly decreased levels of proliferation in vivo and in vitro compared to wild type cells. Blocking antibodies against CD30L and OX40L ameliorated nephrotoxic serum nephritis without affecting pan-effector or memory T cell populations. Thus, our results indicate disease promotion via CD30 and OX40 signaling due to facilitation of exaggerated T cell proliferation and migration of T helper 17 cells in nephrotoxic serum nephritis. Hence, co-stimulation blockade targeting the CD30 and OX40 signaling pathways may provide a novel therapeutic strategy in autoimmune kidney disease. Co-stimulation is a prerequisite for pathogenic activity in T cell-mediated diseases and has been demonstrated to achieve tolerance in organ-specific autoimmunity as a therapeutic target. Here, we evaluated the involvement of the tumor necrosis factor family members CD30 and OX40 in immune-complex mediated kidney disease. In vitro stimulation and proliferation studies were performed with CD4+ cells from wild type and CD30/OX40 double knock-out (CD30OX40-/-) mice. In vivo studies were performed by induction of nephrotoxic serum nephritis in wild type, CD30OX40- /- , CD30-/-, OX40-/-, reconstituted Rag1-/- and C57Bl/6J mice treated with αCD30L αOX40L antibodies. CD30, OX40 and their ligands were upregulated on various leukocytes in nephrotoxic serum nephritis. CD30OX40-/- mice, but not CD30-/- or OX40-/- mice were protected from nephrotoxic serum nephritis. Similar protection was found in Rag1-/- mice injected with CD4+ T cells from CD30OX40-/- mice compared to Rag1-/- mice injected with CD4+ T cells from wild type mice. Furthermore, CD4+ T cells deficient in CD30OX40-/- displayed decreased expression of CCR6 in vivo. CD30OX40-/- cells were fully capable of differentiating into disease mediating T helper cell subsets, but showed significantly decreased levels of proliferation in vivo and in vitro compared to wild type cells. Blocking antibodies against CD30L and OX40L ameliorated nephrotoxic serum nephritis without affecting pan-effector or memory T cell populations. Thus, our results indicate disease promotion via CD30 and OX40 signaling due to facilitation of exaggerated T cell proliferation and migration of T helper 17 cells in nephrotoxic serum nephritis. Hence, co-stimulation blockade targeting the CD30 and OX40 signaling pathways may provide a novel therapeutic strategy in autoimmune kidney disease. Translational StatementThis article describes the applicability of CD30 and OX40 signaling pathways as future therapeutic targets in immune complex–mediated glomerular disease using the murine model of nephrotoxic serum nephritis. The combined blockade of the CD30 and OX40 costimulatory pathways is a way to achieve peripheral tolerance in autoimmunity by reducing pathogenic hyperproliferation in secondary lymphoid organs and the migration of pathogenic T helper cell 17 cells to the kidney. Since traditional treatment options were accompanied by severe adverse effects, combined CD30 and OX40 blockade would possibly result in fewer life-threatening infections compared with traditional immunosuppressive therapies. This article describes the applicability of CD30 and OX40 signaling pathways as future therapeutic targets in immune complex–mediated glomerular disease using the murine model of nephrotoxic serum nephritis. The combined blockade of the CD30 and OX40 costimulatory pathways is a way to achieve peripheral tolerance in autoimmunity by reducing pathogenic hyperproliferation in secondary lymphoid organs and the migration of pathogenic T helper cell 17 cells to the kidney. Since traditional treatment options were accompanied by severe adverse effects, combined CD30 and OX40 blockade would possibly result in fewer life-threatening infections compared with traditional immunosuppressive therapies. Costimulatory signaling is a prerequisite for T-cell activation, differentiation, effector function, and survival.1Chen L. Flies D.B. Molecular mechanisms of T cell co-stimulation and co-inhibition.Nat Rev Immunol. 2013; 13: 227-242Crossref PubMed Scopus (1558) Google Scholar Blocking costimulation has been identified as a strategy to achieve immune tolerance.2Adams A.B. Ford M.L. Larsen C.P. 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T-cell co-stimulatory blockade in kidney transplantation: back to the bench.Kidney Int Suppl. 2011; 1: 25-30Abstract Full Text Full Text PDF Scopus (2) Google Scholar OX40L blockade is currently under clinical evaluation25Gauvreau G.M. Boulet L.-P. Cockcroft D.W. et al.OX40L blockade and allergen-induced airway responses in subjects with mild asthma.Clin Exp Allergy. 2014; 44: 29-37Crossref PubMed Scopus (74) Google Scholar; however, combined targeting of CD30 and OX40 has not been investigated comprehensively so far. CD4-dependent autoimmunity is critically reliant on the interaction of both receptor-ligand pairs, demonstrated by the necessity of combined abrogation of CD30 and OX40 signaling to protect forkhead box P3 (FoxP3)–deficient mice from developing early lethal autoimmune disease.6Gaspal F.M. Withers D. Saini M. et al.Abrogation of CD30 and OX40 signals prevents autoimmune disease in FoxP3-deficient mice.J Exp Med. 2011; 208: 1579-1584Crossref PubMed Scopus (44) Google Scholar,26Nawaf M.G. Ulvmar M.H. Withers D.R. et al.Concurrent OX40 and CD30 ligand blockade abrogates the CD4-driven autoimmunity associated with CTLA4 and PD1 blockade while preserving excellent anti-CD8 tumor immunity.J Immunol. 2017; 199: 974-981Crossref PubMed Scopus (7) Google Scholar Nephrotoxic serum nephritis (NTS) is a murine model of immune-complex glomerulonephritis that has been shown to be strictly dependent on T cells, including T helper cell 1 (Th1) and Th17 cells. Antigen presentation in this model primarily takes place in secondary lymphoid organs, but also locally within the kidney.27Eller K. Weber T. Pruenster M. et al.CCR7 deficiency exacerbates injury in acute nephritis due to aberrant localization of regulatory T cells.J Am Soc Nephrol. 2010; 21: 42-52Crossref PubMed Scopus (38) Google Scholar,28Kurts C. Panzer U. Anders H.-J. et al.The immune system and kidney disease: basic concepts and clinical implications.Nat Rev Immunol. 2013; 13: 738-753Crossref PubMed Scopus (382) Google Scholar Thus, we hypothesized that activation of the CD30 and OX40 costimulatory pathways presumably promotes NTS. CD30OX40–/– double knockout mice deficient in CD30 and OX40 were generated as described previously.5Gaspal F.M.C. Kim M.-Y. McConnell F.M. et al.Mice deficient in OX40 and CD30 signals lack memory antibody responses because of deficient CD4 T cell memory.J Immunol. 2005; 174: 3891-3896Crossref PubMed Scopus (135) Google Scholar CD30OX40–/– and CD30OX40+/+ littermate controls, as well as CD30–/– and OX40–/– were bred in house. CD30–/– mice were also provided by Dr. Illert, Freiburg, Germany. C57BL/6J mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Rag1–/– and B6.Thy1.1 mice were obtained from Jackson Laboratory (Bar Harbor, ME). All mice were on C57BL/6J background. NTS was induced as described previously.29Rosenkranz A.R. Mendrick D.L. Cotran R.S. et al.P-selectin deficiency exacerbates experimental glomerulonephritis: a protective role for endothelial P-selectin in inflammation.J Clin Invest. 1999; 103: 649-659Crossref PubMed Scopus (103) Google Scholar Briefly, mice were s.c. preimmunized with 2 mg/ml rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) dissolved in incomplete Freund adjuvant (Sigma-Aldrich, St. Louis, MO) with desiccated, nonviable Mycobacterium tuberculosis H37a (Difco Laboratories, Detroit, MI) 3 days before administration of nephrotoxic rabbit anti-mouse serum. Evaluation was done after 4 or 14 days after injection of the NTS serum, as indicated. Rag1–/– mice were injected with 1.5 × 106 CD4+ T cells from spleens of either wild-type (WT) or CD30OX40–/– mice or CD90.1 WT cells, and CD90.2 knockout cells were adoptively transferred in a 1:1 ratio. One day after adoptive transfer, NTS was induced and evaluated after 4 or 14 days. CD4+ T cells were isolated from spleens using the mouse CD4+ cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Nephritic C57BL/6J mice were treated with blocking antibodies against mouse OX40L (clone RM134L) and CD30L (clone RM153) either in combination or alone starting on the day of immunization or 3 days after NTS induction. Mice were injected i.p. with either 0.5 mg α-OX40L and/or 0.5 mg α-CD30L monoclonal antibodies or 1 mg control IgG (Sigma-Aldrich) 3 times a week. A double-sandwich enzyme-linked immunosorbent assay (Abcam, Cambridge, MA) was used for determination of urinary albumin excretion, as reported previously (detection range, 0.781–200 ng/ml).30Wolf D. Hochegger K. Wolf A.M. et al.CD4 + CD25 + regulatory T cells inhibit experimental anti-glomerular basement membrane glomerulonephritis in mice.J Am Soc Nephrol. 2005; 16: 1360-1370Crossref PubMed Scopus (157) Google Scholar Urinary creatinine was evaluated photometrically using a commercially available picric acid–based kit (Sigma-Aldrich). Cytokine detection in supernatants was performed as indicated in the Supplementary Methods. Plates were incubated overnight with 100 μg/ml rabbit IgG (Jackson ImmunoResearch Laboratories). Thereafter, serum was incubated in serial-doubling dilutions for the detection of circulating mouse anti-rabbit Ig. Using horseradish peroxidase–conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), mouse anti-rabbit IgG was detected. Blood urea nitrogen (BUN) levels in serum were obtained using a commercially available BUN colorimetric detection kit (Invitrogen). Evaluation of periodic acid–Schiff (PAS)–stained kidney sections was done as described previously31Kitching A.R. Holdsworth S.R. Ploplis V.A. et al.Plasminogen and plasminogen activators protect against renal injury in crescentic glomerulonephritis.J Exp Med. 1997; 185: 963-968Crossref PubMed Scopus (158) Google Scholar,32Chen J. Chen J.-K. Conway E.M. et al.Survivin mediates renal proximal tubule recovery from AKI.J Am Soc Nephrol. 2013; 24: 2023-2033Crossref PubMed Scopus (59) Google Scholar and in the Supplementary Methods. A 3-layered immunoperoxidase staining technique was used for the assessment of tissue infiltrating cells (Supplementary Methods). Frozen tissue samples were stained with fluorescently labeled antibodies (Supplementary Methods) and evaluated on an LSM510 meta (Zeiss, Oberkochen, Germany). Red blood cell lysis using the 1× RBC Lysis Buffer Solution (eBioscience) was performed with blood samples before a single-cell suspension was obtained from blood, spleen, and lymph nodes using 70-μm cell strainers. Cell isolation from kidneys was performed using Percoll density gradient centrifugation after digestion of finely minced kidneys in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific) supplemented with collagenase D and DNAse I at 37 °C, as described previously.33Paust H.-J. Turner J.-E. Steinmetz O.M. et al.The IL-23/Th17 axis contributes to renal injury in experimental glomerulonephritis.J Am Soc Nephrol. 2009; 20: 969-979Crossref PubMed Scopus (186) Google Scholar Flow cytometry stainings and evaluations were performed as described in the Supplementary Methods. CD4+ T cells were incubated with Celltrace Violet (CellTrace Violet Cell Proliferation Kit; Invitrogen), according to the manufacturer's protocol, and seeded at 2.5 × 106/ml for incubation with either 5 μg/ml concanavalin-A (Sigma-Aldrich) or 5 μg/ml phytohemagglutinin (Sigma-Aldrich). After 7 days, flow cytometry was used to evaluate the intensity of Celltrace Violet in these cells. Splenocytes from nephritic mice treated with isotype antibody or αCD30LαOX40L antibodies were harvested after 14 days of NTS. Splenocytes were seeded at 2 × 106/ml in rabbit-IgG precoated wells and restimulated for 24 hours before flow cytometry. Splenocytes from nephritic WT and CD30OX40–/– mice (day 14) were seeded at 2 × 106/ml and stimulated with αCD3 (5 μg/ml) and αCD28 (1 μg/ml) (both from eBioscience) for 3 days before evaluation of cell culture supernatant. CD4+CD62L+ T cells were isolated from spleens using the CD4+CD62L+ isolation kit II (Miltenyi Biotec) and stimulated as indicated in the Supplementary Methods. Total RNA was extracted from lymph nodes and kidneys using TRI Reagent (Sigma-Aldrich) and subsequently cDNA was synthesized by using Superscript III Transcription Kit (Invitrogen) and random primers (Invitrogen) for reverse transcription of 2 μg of total RNA. Real-time polymerase chain reaction was performed in duplicate on a CFX96 Real-Time System (BioRad, Hercules, CA). TaqMan gene expression assays (Applied Biosystems, Foster City, CA) and SYBR green (BioRad) were used (Supplementary Methods). All statistical evaluations (Supplementary Methods) were performed using GraphPad Prism 6.0 for Macintosh (GraphPad Software, La Jolla, CA). All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the Austrian Ministry (BMWFW-66.010/0054-WF/V/3b/2015). CD30 and OX40 expression increased on splenic CD4+ T cells over the time course of NTS (Figure 1a). Both CD30L and OX40L expression levels were significantly increased on splenic CD4+ T cells of nephritic mice after 7 and 14 days (Figure 1a). On CD4+CD25+Foxp3+ regulatory T cells (Tregs), a similar increase of CD30, CD30L, and OX40 expression was found over time (Supplementary Figure S1A). CD30, OX40, and OX40L were significantly increased on Th17 cells (Supplementary Figure S1D) after 14 days of NTS compared with healthy mice. Also, CD30 and OX40 were both significantly increased on CD8+ T cells starting 12 hours after NTS induction compared with healthy mice (Figure 1b). B cells showed a similar expression profile as CD4+ T cells (Figure 1c). OX40L was downregulated already after 12 hours of NTS and remained decreased thereafter on dendritic cells (Figure 1d). Similarly, CD30L and OX40L were downregulated on CD11b+Ly6G+SSChi neutrophils, Ly6G–CD115+Ly6C+, and Ly6G–CD115+Ly6C– monocytes over the time course of NTS (Figure 1e–g). On neutrophils, dendritic cell and monocyte CD30 and OX40 tended to increase in NTS (Figure 1d–g). CD30L was increasingly expressed on type 2 innate lymphoid cells 12 hours after NTS induction, and CD30L and OX40L were also increased after 14 days of NTS (Supplementary Figure S1B). Numbers of leukocytes expressing CD30, CD30L, OX40, and OX40L increased in NTS, which was in line with findings on splenic cells (Supplementary Figure S1E–G). Cd30 and Ox40l mRNA expression was not detectable in kidneys after 7 and 14 days of NTS (Supplementary Figure S1H). However, Cd30l mRNA was increased after 7 and 14 days of NTS, whereas Ox40 mRNA expression remained unchanged after 14 days of NTS in kidneys (Supplementary Figure S1H). We found decreased albuminuria and BUN in nephritic CD30OX40–/– mice compared with WT controls (Figure 2a and b), whereas albuminuria in CD30–/– and OX40–/– mice was comparable to WT control mice (Figure 2a). A significant decrease was also observed in kidney PAS positivity, crescent formation, intraluminar thrombi score, tubular cast formation, and acute tubular injury score in CD30OX40–/– mice compared with WT mice (Figure 2c–i). All these histologic features were not significantly altered in single knockout mice compared with WT controls (Figure 2c and f–i). We found significantly lower numbers of CD68+ macrophages in kidneys of CD30OX40–/– mice compared with WT controls (Figure 2j). There was a trend toward reduced numbers of kidney-infiltrating CD68+ macrophages in CD30–/– and OX40–/– single knockout mice (Figure 2j). Neutrophils and CD8+ T cells were found in significantly reduced numbers in kidneys of CD30OX40–/– mice compared with nephritic WT controls, and CD30–/– as well as OX40–/– mice showed lower numbers of neutrophils and C8+ T cells infiltrating kidneys (Figure 2k and m). CD4+ T cells were found in significantly lower numbers in nephritic kidneys of CD30OX40–/– (Figure 2l). Flow cytometry of kidneys revealed similar results of reduced infiltrating cell numbers (Figure 2p and q). Of note, naïve single and double knockout mice did not show any differences in splenic leukocyte composition compared with healthy WT mice, when CD4+ and CD8+ T cells, neutrophils, and monocytes were evaluated (Supplementary Figure S2). Icos and Gitr expression was reduced in kidneys of nephritic CD30OX40–/– mice (Supplementary Figure S3B). B-cell responses, as measured by mouse anti-rabbit IgG, were reduced in CD30OX40–/– mice compared with WT controls (Figure 2r). Flow cytometry revealed no difference in total lymph node and spleen cellularity (Figure 2n and o) or in numbers of CD4+ cells. Interestingly, we also did not detect any differences in Tregs between WT and CD30OX40–/– mice (Supplementary Figure S3). Of note, cytokines (TNF-α, interleukin-6, interferon-γ, interleukin-17, interleukin-4, and interleukin-10) measured in supernatants of restimulated splenocytes isolated from WT and CD30OX40–/– mice 14 days after NTS did not show a difference (data not shown). To study the role of CD30 and OX40 expression on CD4+ cells in NTS, Rag1–/– were injected with CD4+ cells (Supplementary Figure S4) from spleens of either WT or CD30OX40–/– mice. Total splenic CD4+ cell counts and rates of dead cells were comparable on day 4 after NTS induction (Supplementary Figure S5). Although Rag1–/– mice receiving CD4+ cells from WT mice developed significant albuminuria and BUN after 14 days of NTS, Rag1–/– mice reconstituted with CD4+ cells from CD30OX40–/– mice (Figure 3a) did not. Histologic damage was significantly reduced in Rag1–/– mice receiving CD4+ T cells from CD30OX40–/– mice when compared with WT cells, as determined by PAS scoring, crescent score, intraluminar thrombi score, acute tubular injury score, and tubular casts (Figure 3b–g). Cells infiltrating kidneys of Rag1–/– mice, including CD4+ cells, CD68+ macrophages, and Ly6G+ neutrophils, were also significantly reduced if transferred CD4+ cells did not express CD30 and OX40 (Figure 3h–k). CD4+ T cells isolated from WT mice expressing CD90.1 and CD30OX40–/– mice expressing CD90.2 were further adoptively transferred to Rag1–/– mice in a 1:1 ratio. Herein, absolute numbers of CD90.2 expressing CD30OX40–/– CD4+ T cells were decreased in the spleen compared with CD90.1 WT CD4+ T cells (Figure 4t and u), but percentages of Ifn-γ (Figure 4a and b), Il4 (Figure 4d and e), and Il17 (Figure 4g and h) producing cells were significantly higher in CD90.2 expressing knockout cells. However, at the same time, decreased percentages of CD90.2 knockout cells were found to have a Th17 phenotype in kidneys compared with CD90.1 WT cells (Figure 4i), whereas the opposite was found for Th1 and T helper cell 2 (Th2) cells in kidneys (Figure 4c and f). Still, absolute numbers of transferred CD90.2 knockout cells in the kidney were significantly lower compared with CD90.1 WT cells (data not shown).Figure 4CD30OX40–/– mice show a T helper cell 17 (Th17) trafficking defect. After adoptive transfer of CD4+ T cells from CD90.1 wild-type (WT) mice and CD90.2 CD30OX40–/– mice in a 1:1 ratio in Rag1–/– mice (n = 5 mice), mice were subjected to nephrotoxic serum nephritis and followed up for 4 days. (a–c) Percentages of T helper cell 1 (Th1), (d–f) T helper cell 2 (Th2), and (g–i) Th17 cells of either CD90.1 WT or CD90.2 CD30OX40–/– mice were evaluated in (a,b,d,e,g,h) spleens (c,f,i) and kidneys. Expression of chemokine receptors (j,k) CXC chemokine receptor (CXCR) 3, (l,m) CXCR6, and (n,o) CC chemokine receptor (CCR) 6 on CD4+ splenic cells from CD90.1 WT or CD90.2 CD30OX40–/– mice is shown. (p,q) Ki-67 and (r,s) CD69 expression of CD90.1 WT and CD90.2 CD30OX40–/– cells was evaluated. (t,u) Total numbers of CD4+ CD90.1 WT cells and CD90.2 CD30OX40–/– cells in lymph nodes and spleens. All data show mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001. Ifn, interferon; Il, interleukin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) CXC chemokine receptor (CXCR) 3 (Figure 4j and k) was increased on CD90.2 CD30OX40–/– cells compared with CD90.1 WT cells, whereas no differences were detected for CXCR6 (Figure 4l and m) expression. More important, we found a significant decrease of CC chemokine receptor (CCR) 6 on CD90.2 knockout cells (Figure 4n and o). In a next step, naïve CD4+ T cells were stimulated in vitro under Th1 or Th17 differentiation conditions (Supplementary Figure S6). We observed a trend toward a greater potential for Th17 (Figure 5a and b) and Th1 differ