The pleiotropic cytokine IL-21 is implicated in the pathogenesis of human systemic lupus erythematosus by polymorphisms in the molecule and its receptor (IL-21R). The systemic lupus erythematosus-like autoimmune disease of BXSB.Yaa mice is critically dependent on IL-21 signaling, providing a model for understanding IL-21/IL-21R signaling in lupus pathogenesis. In this study, we generated BXSB.Yaa mice selectively deficient in IL-21R on B cells, on all T cells, or on CD8+ T cells alone and examined the effects on disease. We found that IL-21 signaling to B cells is essential for the development of all classical disease manifestations, but that IL-21 signaling also supports the expansion of central memory, CD8+ suppressor cells and broadly represses the cytokine activity of CD4+ T cells. These results indicate that IL-21 has both disease-promoting and disease-suppressive effects in the autoimmune disease of BXSB.Yaa mice.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease in which a loss of tolerance to nucleic acids results in an abundance of autoantibodies that form pathogenic immune complexes and cause multisystem clinical disease. CD4+ T cells and B cells acting in a cooperative manner are critical drivers of all forms of SLE, but debate continues over their mechanisms of action. Further complicating the issue is the recognition that regulatory cells, including CD4+ regulatory T cells (Tregs) and CD8+ Tregs/suppressor T cells (Ts), are able to slow the development of disease. Thus, the balance between disease drivers and controllers determines the course and outcomes of SLE.

IL-21, a member of the common γ-chain cytokine family, has gained increasing interest because of its potent effects on normal immunological homeostasis and disease (reviewed in Refs. 1, 2). This cytokine is a major product of CXCR5hiICOShiCD4+ follicular Th cells (TFH) as well as a CD4+CXCR5lo/−ICOShi Th subset, termed extrafollicular Th cells (TEFH), that is involved in extrafollicular differentiation of plasmablasts and plasma cells (35). IL-21 is emerging as a critical driver for the development of many forms of autoimmune disease, including SLE (1, 2, 610). IL-21 signals through its receptor, IL-21R, which is expressed by many types of lymphoid and myeloid cells (1, 2, 11). In keeping with this expression pattern, there is evidence for the need for both CD4+ T cell–intrinsic and B cell–intrinsic IL-21 signaling in the promotion of SLE-like autoimmune diseases (12). IL-21 can act in an autocrine manner to promote the differentiation of naive CD4+ T cells to TFH (1315) and suppress the functions of CD4+ Tregs (16). IL-21 can also potently promote Ag-experienced B cells through follicular and extrafollicular development, class-switch recombination, and plasma cell differentiation (1722). IL-21 is also a powerful inducer of the cytotoxic arm of immunity, stimulating the maturation and enhancing the activity of NK and CD8+ T cells, as well as acting with IL-15 to support the maintenance of memory CD8+ T cells (2326). The downstream consequences of IL-21 signaling can therefore be complex.

BXSB male mice carrying the Y-linked autoimmune accelerator mutation Yaa recapitulate key facets of severe human SLE (4, 2730). Yaa is a duplication of genes, most importantly TLR7 (Tlr7), that normally reside on the X chromosome but were translocated to the Y chromosome (31, 32). Therefore, male mice bearing Yaa express two copies of Tlr7, whereas females express only one copy due to X inactivation. Heightened TLR7 signaling in response to ligation of ssRNA is a primary driver of SLE in BXSB.Yaa mice (31, 32). In cooperation with autosomal SLE predisposition alleles, TLR7 signaling promotes interactions among dendritic cells, activated B cells, and CD4+ T cells, ultimately leading to the production of copious amounts of anti-nuclear Abs (ANA), resulting in severe clinical disease (31, 33). Recent studies have also identified an expansion of CD8+ T cells with a central memory (CM) phenotype that act to substantially retard the onset and severity of disease in BXSB.Yaa and B6.Yaa mice (27, 34).

The pathogenesis of disease in BXSB.Yaa mice is characterized by an expansion of TFH as well as TEFH, both of which express high levels of IL-21 (4). In contrast, Th17 cells, which have been identified as another source of IL-21 (35, 36), are not expanded in this strain (4, 27). BXSB.Yaa mice lacking the IL-21R are highly resistant to lupus (4, 27). Despite this, the administration of an IL-21R–Fc fusion protein known to bind and remove IL-21 was not an effective treatment (37). Early in the course of treatment, some mice developed more severe disease but, as treatment continued, the disease became less severe and a protective effect was observed. It was suggested that the biphasic response to this intervention could be ascribed to an early negative impact of reduced IL-21 signaling on CD8+ Ts, whereas the later protective effect could be attributed to limiting IL-21–driven B cell proliferation and differentiation. In this study, we directly address the lymphocyte populations targeted by IL-21 that drive or restrict the lupus-like disease of these mice.

Mice were bred and maintained in a specific pathogen-free mouse colony at The Jackson Laboratory, including BXSB/MpJ (designated BXSB.Yaa) and BXSB.B6-Yaa+/J (designated BXSB.B6Y). All experiments were performed under protocol 01022 approved by the Institutional Animal Care and Use Committee. BXSB.Yaa mice carrying the Il21r null allele were bred to N11 (4). BXSB-Cd8a−/− mice were as described (27). B cell–deficient BXSB-Ighmtm1Cgn (38) and lymphocyte-deficient BXSB.Rag1tm1Mom mice (both N11) were produced similarly. T cell–deficient BXSB-Tcratm1Mjo/TheoJ mice (N8) were generously provided by Dwight Kono (Scripps Institute) (39).

Mixed bone marrow (BM) chimeras were generated and validated as described in Supplemental Table I and Supplemental Fig. 1. Recipient mice were irradiated twice with 550–600 cGY 3 to 4 h apart and were then injected i.v. with a 1:1 mixture of 2.5 × 106 BM cells from each donor.

Flow cytometry was performed using established procedures and Abs against B220, CD8, ICOS, CD11b, IL-21R, programmed cell death 1 (PD-1) (103227, 100730, 313516, 101216, 131906, and 109109; BioLegend), CD4 (MCD0430; Invitrogen), CD44, CD62L (27-0441, 25-0621; eBioscience), and CD122 and FAS (552818, 557653; BD Pharmingen). Studies were performed on a two-laser/four-color FACSCalibur or a four-laser/13-color BD LSRII analytical cytometer (both from BD Biosciences) and analyzed with FlowJo software (Tree Star).

IgG1, IgG2b, IgG2c, and IgM serum levels were quantified by ELISA techniques with data expressed as concentration of Ig per milliliter by interpolation using a standard curve based on the titration of purified mouse Igs as described (40). Anti-DNA Abs were quantified by ELISA as described (41). ANA abundance was determined by their binding to Hep-2 cells (Antibodies, Inc.) using 15 μl 1:40 dilution of serum following the supplier’s protocol. Intensity was quantified based on a scale of 0–4 as described (4, 27).

Formalin-fixed, paraffin-embedded kidneys were stained with H&E and periodic acid-Schiff. The sections were scored in a blinded manner using criteria described in Weening et al. (42). For immunohistochemistry, kidney sections were incubated with anti-IgG (1030-01; Southern Biotechnology Associates) as previously described (27). Slides were scanned on a Nanozoomer slide scanner at ×40, and images were obtained using NDP.view2 software (Hamamatsu).

Spleen cells were isolated and stained with anti-CD4, IL-21R, and B220 (clones denoted above), and propidium iodine was used to assess viability. Using an FACSAria (BD Biosciences), cells were gated and sorted for viable B220CD4+IL-21R+ and B220CD4+IL-21R. Total RNA was extracted with the RNeasy Micro kit (Qiagen) followed by cDNA synthesis using the QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer’s protocol. The cDNA was combined with SYBR Green RT-PCR master mix (Applied Biosystems) and oligonucleotide primers. Oligonucleotide primers for RT-quantitative PCR (qPCR): Il2, forward 5′-AGCAGGCCACAGAATTGAAAG-3′ and reverse 5′-CTCATCATCGAATTGGCACTCA-3′; Il6, forward 5′-TAGTCCTTCCTACCCCAATTT CC-3′ and reverse 5′-TTGGTCCTTAGCCACTCCTTC-3′; IL10, forward 5′-CCAAGCCTTATCGGAAATGAT C-3′ and reverse 5′-TTTCTGGGCCATGCTTCTCT-3′; IL21, forward 5′-GAAGATGGCAATGAAAGCCTG T-3′ and reverse 5′-AGGATGTGGGAG AGGAGACTGA-3′; IL21R, forward 5′-CCTTCTCAGGACGCTATGATATCTC-3′ and reverse 5′-CTTGCCCCTCAGCACGTAGT-3′; Ifng, forward 5′-CAAGCGGCTGACTGAACTCA-3′ and reverse 5′-CACTGCAGCTCTGAATGTTTCTTATT-3′′; Tnfa, forward 5′-TATGGCTCAGGGTCCAACTC-3′ and reverse 5′-CTCCCTTTGCAGAACTCAGG-3′; IL17a, forward 5′-GAAGAT GCTGGTGGGTGTGG-3′ and reverse 5′-AGCCGCGGGTCTCTGTTTAG-3′; Stat3, forward 5′-GAC CCGCCAACAAATTAAGA-3′ and reverse 5′-TCGTGGTAAACTGGACACCA-3′; Stat5a, forward 5′-GTGAAGCGCTCAACATGAAA-3′ and reverse 5′-ACTGGGACCAGGACACAGAC-3′; Stat5b, forward 5′-CCAGATGCAGGCCTTGTACG-3′ and reverse 5′-AGGCTTGGCTTTCGATCCAC-3′; 18sRNA, forward 5′-CCGCAGCTAGGAATAATGGAAT-3′ and reverse 5′-CGAACCTCCGACTTT CGTTCT-3′; and Gapdh, forward 5′-TGTGTCCGTCGTGGATCTGA-3′ and reverse 5′-TTGCTGTTG AAGTCGCAGGAG-3′. RT-qPCR gene expression quantification (43) was performed in 384-microwell plates (Applied Biosystems) using ViiA7 Real-Time PCR system (Applied Biosystems).

The two-tailed heteroscedastic Student t test was used for two sample comparisons. The log-rank test was used for survival analysis.

BXSB.Yaa mice represent a unique model of SLE in that their CD4+ T cells express high levels of IL-21, a cytokine absolutely required for the lupus-like disease that characterizes this strain (4, 17, 27). Importantly, however, the cell types responsive to IL-21 in this disease have not been defined. As IL-21 can induce target cells to upregulate their IL-21Rs (44), we quantified levels of IL-21R expression on subpopulations of spleen cells from 6- and 14-wk-old BXSB.Yaa and control BXSB mice carrying a C57BL/6-derived Y chromosome (BXSB.B6Y) (Fig. 1A, 1B). The elevated expression of the IL-21R was seen on all subsets of B220+ B cells examined, including the dominant follicular B cell subset (Fig. 1A, 1B) as well as T1 B cells, marginal zone (MZ) B cells, and germinal center B cells (not shown). In contrast, Yaa was not associated with heightened expression of IL-21R on total CD4+ T cells or the ICOShi subsets that are the primary sources of IL-21. However, Yaa consistently caused the upregulation of IL-21R on CD8+ T cells from mice of both ages. These data suggest that elevated expression of IL-21R on Yaa B cells and CD8+ T cells may position them for heightened responses to IL-21.

FIGURE 1.

Yaa acts early to cause the upregulation of IL-21R on B cells and CD8+ T cells but not on CD4+ T cells. (A) Results compare IL-21R expression of spleen cells from BXSB.Yaa and BXSB.B6Y control mice (n = 5) at 6 and 14 wk of age. The mean fluorescence intensity (MFI) + SEM of spleen cells from IL-21r−/− BXSB controls (<100 MFI in this experiment) is shown. (B) Representative histograms of 14-wk-old BXSB.Yaa and BXSB.B6Y splenic B and T cells. *p < 0.05, **p < 0.01.

FIGURE 1.

Yaa acts early to cause the upregulation of IL-21R on B cells and CD8+ T cells but not on CD4+ T cells. (A) Results compare IL-21R expression of spleen cells from BXSB.Yaa and BXSB.B6Y control mice (n = 5) at 6 and 14 wk of age. The mean fluorescence intensity (MFI) + SEM of spleen cells from IL-21r−/− BXSB controls (<100 MFI in this experiment) is shown. (B) Representative histograms of 14-wk-old BXSB.Yaa and BXSB.B6Y splenic B and T cells. *p < 0.05, **p < 0.01.

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The disease of BXSB.Yaa mice, like other systemic humoral autoimmune disorders, is characterized by expanded numbers of activated B cells, hypergammaglobulinemia, and high levels of circulating autoantibodies clearly defining a pathogenic role for B cells. These mice also develop a characteristic monocytosis and substantially increased numbers of ICOShi TFH/TEFH that are considered to be critical cellular drivers of this disease (4, 27). We first asked if B cells are necessary for development of disease. To do this, we introduced a null allele (Ø) allele of Ighm onto the BXSB genetic background to generate B cell–deficient Yaa mice. Survival studies demonstrated that BXSB.Yaa mice lacking B cells were highly resistant to the lethal effects of BXSB.Yaa disease (Fig. 2A). Resistance to disease in the absence of B cells was associated with remarkably low numbers of CD4+ T cells and CD11b+ monocytes (Fig. 2B). Importantly, there was no increase in the numbers of ICOShi CD4+ T cells that are elevated in B cell–competent BXSB.Yaa mice, suggesting that B cells are required for the development of TFH/EFH cells (Fig. 2B). These results demonstrated that B cells are central to several cellular manifestations as well as the lethal aspects of the autoimmune disease characteristic of BXSB.Yaa mice.

FIGURE 2.

BXSB.Yaa mice lacking B cells are resistant to autoimmune disease. (A) Survival of Ighm−/− and Wt BXSB.Yaa mice (n > 42). (B) The lack of B cells was associated with substantial reductions in the numbers of splenic CD4+ T cells and CD11b+ cells and the expression of ICOS on CD4+ T cells. n = 5 at 17 wk of age. *p < 0.05, **p < 0.01.

FIGURE 2.

BXSB.Yaa mice lacking B cells are resistant to autoimmune disease. (A) Survival of Ighm−/− and Wt BXSB.Yaa mice (n > 42). (B) The lack of B cells was associated with substantial reductions in the numbers of splenic CD4+ T cells and CD11b+ cells and the expression of ICOS on CD4+ T cells. n = 5 at 17 wk of age. *p < 0.05, **p < 0.01.

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In view of the critical requirement for B cells in disease development, their heightened expression of the IL-21R, and the requirement of IL-21 signaling for all aspects of disease, we asked if expression of the IL-21R on B cells contributed to the development of disease. Mixed BM chimeras were produced in which lethally irradiated female BXSB mice were reconstituted with a 1:1 mixture of B cell–deficient (Ighm−/−) and IL-21R–deficient (Il21r Ø) BXSB.Yaa BM cells or with a mixture of Il21r Ø Ighm wild-type (Wt) and Il21r Wt BXSB.Yaa BM cells. The former pairing resulted in donor-derived splenic populations in which B cells lacked the IL-21R, with all other cell types being a mixture of Il21r Wt and Il21r Ø genotypes, whereas the latter pairing resulted in chimeras with all donor BM-derived compartments being comprised of both the Il21r Wt and Ø genotypes (Supplemental Fig. 1A, Supplemental Table I). Survival studies showed that by 32 wk after reconstitution, 79% of mice with IL-21R–competent B cells had died. In contrast, only 9% of mice reconstituted with donor IL-21R–deficient B cells succumbed at late time points (Fig. 3A). The late deaths in this group were likely due to the fact that a similar percentage of this cohort exhibited partial reconstitution by radioresistant host IL-21R+ B cells (up to 5%; data not shown). Even considering partial host cell reconstitution in some mice, this cohort had significantly reduced serum levels of IgM, IgG1, IgG2b, IgG2c, anti-DNA Abs, and ANA as compared with chimeras that received IL-21R B cell–competent BM (Fig. 3B). Histological analysis of kidneys from surviving 32-wk chimeric mice were consistent with an IL-21 dependence for glomerular pathology in that mice lacking IL-21R+ B cells had minimal glomerular deposition of IgG and glomerulonephritis (Supplemental Fig. 2).

FIGURE 3.

IL-21R+ B cells are required for development of BXSB.Yaa disease. (A) Survival of mixed BM chimeras with IL-21R expression varied only in B cells (n = 19–23). (B) Expression of IL-21R on B cells is required for the elevation of serum Igs, anti-ssDNA and ANA. n = 5–8, 32 wk after BM transplantation (BMT). (C) Total numbers of spleen cells, CD19+ B cells, MZ B cells (B220+, CD21hi, CD23lo), and percentages of splenic monocytes (CD11b+F4/80) are affected by the expression of IL-21R on B cells. n = 5, 23 wk after BMT. (D) Expression of IL-21R on B cells has a small effect on the expression of TFH markers. n = 7–10, 32 wk after BMT. Results from spleen cells from nonreconstituted Il21r Wt (n = 7) and Il21r−/− (n = 9) BXSB.Yaa mice at 30 wk of age and evaluated in parallel with the BMT samples are shown for comparison. *p < 0.05, **p < 0.01.

FIGURE 3.

IL-21R+ B cells are required for development of BXSB.Yaa disease. (A) Survival of mixed BM chimeras with IL-21R expression varied only in B cells (n = 19–23). (B) Expression of IL-21R on B cells is required for the elevation of serum Igs, anti-ssDNA and ANA. n = 5–8, 32 wk after BM transplantation (BMT). (C) Total numbers of spleen cells, CD19+ B cells, MZ B cells (B220+, CD21hi, CD23lo), and percentages of splenic monocytes (CD11b+F4/80) are affected by the expression of IL-21R on B cells. n = 5, 23 wk after BMT. (D) Expression of IL-21R on B cells has a small effect on the expression of TFH markers. n = 7–10, 32 wk after BMT. Results from spleen cells from nonreconstituted Il21r Wt (n = 7) and Il21r−/− (n = 9) BXSB.Yaa mice at 30 wk of age and evaluated in parallel with the BMT samples are shown for comparison. *p < 0.05, **p < 0.01.

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Further comparisons showed that a deficit in IL-21R–expressing B cells was associated with substantially reduced numbers of total spleen cells and CD19+ B cells (Fig. 3C). Previous studies showed that the low frequencies of splenic MZ B cells characteristic of BXSB.Yaa mice were normalized in BXSB.Yaa mice globally lacking the IL-21R (23). The frequencies of splenic MZ B cells were significantly increased in mice with a deficit restricted to IL-21R–expressing B cells, indicating that direct signaling by IL-21 to B cells is a requirement for the reduced numbers of MZ B cells in BXSB.Yaa mice (Fig. 3C). We also found that the frequencies of splenic monocytes, which progressively increase in BXSB.Yaa mice, were significantly lower in mice with a deficit in IL-21R–expressing B cells, indicating that monocytosis is also dependent on B cells responding to IL-21 (Fig. 3C). In contrast, this deficit had no effect on the total numbers of splenic CD4+ T cells (Fig. 3C), the levels of ICOS expressed by these cells were only minimally reduced, and the expression of PD-1, another marker for TFH, was unchanged (Fig. 3D). These patterns contrast with the consistently reduced expression of ICOS and PD-1 typically observed for CD4+ T cells from BXSB.Yaa fully lacking the IL-21R compared with Il21R Wt BXSB.Yaa mice (Fig. 3D). Taken together, these results support a critical role for IL-21 signaling to B cells in the development of multiple pathological manifestations of BXSB.Yaa disease, including their greatly shortened survival, while still enabling a high state of CD4+ T cell activation.

Observations indicating that IL-21 promotes the differentiation of TFH suggest that IL-21 may act in an autocrine manner to support the expansion and function of these cells and thereby contribute to disease (1315, 45). To examine this possibility, we generated mixed BM chimeras distinguished only by the presence or absence of IL-21R–competent T cells. This was accomplished by the transfer of mixtures of Tcra Ø and Il21r Ø BXSB.Yaa BM cells into lethally irradiated BXSB female recipients that were also Rag1 Ø to negate the possibility of host lymphocyte repopulation (Supplemental Fig. 1B, Supplemental Table I). Survival studies revealed no significant differences between these cohorts through 34 wk of observation (Fig. 4A). Despite the fact that the survival of these cohorts was essentially identical, we unexpectedly found that serum IgG1, IgG2b, and ANA levels were significantly increased in mice lacking IL-21R+ T cells (Fig. 4B). However, these readouts of heightened B cell activation in mice with IL-21R–deficient T cells were not accompanied by manifestations of accelerated systemic disease as the cohorts did not differ significantly for renal scores or the extent of glomerular Ig deposition. (Supplemental Fig. 2). In addition, they did not differ significantly in studies of PBL for expression of FAS on B cells (another marker of activation), levels of ICOS on CD4+ T cells (Fig. 4C), or the frequencies of monocytes (Fig. 4C), B cells, or CD4+ T cells (Fig. 4D). The only significant difference in T cells between the cohorts was a reduction in the frequencies of CD8+ T cells in mice lacking expression of IL-21R on all T cells (Fig. 4D). As these data provided evidence to suggest that a failure of CD4+ T cells to signal through the IL-21R promoted certain disease biomarkers, we sought to determine the consequences of expression of the IL-21R by CD4+ T cells more directly. To do so, we set up a BM reconstitution experiment in which lethally irradiated BXSB.Yaa Rag1 Ø females were reconstituted with 1:1 mixture of Il21r Ø and Il21r Wt BXSB.Yaa BM. The CD4+ T cells that develop in these mixed BM chimeras will have comparable exposure to autoimmune processes with the only differential being their ability to receive signals through the IL-21R. Fourteen weeks after reconstitution, IL-21R–positive and –negative CD4+ T cells were purified by FACS (Fig 5A). RNA was isolated and analyzed by RT-qPCR to determine the effects of IL-21 signaling on expression of a selected set of genes (Fig. 5B). Surprisingly, transcripts for Il2, Il10, Il21, Ifng, Tnfa, Stat3, and Stat5a were significantly increased in IL-21R–deficient CD4+ T cells. These results indicated that IL-21 signaling to CD4+ T cells broadly represses the expression of cytokines, including those considered to promote (IL-21, IFN-γ, and TNF-α) and limit (IL-2/IL-10) autoimmune disease (46, 47).

FIGURE 4.

IL-21R+ T cells are not required for disease progression in BXSB.Yaa mice. (A) Survival of mixed BM chimeras with IL-21R expression varied only on T cells (n = 13 to 14). (B) Serum Ig and ANA levels 15 wk after BM transplantation (BMT) (n = 18–20). (C) Percentages of monocytes and activated CD4+ T and B cells in peripheral blood at 16 wk following BMT (n = 9–13). Data from PBL of Yaa (n = 4), B6Y (n = 6), and Il21r−/− Yaa BXSB mice (n = 2) 14 wk of age evaluated in parallel with the BMT samples are shown for comparison. (D) Relative proportions of B220+ B cells, CD4+ T cells, and CD8+ T cells among PBL at 16 wk after BM transfer (n = 10–13). (A–D) Data are representative of two experiments. *p < 0.05, **p < 0.01.

FIGURE 4.

IL-21R+ T cells are not required for disease progression in BXSB.Yaa mice. (A) Survival of mixed BM chimeras with IL-21R expression varied only on T cells (n = 13 to 14). (B) Serum Ig and ANA levels 15 wk after BM transplantation (BMT) (n = 18–20). (C) Percentages of monocytes and activated CD4+ T and B cells in peripheral blood at 16 wk following BMT (n = 9–13). Data from PBL of Yaa (n = 4), B6Y (n = 6), and Il21r−/− Yaa BXSB mice (n = 2) 14 wk of age evaluated in parallel with the BMT samples are shown for comparison. (D) Relative proportions of B220+ B cells, CD4+ T cells, and CD8+ T cells among PBL at 16 wk after BM transfer (n = 10–13). (A–D) Data are representative of two experiments. *p < 0.05, **p < 0.01.

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FIGURE 5.

IL-21 signaling prevents CD4+ T cells from acquiring a more pathogenic phenotype. Lethally irradiated Rag−/− BXSB female mice were reconstituted with a 1:1 mixture of BM from BXSB.Yaa Il21r Ø and BXSB.Yaa Il21r Wt mice. (A) Fourteen weeks postreconstitution, IL-21R–positive and –negative CD4+ T cells were sort-purified by FACS, and gene expression analysis of RNA was performed by RT-qPCR procedures. (B) ΔΔ cycle threshold (CT) values of samples run in technical triplicates of three to four independently analyzed recipient mice using 18S RNA as a normalizer. Similar results were found when GAPDH was used as a normalizer (not shown). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

IL-21 signaling prevents CD4+ T cells from acquiring a more pathogenic phenotype. Lethally irradiated Rag−/− BXSB female mice were reconstituted with a 1:1 mixture of BM from BXSB.Yaa Il21r Ø and BXSB.Yaa Il21r Wt mice. (A) Fourteen weeks postreconstitution, IL-21R–positive and –negative CD4+ T cells were sort-purified by FACS, and gene expression analysis of RNA was performed by RT-qPCR procedures. (B) ΔΔ cycle threshold (CT) values of samples run in technical triplicates of three to four independently analyzed recipient mice using 18S RNA as a normalizer. Similar results were found when GAPDH was used as a normalizer (not shown). *p < 0.05, **p < 0.01, ***p < 0.001.

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We recently identified a potent regulatory axis comprised of CD8+ T cells and NK cells that actively suppresses disease progression in BXSB.Yaa mice (27). A similar population of CD8+ Ts with a CM phenotype was shown to be active in B6.Yaa mice (34). The fact that the expression of IL-21R is increased on CD8+ T cells in BXSB.Yaa mice as young as 6 wk (Fig. 1) and that the frequencies of CD8+ T cells were selectively reduced in PBL of BM chimeras lacking IL-21R on T cells (Fig. 4C) prompted us to directly test whether IL-21R signaling of CD8+ T cells can influence BXSB.Yaa disease. To do this, we generated BM chimeras distinguished only by the presence or absence of the IL-21R on CD8+ T cells (Supplemental Fig. 1C, Supplemental Table I). In contrast to chimeras in which all T cells lacked the IL-21R, the survival of chimeric mice was significantly reduced when a deficiency in the IL-21R was restricted to CD8+ T cells (Fig. 6A). Consistent with observations made for chimeras in which all T cells lacked the IL-21R, the lack of expression of the IL-21R on CD8+ T cells was associated with increased levels of serum IgGs and IgM, whereas ANA levels were also elevated but to a lesser extent (Fig. 6B). The two cohorts expressed similar levels of ICOS on CD4+ T cells and FAS on B cells in PBL (Fig. 6C). Similarly, the frequencies of B cells and CD4+ T cells in PBL were not significantly different, but the frequency of CD8+ T cells was significantly lower in mice having only IL-21R–deficient CD8+ T cells (Fig. 6D). An expanded population of CD122hiCD44hiCD62Lhi CM CD8+ T cells is correlated strongly with splenic CD8+ Ts in BXSB.Yaa as well as B6.Yaa mice (27, 34). However, although some mice with IL-21R–competent CD8+ T cells showed a considerable elevation in the expression of CD122, the levels were not significantly different from those of mice lacking IL-21R–expressing CD8+ T cells (Fig. 6E). The lack of IL-21R–expressing CD8+ T cells did, however, result in a significant shift in the relative frequencies of circulating CD8+ T cell subsets, evidenced by reductions in CM and naive CD8+ T cells and an increase in effector memory (EM) cells (Fig. 6F). Taken together, these results are consistent with the proposition that a population of CM CD8+ Ts that retard the development of lethal autoimmune disease in BXSB.Yaa mice is IL-21R dependent.

FIGURE 6.

CD8+ T cells in BXSB.Yaa mice require IL-21R expression to suppress lethal disease and to expand CM CD8+ T cells. (A) Survival of mixed BM chimeras with IL-21R varied only on CD8+ T cells (n = 18–20). (B) Serum IgM, IgG, and ANA levels 13 wk after BMT (n = 10–20). (C) Percentages of B220+ B cells, CD4+, CD8+ T cells from PBL of chimeras with IL-21R varied only on CD8+ T cells. (D) Expression levels of FAS on B cells and ICOS on CD4+ T cells in PBL. (E) MFI of CD122 on CD8+ T cells in PBL. (F) Relative frequencies of CM (CD44hiCD62Llo), EM (CD44hiCD62Llo) and naive (CD44lo CD62Lhi) CD8+ T cells in PBL of chimeric mice. (C–F) Results from 15 wk after BMT (n = 18–20). (B–E) Data are representative of two experiments. *p < 0.05, **p < 0.01.

FIGURE 6.

CD8+ T cells in BXSB.Yaa mice require IL-21R expression to suppress lethal disease and to expand CM CD8+ T cells. (A) Survival of mixed BM chimeras with IL-21R varied only on CD8+ T cells (n = 18–20). (B) Serum IgM, IgG, and ANA levels 13 wk after BMT (n = 10–20). (C) Percentages of B220+ B cells, CD4+, CD8+ T cells from PBL of chimeras with IL-21R varied only on CD8+ T cells. (D) Expression levels of FAS on B cells and ICOS on CD4+ T cells in PBL. (E) MFI of CD122 on CD8+ T cells in PBL. (F) Relative frequencies of CM (CD44hiCD62Llo), EM (CD44hiCD62Llo) and naive (CD44lo CD62Lhi) CD8+ T cells in PBL of chimeric mice. (C–F) Results from 15 wk after BMT (n = 18–20). (B–E) Data are representative of two experiments. *p < 0.05, **p < 0.01.

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Recent studies defined three cardinal features of the severe SLE-like disease of BXSB.Yaa mice: the mice express high levels of IL-21 (17); IL-21 signaling is critically required for the development of all measured parameters of disease (4, 27); and a regulatory axis comprised of CM CD8+ Ts and NK cells retards early disease development and progression (27, 34). The data presented in this study unify these observations by showing that IL-21 functions as a double-edged sword in this disease, driving the expansion of autoreactive B cells while also retarding the disease by promoting the development of CD8+ Ts with a CM phenotype.

First, we found that the Yaa mutation results in enhanced expression of the IL-21R on splenic B cells and CD8+ T cells but not on CD4+ T cells in mice as young as 6 wk, well before the development of overt signs of autoimmunity. This suggests that heightened expression of the IL-21R may serve as a novel early biomarker of impending disease. The early increase in IL-21R expression on CD8+ T cells is unlikely to be a direct consequence of cell-intrinsic effects of the Yaa mutation because among lymphocytes, TLR7 expression is limited to B cells. Enhanced expression of IL-21R both on B cells and CD8+ T cells is more likely to be induced by cell-extrinsic stimuli, potentially including a paracrine response to IL-21 (44).

Second, our results show that direct signaling of IL-21 to B cells is the primary mechanism by which IL-21 drives the autoimmune disease of BXSB.Yaa mice. BXSB.Yaa mice that lack B cells were highly resistant to disease and failed to exhibit hematopoietic markers of disease including expansions of ICOShi TFH/TEFH cells and monocytes. Thus, B cells contribute globally to the pathogenesis of this disease. To a surprising degree, the selective absence of the IL-21R on B cells resulted in the same disease-protective effects. BXSB.Yaa mice lacking IL-21R–positive B cells experienced greatly prolonged survival, even including the small number of mice in which low levels of host cell chimerism with IL-21R–positive B cells was evident. In keeping with disease resistance, the lack of IL-21R–expressing B cells broadly impacted and normalized the cellular and serological features that characterize the SLE-like disease of BXSB.Yaa mice. The effects of a selective lack of the IL-21R on B cells parallels findings made in BXSB.Yaa mice with a global deficit in expression of the IL-21R (4, 27), with the exception that it did not limit the development of ICOShi TFH/TEFH cells, which are the primary sources of IL-21. These results serve to highlight the centrality of B cell–intrinsic IL-21 signaling in the development of this lethal SLE-like autoimmune disease.

Third, our studies, to our knowledge, provide the first evidence to support an immunosuppressive role for IL-21 signaling of CD8+ T cells in autoimmune diseases. In contrast to the stringent requirement for IL-21 signaling to B cells, the selective ablation of IL-21R expression on CD8+ T cells was disease protective. Mice lacking IL-21R selectively on CD8+ T cells showed increases in serum Ig levels and ANA, and, most importantly, accelerated morbidity. Although this selective deficiency had little impact on the activation status of CD4+ T cells and B cells, it resulted in a reduction in the frequency of CD8+ T cells with a CM phenotype and naive CD8+ cells, whereas the proportion of EM CD8+ T cells was increased. Other studies have clearly demonstrated the importance of IL-21 in maintaining CM CD8+ T cell populations (23, 25, 26). Additional studies, including results from BXSB.Yaa mice, strongly support a regulatory role for CM CD8+ cells that retard the development of autoimmune disease by eliminating or otherwise controlling autoreactive B cells and/or TFH (27, 34). Although unlikely and not in keeping with the literature, the increase in naive CD8+ T cells seen in the presence of IL-21 signaling to CD8+ T cells could also be exerting a suppressive effect. Although the exact nature of these CD8+ suppressor T cells remains to be determined, our results provide strong evidence that they require IL-21 to mediate their immunosuppressive effect.

Fourth, our findings strongly argue that IL-21 signaling to CD4+ T cells does not contribute significantly to the overall pathogenesis of autoimmune disease in BXSB.Yaa mice. These results obtained using the TLR7-dependent BXSB.Yaa model differ from recent studies of an alloantigen-induced chronic graft-versus-host disease model in which IL-21 signaling by CD4+ T cells contributed significantly to disease pathogenesis (12). Although the lack of IL-21R on all T cells in BXSB.Yaa mice did not result in accelerated mortality, it was associated with increased serum levels of IgGs and ANA, tendencies toward monocytosis, and increased expression of activation markers on B and T cells in peripheral blood. Moreover, gene expression analyses of IL-21R–positive and IL-21R–negative CD4+ T cells isolated from mixed BM chimeric BXSB.Yaa hosts clearly indicated that IL-21 signaling to CD4+ T cells broadly reduced the levels of transcripts of both immune-regulatory (Il2 and Il10) and -promoting (Il21, Ifng, and Tnfa) cytokines. Autocrine IL-21 signaling to CD4+ T cells may thus broadly restrain the amount and types of cytokines they produce and thereby alter the balance of downstream immunological processes both positively and negatively. Further studies are required to unravel the mechanisms by which IL-21 signaling to CD4+ T cells impacts the overall pathogenesis of this disease.

Although genetic and functional studies support the involvement of IL-21 signaling in the pathogenesis of multiple autoimmune diseases of humans and mice, our findings interject a note of caution for designing therapeutic interruptions of this signaling pathway. Blockade may lead to unpredicted effects, inhibiting the development of autoreactive B cells and autoantibody-secreting plasma cells, while at the same time impairing the development of naturally occurring CD8+ Ts that limit disease progression and broadly restraining the spectrum of cytokines produced by CD4+ T cells.

We thank Rosalinda Doty for histological interpretations.

This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases (to H.C.M.) and by the Alliance for Lupus Research (to D.C.R.). C.G.M. was supported in part by a National Institutes of Health Training Grant. J.A.B. was supported by the Arthritis Foundation.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ANA

anti-nuclear Ab

BM

bone marrow

CM

central memory

EM

effector memory

IL-21r Ø

IL-21R–deficient

MZ

marginal zone

PD-1

programmed cell death 1

qPCR

quantitative PCR

SLE

systemic lupus erythematosus

TEFH

extrafollicular Th cell

TFH

follicular Th cell

Treg

regulatory T cell

Ts

suppressor T cell

Wt

wild-type.

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The authors have no financial conflicts of interest.