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Expression of CD30 and OX40 is indispensable for the proliferation of disease-mediating CD4+ T cells CD90.2 CD30OX40 cells showed a significant decrease in proliferation(Figure 4p and q) and activation(Figure 4r and s). The proliferation of CD30OX40- CD4+ T cells stimulated with Concanavalin A(Figure 6a and b)or phytohemagglutinin(data not shown) was equally reduced by 50% compared with CD4+T cells from WTmice, although this difference did not reach statistical significance because of some outliers. Positivity for cleaved caspase3 in lymph nodes was comparable in CD4+ T cells of healthy and nephritic WT and CD30OX40~-mice(Figure 6c).Ki-67 was sparse in CD4+ T mice; however, their cells of healthy WT and CD30OX40-r were pronounced Ki-67 positivity in lymph nodes of nephritic WT mice(Figure 6d). Lymph nodes of nephritic CD30OX40-/ mice had a staining pattern comparable to healthy mice(Figure 6d). CD30OX40-/- nephritic mice showed lower percentages of proliferating Ki-67+ central memory cells, effector T cells, and Tregs(Figure 6e) in the lymph node.

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Treatment with αCD30LcOX40L antibodies ameliorates NTS indexes

The therapeutic potential of targeting CD30 and OX40 was evaluated by treating mice with aCD30LxOX40L, aCD30L, or aOX40L antibodies starting 3 days before NTS induction. There was a reduction in albuminuria(Figure 7a), PASscores, crescent scores, and tubular casts(Figure 7b,c, and e) in mice treated with xCD30LaOX40L antibodies compared with isotype-treated mice(Figure7a and b). We found a significant decrease of intraluminal thrombi score(Figure 7d) and acute tubular injury score (Figure 7f) in mice preemptively treated with xCD30LaOX40L antibodies compared with isotype-treated mice, whereas no effect was observed after a single blockade of the ligands. Kidney-infiltrating CD4+ T cells and neutrophils(Figure 7g, h, and j) were significantly decreased in mice treated with aCD30LxOX40L antibodies. No changes in numbers of kidney-infiltrating CD8+ T cells and CD68+macrophages(Figure 7i and k) were detected.

If treatment with xCD30LxOX40L antibodies was initiated 3 days after established disease, albuminuria and glomerular PAS scores tended to decrease, but no difference in serum BUN was detected (Supplementary Figure7A-C). Herein, infiltration of renal CD4+ T cells and neutrophils was also significantly decreased (Supplementary Figure S7F). There was no difference in total CD4+ T-cell numbers in the peripheral blood of mice treated with either isotype control antibody or aCD30LaOX40L antibodies (Supplementary Figure 7G) or in the expression of CD44 and CD62L in CD4+T cells in lymph nodes(Supplementary Figure 6H). Also, Tregs were found in comparable numbers in both groups (data not shown). Th1, Th17, and Treg markers on mRNA level were comparable in lymph nodes of mice treated with either isotype control antibody or xCD30LaOX40L anti-bodies(Supplementary Figure S7I). When splenic cells of nephritic mice treated with aCD30LxOX40L antibodies were restimulated with rabbit IgG, however, a significantly reduced proliferation capacity of CD4+ T cells was found compared with isotype-treated mice, without apoptosis being affected (Figure 7). No differences in C3 titers were detected on kidney slides, and no difference in mouse anti-rabbit IgG was detected in the serum of mice treated with xCD30LoOX4OL starting 3 days after NTS induction compared with isotype control-treated mice(Supplementary Figure S7D and E).

DISCUSSION

In this study, we demonstrate a significant contribution of costimulatory signaling via TNF superfamily members CD30 and OX40 to CD4+ T-cell-dependent murine immune-complex glomerulonephritis. We show that preemptive blockade of CD30 and OX40 ligands significantly ameliorates the disease by induction of peripheral tolerance via a prominent reduction of CD4+ effector T-cell proliferation and defective CCR6-mediated migration of Th17 cells to the kidney.

Based on current knowledge on the involvement of CD30 and OX40 in autoimmunity,° we examined whether these receptors are involved in the pathogenesis of NTS. CD30 and OX40 are upregulated on T cells on antigen encounter. Expression of both CD30 and OX40, as well as CD30L and OX40L, was increased on CD4+ T cells in our NTS model, which has also been found previously in SLE patients.16-20 CD30 and OX40 increased most markedly on Th17 cells and Tregs, whereas CD30L was increased on Tregs and OX40L showed increased expression on both. Interestingly, the ligands CD30L and OX40L were downregulated on dendritic cells, neutrophils, and monocytes during the later course of NTS. We hypothesize that this downregulation occurs counteractively after prolonged activation.

CD30OX40-/mice were found to be completely protected from the disease, whereas NTS phenotype in CD30~and OX40 was comparable to WT controls. Neither healthy CD30OX40-/- mice nor single knockout mice showed changes in leukocyte phenotype or changes in relative cell numbers in secondary lymphoid organs.

Distinct CD4+ T-cell subpopulations, such as Th1 and Th17 cells, have been shown to critically affect NTS via recruitment of macrophages and neutrophils.28,5

Figure 4| CD30OX40-/mice show a Thelper cell 17 (Th17) trafficking defect.After adoptive transfer of CD4* Tcells from CD90.1 wild-type (W) mice and CD90.2 CD30OX407mice in a 1:1 ratio in Rag1mice (n =5 mice), mice were subjected to nephrotoxic serum nephritis and followed up for 4 days.(a-c) Percentages of Thelper cell 1 (Th1), (d-f) T helper cell 2 (Th2),and (g-iTh17 cells of either CD90.1 WT or CD90.2 CD30OX40-/mice were evaluated in (a,b,de,g,h) spleens (c,f,i) and kidneys. Expression of chemokine receptors (k) CXC chemokine(continued)

Figure 5|CD30OX40 deficiency does not affect the differentiation of CD4+ T cells. (a)Representative dot plots and (b) proportions of CD4+T cells with T helper cell 17 (Th17) phenotype after stimulation of CD4* T cells from wild-type (WT) and CD30OX40~7mice under Th17 polarizing conditions (n =5 mice per group). (c) Representative dot plots and(d) proportions of CD4*T cells with the T helper cell 1 (Th1)phenotype after the stimulation of CD4+ T cells from WT and CD30OX40~-mice under Th1 polarizing conditions (n =5 mice per group). All data show mean ± SEM from 2 independent experiments. Ifn, interferon;Il, interleukin.

We demonstrated that protection from disease is CD4+ T-cell dependent by inducing NTS in Rag1-/mice reconstituted with CD4+ T cells. Comparable numbers of CD4+ cells in spleens of both groups early after T-cell transfer excluded defective reconstitution of CD30OX40-- cells. During disease, numbers of CD4+ T cells were significantly reduced in spleens of Rag1- mice receiving CD4+ T cells from CD30OX40~-mice. Rag1-/mice showed significantly milder disease after 14 days when reconstituted with CD30OX40~- CD4+ T cells compared with WT CD4+ T cells. CD30OX40-/- CD4+ T cells(namely, memory cells, effector CD4+ T cells, and Tregs)were found to have a reduced proliferative capacity both in vitro and in vivo in lymph nodes.

Adequate immune responses on encountering an antigen require rapid proliferation, differentiation, and migration of T cells. T-cell proliferation in secondary lymphoid organs is not only associated with responses directed against an antigen, but might also favor autoimmunity when homeostatic proliferation takes place.7 Although numbers of kidney-infiltrating T cells, macrophages, and neutrophils were reduced in CD30OX40-/mice subjected to NTS, total cellularity in lymph nodes was stable. However, T-cell pro-liferation in lymph nodes of nephritic CD30OX40- mice was reduced to the level of healthy WT mice, suggesting a potential reduction of pathogenic T-cell clones while pre-serving steady-state conditions. Thereby, a defect in T-cell repertoire as a cause for reduced kidney infiltrating CD4+cells was excluded.

Although signaling via CD30 and OX40 has been connected to T-cell differentiation,8loss of CD30 and OX40—in our hands—did not negatively affect the potential of CD4+T cells to differentiate toward either a Th1 or a Th17 phenotype under polarizing conditions in vitro. However,

data on the role of OX40 for Th17 promotion seem to be dichotomous.38,40

In line with our in vitro data, adoptively transferred CD90.2 CD30OX40-- cells showed increased percentages of Th1, Th2, and Th17 cells in spleens compared with CD90.1 WT cells. Our finding might be explained by compensatory upregulation of CD28 signaling under the nonphysiologic in vitro and adoptive transfer conditions, where CD30 and OX40 are unavailable on CD4+ cells. Accordingly, CD4+ T cells expressing CD28 were found in significantly increased numbers in lymph nodes of nephritic CD30OX407/- mice compared with WT mice. Cotransferred CD30OX40-/cells had increased percentages of Th1 and Th2 cells in kidneys compared with WT cells, mimicking the increased Th1 and Th2 phenotype of CD4 CD30OX40~-cells in the spleen. These findings are in accordance with increased CXCR3 expression on CD30OX40-- cells in the spleen because CXCR3 is known to mediate Th1 migration to the kid-ney.1 CCR6 mediates Th17 migration to the kidney.2 Herein, the significant decrease of CCR6 expression on CD30OX40-- clls results in reduced migration of CD30OX40- Th17 cells to the kidney and finally decreases tissue injury. Still, we need to keep in mind that absolute numbers of infiltrating CD4+ T cells into the kidney were massively reduced when CD30OX40~/- cells were transferred, which explains the protection of those mice even

Figure 7 | Preemptive treatment with aCD30LoOX40L antibodies reduces histologic damage and disease indexes in nephrotoxic serum nephritis (NTS). Treatment with xCD30LaOX40L antibodies was initiated 3 days before NTS induction. (a) Albuminuria was evaluated in mice treated with isotype, aCD30LxOX40L, αCD30L,or xOX40L after 14 days of NTS (n =5 mice per group),(b) Glomerular periodic acid-Schiff (PAS)score, (c) crescent score, (d) capillary intraluminal thrombi score, (e) tubular casts, and (f) acute tubular injury score were evaluated. Numbers of (g,h)CD4+ T cells, (i) CD8+T cells, (i)neutrophils, and (k) CD68+ macrophages were evaluated.() Splenocytes of nephritic mice treated with aCD30L and aOX40L antibodies (n =4 mice per group) or isotype antibody (n =4)starting 3 days after established disease were challenged with rabbit lgG for 24 hours. Percentages of late apoptotic CD4+ cells (annexin V propidium iodide [PI) and proliferating (Ki-67) CD4*cells are given.Data show (a,l) mean± SEM;(b-g,i-k) medians are indicated by a horizontal line. Data are representative of 2 independent experiments.*P<0.05,**P<0.01. HPF, high-power field. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

though the few infiltrating cells showed a prominent Th1 phenotype.

Tregs limit NTS in regional draining lymph nodes,7,30 but also within the kidney in the prolonged phase of the disease. Odobasic et al. postulated that OX40L enhances Treg function in NTS. Previous data, however, have further shown that even in the absence of Tregs, combined abrogation of CD30 and OX40 signals saved mice from lethal autoimmune disease. In our hands, no alterations were found in percentages of Tregs in nephritic CD30OX40mice compared with WT mice, but effector and memory T cells, as well as Tregs, proliferated less in CD30OX40-- mice. Thus, peripheral tolerance, induced by blocking CD30 and OX40 signaling, seems to be induced by blocking pathogenic T-cell effector clones rather than increasing Treg numbers. Still, further experiments are needed to examine whether Treg function is influenced in our setting.

Preemptive combined application of xCD30L and xOX40L blocking antibodies in mice with NTS demonstrated that CD30 and OX40 costimulatory pathway blockade re-duces renal albumin excretion and, most important, histopathologic indexes as well as cell infiltration of CD4+ T cells and neutrophils into the kidney. When combined antibody blockade was started 3 days after disease onset, disease indexes were reduced as well, although effects were not as pronounced. Total CD4blood and lymph node T cells did not differ in numbers, and proportions of effector T cells, central memory T cells, effector memory T cells, and naive T cells were comparable between treated and untreated mice. This is important for a possible future clinical application because it shows that systemic immune response is not altered. Thereby, the costimulatory blockade is an attractive therapeutic option in T-cell-mediated autoimmune diseases. This therapeutic approach has the advantage of selectively blocking the expansion and migration of antigen-specific T cells, thereby leading to tolerance against foreign and self-antigens. 4-45"

Separate blockade of either CD30L or OX40L using antibodies preemptively in nephritic mic did not show significant amelioration, as seen with double blockade of CD30L and OX40L. As for the role of OX40, diverging results have been found in experimental models of nephritis. Although in line with our findings, agonistic OX40 monoclonal antibodies exacerbated renal disease in a mouse model of SLE," a protective effect of OX40L was reported recently in crescentic glomerulonephritis. In analogy to previous findings, CD30 and OX40 appear to work synergistically in our NTS model. Still, also experimental setup and choice of models, such as the antiserum host, timing, and amount of antibody application, might contribute to different outcomes.

This study proposes the potential of combined blockade of the CD30 and OX40 costimulatory pathways as a novel therapeutic strategy to achieve peripheral tolerance in autoimmunity by reducing pathogenic hyperproliferation in secondary lymphoid organs and migration of disease-promoting Th17 cells. In addition, it is likely that combined CD30 and OX40 blockade would result in fewer life-threatening infections than traditional immunosuppressive therapies.

DISCLOSURE

All the authors declared no competing interests.

ACKNOWLEDGMENTS

This work was supported by the Austrian Science Funds to PE (P27537-B26) and the Austrian National Bank OeNB (number 17212 to KE) as well as the PhD programs MOL MED and MOLIN (W1241)of the Medical University of Graz, which are supported by the Austrian Science Funds. KS is funded by an investigator-initiated research grant by Baxter. Visual abstract was created with BioRender.com.

AUTHOR CONTRIBUTIONS

KA, AHK, PE, ARR, ALl, HY, JPL, and KE designed the study; KA, AHK, AAM, DJC, IA, CS, KAK, KS, MS, and KE performed experiments; KA, AHK, DJC, and KE analyzed the data; KA made the figures; KA and KE drafted and revised the article; all authors approved the final version of the article.

SUPPLEMENTARY MATERIAL Supplementary File (PDF)

Figure S1.Expression of CD30, CD30L, OX40, and OX40L in spleen, lymph node, and kidneys.

Figure S2. Leukocyte phenotype of healthy CD30-/-, OX40-/, and CD30OX40-/-

Figure S3. Tregs in lymph nodes of WT and CD30OX40-/mice. Figure S4. Purity of isolated CD4+ T cells.

Figure S5.Rag1-mice are reconstituted efficiently with CD4+Tcells from WT and CD30OX40-/- mice.

Figure S6.Gating strategies for Th1 and Th17 flow cytometry, Figure S7. Treatment start with xCD30LxOX40L antibodies after established disease ameliorates renal cell infiltration in NTS. Supplementary Methods.

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