Gr1⁺ Cells Suppress T-Dependent Antibody Responses in (NZB × NZW)F1 Male Mice through Inhibition of T Follicular Helper Cells and Germinal Center Formation

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoreactive Abs resulting in a variety of symptoms including fatigue, skin rashes, arthritis, and nephropathy leading to renal failure (1, 2). One mouse model used to study lupus is the (New Zealand black [NZB] × New Zealand white [NZW])F1 strain. Lupus-like disease in the (NZB × NZW)F1 mice is characterized by hyperactive B cells, abnormal autoantibody production, and ectopic germinal center (GC) formation, and has been shown to depend on CD4⁺ T cell help (3–6). Upon activation, CD4⁺ T cells can differentiate into several effector cell subsets characterized by their unique production of effector cytokines, that is, Th1 (IFN-γ), Th2 (IL-4, IL-5), Th9 (IL-9), Th17 (IL-17), or T follicular helper cells (T_FH; IL-21). Although all of these subsets can influence B cell activation and/or differentiation, IL-21–producing T_FH cells are critically required for GC formation and plasma cell (PC) differentiation (7–9). Consistent with lupus being a B cell–dependent, autoantibody-mediated disease, T_FH cells and IL-21 have been found to be involved in at least some aspects of lupus-like disease development in several mouse models (10–12). Importantly, studies have also demonstrated dysregulated T_FH cells in SLE patients (13–15).

Like many other autoimmune diseases, SLE disproportionately affects women more than men (16). Hormonal therapy has been successfully applied in severe cases of SLE, suggesting that sex hormones play a major role in determining disease development in genetically predisposed individuals (17–19). Likewise, female (NZB × NZW)F1 mice develop lupus-like disease with a much higher incidence than male (NZB × NZW)F1 mice (20, 21). The female predominance observed in the (NZB × NZW)F1 mouse model is known to be controlled in part by sex hormones (21–25). Interestingly, it has been suggested that sex hormones also partake in defining the magnitude of immune activation during infections and after immunizations in healthy individuals (26 and reviewed in Ref. 27).

Our laboratory recently described the presence of a population of testosterone-regulated immunosuppressive Gr1⁺CD11b⁺ cells in male (NZB × NZW)F1 mice (28). Particularly, we found that depletion of Gr1-expressing cells augmented spontaneous autoantibody production in male (NZB × NZW)F1 mice, whereas a similar strategy had no effect on autoantibody production in female mice. In this study, we tested whether the immune-suppressive function of Gr1⁺CD11b⁺ cells observed in male lupus-prone (NZB × NZW)F1 mice also affected immune activation in response to exogenous Ag. As expected, male (NZB × NZW)F1 mice responded less vigorously to immunization with a T-dependent Ag, whereas depletion of Gr1-expressing cells augmented the Ag-dependent Ab response in males, but not in females. In correlation, numbers of both T_FH cells and GC B cells were elevated after Gr1⁺ cell depletion. Thus, manipulation of analogous human cells might offer a new potential target for improving vaccination efficacy in individuals with low Ab responses, as well as repressing Ab production in systemic autoimmunity.

Three-week-old male and female (NZB × NZW)F1 mice were obtained from The Jackson Laboratories and kept at the Biological Resource Unit at Lerner Research Institute, Cleveland Clinic. All mouse experiments were approved by the local Institutional Animal Care and Use Committee. Mice were bled at 8 wk of age, and PBMCs were analyzed for the presence of Gr1⁺CD11b⁺ cells by flow cytometry, allowing for stratification of the mice into equal groups based on their basal levels of Gr1⁺CD11b⁺ cells. At 9 wk of age, mice were injected i.p. with 20 μg (4-hydroxy-3-nitrophenylacetyl)₂₇ conjugated chicken γ-globulin (NP₂₇-CGG) or NP₂₇-FICOLL [both from Biosearch Technologies, Petaluma, CA]), in a ratio of 1:1 with CFA (Sigma-Aldrich, St. Louis, MO), or with 1× PBS. Each injection was done in a total volume of 200 μl. Ab-treated mice were additionally injected i.p. every 3 d starting on day −1 with either 500 μg rat anti-mouse Gr1 (clone RB6-8C5, a kind gift from Dr. Pearlman, Case Western Reserve University, Cleveland, OH), 500 μg control rat IgG, or sterile-filtered PBS, each in a total volume of 200 μl as previously described (28). This treatment strategy was based on data showing that Gr1-expressing cells were fully depleted from BM, spleen, and blood for 2 d, and only began to reappear on day 3 after injection with anti-Gr1 Ab (data not shown). Ab treatment was continued for up to 28 d as stated in the text. Mice were bled for serum by tail-vein bleeding on day −1, day 7, and every 7 d thereafter as applicable.

Flow cytometry was performed using a FACSCalibur (BD Biosciences, San Jose, CA), and all analyses were done using FlowJo Version 9.3. Abs with the following specificities were used for all analyses: FITC-conjugated anti-CD3 (clone 145-2C11), PE- or allophycocyanin-conjugated anti-CD4 (clone L3T4), PerCP-conjugated anti-CD8 (clone 53-6.7), PE- or allophycocyanin-conjugated anti-CD11b (clone M1/70), PE-conjugated anti-CD38 (clone 90), FITC- or PerCP-conjugated anti-B220 (CD45R; clone RA3-6B2), FITC-conjugated anti-CD62L (clone MEL-14), PerCP-conjugated anti-Gr1 (Ly6G/6C; clone RB6-8C5), biotin-conjugated anti–GL-7 (clone GL-7), PE-conjugated anti-CD278 (ICOS; clone 7E.17G9), PE-conjugated anti-CD279 (PD-1; clone MIH4), and PE-conjugated streptavidin (all from eBioscience, San Diego, CA). PE-conjugated anti-CD138 (clone 281-2) and biotin-conjugated anti-CXCR5 (clone 2G8) Abs were purchased from BD Biosciences. All flow-based analyses were based on the gating of live cells as given by forward and side scatter properties.

Serum was obtained from NP₂₇-CGG immunized anti-Gr1 Ab and control Ab-treated animals before, during, and after treatment. 2HB (Immulon) 96-well microtiter plates were coated with 5 μg/well NP₅ conjugated to BSA in PBS overnight and blocked for 2 h at room temperature with 5% gelatin in PBS. Serum was diluted 1:10,000 in serum diluent (5 mg/ml bovine γ-globulin, 5% gelatin, 0.05% Tween in PBS). A total of 100 μl/well diluted serum was plated and incubated for 2 h at room temperature. All washes were done using 0.05% Tween in PBS. Plates were developed using HRP-conjugated anti-IgG1 secondary Abs and 10 mg/ml 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) in Mcilwain’s buffer (0.09 M Na₂HPO₄, 0.06 M citric acid, pH 4.6), and colorimetric readings were done at 450 nm on a Victor 3 plate reader (Perkin Elmer, Waltham, MA). All samples per experiment were run at the same time, allowing for direct comparisons of OD values to determine Ab levels between samples.

Spleen single cells were prepared from 9-wk-old male and female (NZB × NZW)F1 mice. Gr1^highCD11b⁺ and Gr1^lowCD11b⁺ cells were sorted by high-speed cell sorting (FACSAria II; BD Biosciences). Total T cells were isolated from female (NZB × NZW)F1 age-matched mice by MACS separation using CD90.2 microbeads (Miltenyi Biotech, CA). After isolation, T cells were labeled with CFSE (Sigma-Aldrich). After labeling, cells were washed and reconstituted in cell culture media (RPMI 1640, 10% FBS, 1% Penicillin/Streptomycin, 1 mM Na-pyruvate, 1× MEM). A total of 2 × 10⁵ T cells/well were plated in 96-well microtiter plates in the presence or absence of plate-bound anti-CD3 and anti-CD28 Abs (10 μg/ml each; both from eBioscience). Where noted, 5 × 10⁴ flow-sorted Gr1^highCD11b⁺ or GR1^low CD11b⁺ cells were added to the cultures at time 0 h. After 48 h, cells were harvested and stained with allophycocyanin-conjugated anti-CD4 and PerCP-conjugated anti-CD8 Abs (eBioscience), allowing for separate analysis of proliferation of CD4⁺ and CD8⁺ T cell subsets.

Naive CD90.2⁺CD62L^high T cells and Gr1⁺CD11b⁺ cells were sorted by high-speed cell sorting (FACSAriaII; BD Biosciences) from spleens of 9-wk-old male (NZB × NZW)F1 mice. A total of 3 × 10⁵ naive T cells was plated in the presence or absence of Gr1⁺CD11b⁺ cells at different ratios (Gr1⁺/T cell ratio = 1:4, 1:8, and 1:16). Cells were cultured in cell culture media (RPMI 1640, 10% FBS, 1% Penicillin/Streptomycin, 1 mM Na-pyruvate, 1× MEM) in the presence of plate-bound anti-CD3 and anti-CD28 Abs (3 μg/ml each), 10 μg/ml anti–IL-4 Ab (eBioscience), 10 μg/ml anti–IFN-γ Ab (eBioscience), 20 μg/ml anti–TGF-β Ab (R&D Systems, Minneapolis, MN), 50 ng/ml rIL-21, and 100 ng/ml IL-6 (both from Biolegend, San Diego, CA) (29). The media were replaced with fresh media containing the same amounts of cytokines on day 3. After 5 d, cells were harvested and stained using the following Abs and conjugates: anti–PD-1 (FITC), biotinylated anti-CXCR5 and streptavidin-PE conjugate, anti-Gr1 (PerCP), and anti-CD4 (allophycocyanin; all from eBioscience). T_FH cells were identified as CD4⁺Gr1⁻CXCR5⁺PD-1⁺ by flow cytometry as previously described (29).

Two-millimeter slices of spleens were taken from mice on days 3, 5, 7, and 14 postimmunization and quick-frozen horizontally in OCT (Tissue-Tek, Sakura, CA). Five-micrometer sections were prepared and stained with biotinylated anti–GL-7/streptavidin-conjugated allophycocyanin and Pacific Blue–conjugated anti-B220 (all from eBioscience). Images were collected using an HC Plan Apo 10×/0.7NA objective lens on a Leica DMR upright microscope (Leica Microsystems) equipped with a Retiga EXi Cooled CCD Camera (QImaging). Numbers of GCs were counted from three distinct fields per spleen and the average number was used for statistical analyses.

All statistical analyses were performed using GraphPad Prism version 4.00 for Windows, GraphPad Software (San Diego, CA; http://www.graphpad.com). Statistical significance between the means of two groups was determined using Student t test or the nonparametric Mann–Whitney U test as applicable. Analyses of in vivo kinetics of T_FH and GC B cells were done by two-way ANOVA tests. Analysis of Gr1⁺ cell–mediated suppression of T_FH differentiation in vitro was done using the one-way ANOVA test.

To determine whether male and female (NZB × NZW)F1 mice responded differently to Ag challenge, 9-wk-old mice were immunized i.p. with NP-CGG/CFA known to elicit a strong T-dependent IgG₁ response (30). Four weeks postimmunization (day 28), female (NZB × NZW)F1 mice produced significantly higher titers of anti-NP–specific IgG₁ Abs (p < 0.001) than male (NZB × NZW)F1 mice (Fig. 1A). In a separate study, we determined the numbers of splenic GC B cells and PCs in male and female (NZB × NZW)F1 mice 2 wk postimmunization (day 14). Female (NZB × NZW)F1 mice expressed significantly elevated numbers of splenic GL-7⁺B220⁺CD38^low GC B cells (p < 0.05) and CD138⁺B220^lowIgM^low PCs (p < 0.05) as compared with male (NZB × NZW)F1 mice (Fig. 1B, 1C).

We have previously shown that male (NZB × NZW) F1 mice have constitutively higher levels of Gr1⁺CD11b⁺ cells, and that depletion of Gr1⁺ cells in vivo reduces the levels of antinuclear Abs in male but not female (NZB × NZW)F1 mice (28). To determine whether Gr1⁺ cells play a similar role in the NP-CGG/CFA–induced Ab response, we treated male and female (NZB × NZW)F1 mice with anti-Gr1–depleting Abs or control rat IgG Abs continuously every 3 d starting 1 d before immunization. Treatment with anti-Gr1–depleting Ab did not significantly affect other cell populations (Supplemental Fig. 1). As expected, depletion of Gr1-expressing cells elevated the anti-NP–specific IgG₁ Ab titers in male mice as compared with control rat IgG-treated males (p < 0.001; Fig. 2A), whereas depletion of Gr1-expressing cells had no significant effect on the Ab response in female mice (p = 0.17; Fig. 2B). When analyzing the splenic compartment at 2 wk postimmunization, we found that depletion of Gr1-expressing cells resulted in higher numbers of both GC B cells and PCs in male mice (p < 0.05), but again with no effect in female mice (Fig. 2C, 2D). Notably, consistent with their autoimmune phenotype, unimmunized female (NZB × NZW)F1 mice expressed continuously increased numbers of PCs as compared with male (NZB × NZW)F1 mice (p < 0.05; Fig. 2D). Thus, Gr1-expressing cells suppress immunization-induced Ab responses in male, but not female, mice. To determine whether the suppressive effect was dependent on T cells, an additional cohort of male (NZB × NZW)F1 mice was immunized with NP-Ficoll, a T-independent Ag, and treated with either depleting anti-Gr1 Ab or rat IgG control Ab. Depletion of Gr1-expressing cells had no effect on the Ab response and B cell differentiation induced after T-independent Ag immunization (Supplemental Fig. 2). Thus, the in vivo suppressive effect of Gr1⁺ cells is dependent on T cells.

Gr1-expressing cells consist of two major cell subsets expressing high and low levels of Gr1, respectively. Both subsets have been shown to have suppressive functions in cancer studies (reviewed in Ref. 31); however, Gr1^lowLy6C⁺CD11b⁺ cells have been specifically associated with T cell suppression in another model of lupus (32). We hypothesized that Gr1^lowCD11b⁺ cells from male (NZB × NZW)F1 mice could have a similar suppressive effect on T cells and hence be involved in the in vivo effect of Gr1⁺ cells during T-dependent Ab responses. Gr1^highCD11b⁺ and Gr1^lowCD11b⁺ cells were sort purified from male and female 4-wk-old (NZB × NZW)F1 mice (Fig. 3A) and added to CFSE-labeled MACS-purified T cells in the presence or absence of anti-CD3 and anti-CD28 cross-linking Abs. Gr1^lowCD11b⁺ cells, but not Gr1^highCD11b⁺ cells, suppressed T cell proliferation in vitro (Fig. 3B, 3C). Although both male- and female-derived Gr1^lowCD11b⁺ cells from 4-wk-old mice functionally suppressed T cell proliferation, only suppression by male-derived cells reached statistical significance (male: p < 0.01; female: p = 0.056; Fig. 3D). This difference became even more apparent when we compared the suppressive effect of male and female Gr1^lowCD11b⁺ cells obtained from 9-wk-old (NZB × NZW)F1 mice (Fig. 3E), and is consistent with the differential effect of in vivo Gr1⁺ cell depletion in 9-wk-old male and female (NZB × NZW)F1 mice as described earlier (Fig. 2).

Because the suppressive effect of Gr1-expressing cells after immunization is dependent on T cells, and Gr1^lowCD11b⁺ cells specifically suppress T cell proliferation in vitro, we expected T cells to be differentially expressed in immunized male (NZB × NZW)F1 mice treated with anti-Gr1–depleting Ab or control rat IgG. Contrary to our prediction, total numbers of both CD4⁺ and CD8⁺ T cells were unaffected by the absence of Gr1⁺ cells (Fig. 4A, 4B). Further analyses showed that the percentage of CD4⁺ T cells expressing markers consistent with memory-effector T cells (CD44^highCD62L^low), T_FH cells (ICOS⁺PD-1⁺CD4⁺) were significantly increased in the absence of Gr1-expressing cells (p < 0.001, p < 0.01, respectively; Fig. 4C). T regulatory cells (Foxp3⁺CD4⁺CD25⁺) were also increased in Gr1⁺ cell–depleted mice as compared to nondepleted mice, but the difference failed to reach statistical significance (p = 0.056). In contrast, the percentage of naive CD4⁺ T cells (CD44^lowCD62L^high) significantly decreased (p < 0.01) after Gr1⁺ cell depletion (Fig. 4C). Thus, Gr1⁺ cells appear to regulate differentiation of CD4⁺ T cells during thymus-dependent (TD) Ab responses in vivo.

GC formation is crucial for class switching and production of Abs after immunization, and is dependent on T cell help, specifically T_FH cells. The increased number of GC B cells (Fig. 2C) and T_FH cells (Fig. 4C) in anti-Gr1 Ab–treated male (NZB × NZW)F1 mice 14 d postimmunization suggest that Gr1-expressing cells may also affect GC reactions. We tested this by comparing the number of T_FH cells and GC B cells, and the appearance of GCs in mice immunized with NP-CGG/CFA and treated with either depleting anti-Gr1 Ab or control rat IgG. Already at 5 d postimmunization, mice depleted of Gr1-expressing cells expressed elevated levels of T_FH cells, and at 7 and 14 d postimmunization, the levels were significantly higher in anti-Gr1 Ab–treated mice (Fig. 5A). Numbers of splenic GC B cells were similarly elevated in mice depleted of Gr1-expressing cells 14 d postimmunization (p < 0.01; Fig. 5B). These data were further supported by immunofluorescence stainings of spleen sections showing significantly elevated numbers and increased sizes of GCs in anti-Gr1 Ab–treated mice (Fig. 5C, 5D).

T_FH cells play a major role in the formation of GC. Because numbers of T_FH cells, GC B cells, GCs, and PCs were all significantly upregulated in the absence of Gr1-expressing cells, we asked whether Gr1⁺ cells can directly suppress differentiation of naive T cells into T_FH cells in vitro. Sort-purified male (NZB × NZW)F1 naive T cells (CD90.2⁺CD62L^high) were cocultured with increasing concentrations of sort-purified male Gr1⁺ cells for 5 d in T_FH cell-skewing media (Fig. 6A, 6B) (29). Upon harvest, cells were stained for Gr1, CD4, CXCR5, and PD-1, and the percentage of CXCR5, PD-1, double-positive CD4⁺ cells was determined as previously described (29). Gr1⁺ cells significantly suppressed the differentiation of naive T cells into T_FH cells. The suppression was dose dependent and reached statistical significance at 4:1 and 8:1 ratios of T cells to Gr1⁺ cells (p < 0.01). The suppression of differentiation was not due to reduced survival of T cells, as the number of CD4⁺ T cells recovered on day 5 improved significantly and in a dose-dependent manner with the addition of Gr1⁺ cells (p < 0.05–0.001; data not shown). Thus, immunosuppressive Gr1⁺ cells appear to play a hitherto unrecognized role in controlling naive T cell differentiation during T-dependent Ag exposure.

The sex bias in SLE and other autoimmune diseases is not well understood. There is evidence that sex hormones, both estrogen and testosterone, are involved in the differential prevalence of SLE and mouse lupus-like disease among males and females (17, 18, 21, 23–25). The protective function of testosterone, although well established, has not been studied in detail, and the molecular mechanism through which male sex hormone may act is not known. Interestingly, Ab responses to immunization are often also higher in females than in males (reviewed in Ref. 27), suggesting that a generally overactive immune system in females might exist and helping to explain the female sex bias observed in many autoimmune disorders, including SLE.

We have recently shown that male lupus-prone (NZB × NZW)F1 mice have higher levels of immunosuppressive Gr1⁺ cells than females, that these cells are driven by testosterone, and that depletion of the cells in males resulted in increased spontaneous autoantibody production (28). Using the same model, in this article, we describe how male (NZB × NZW)F1 mice respond less vigorously than females to TD exogenous Ag immunization. Furthermore, depletion of Gr1⁺ cells in (NZB × NZW)F1 males increased the Ab response to Ag challenge, along with an underlying expansion of splenic GC B cells, PCs, and T_FH cells. The main function of T_FH cells is to facilitate GC reactions and the differentiation of naive B cells into PCs. Interestingly, T_FH cells have been associated with disease severity in lupus patients, as well as several murine models (7, 15, 33–37). Thus, we hypothesized that Gr1⁺ cells control Ab production primarily via modulation of T_FH cells. In support hereof, we found that Gr1⁺ cells inhibited the differentiation of T_FH cells in vitro in a dose-dependent manner, although the mechanism of suppression remains unresolved.

Gr1⁺ cells are a heterogeneous population of cells consisting of mature and immature neutrophils, immature monocytes, and some dendritic cell subsets. Both proinflammatory and suppressor functions have been associated with such cells. In cancer, immunosuppressive Gr1⁺ cells are widely known as myeloid-derived suppressor cells (MDSCs; reviewed in Ref. 31). Despite the identification of immunosuppressive Gr1⁺ cells in several models of autoimmunity (38–42), during viral infections (43), and graft rejection (44), the term MDSC is only sporadically used in such models. Whether testosterone-induced Gr1-expressing cells from male (NZB × NZW)F1 mice can be classified as MDSCs or represent a different population of immunosuppressive Gr1-expressing cells in this lupus model remains to be determined. The observation that male Gr1^highCD11b⁺ cells suppress B cells independent of reactive oxygen species, NO, IL-10, and TGF-β (28) suggests the cells may not reflect tumor-induced MDSCs (45, 46), although further molecular and functional studies directly comparing these cell subsets are needed to confirm such distinction.

In nonautoimmune C57BL/6 mice, Gr1-expressing cells have also been shown to affect the activation of adaptive immunity after footpad immunizations (47). Specifically, it was shown that neutrophils (defined as Gr1⁺Ly-6G⁺CD11b⁺) interfered with the activation of T cells through the control of Ag uptake by professional APCs, thereby limiting dendritic cell–T cell contact time in draining lymph nodes (47). Interestingly, analyses of Gr1⁺ cells in nonautoimmune mouse strains revealed similarly elevated levels in males of most strains, including C57BL/6 (B6), BALB/c, and 129/Sv (data not shown). Upon immunization with NP-CGG in CFA, we detected increased serum Ab responses in female as compared with male B6 mice (data not shown). Interestingly, preliminary analyses showed that Ab-mediated depletion of Gr1⁺ cells in B6 mice drove expansions of GC and memory B cells in spleens of both male and female C57BL/6 mice 4 wk after immunization, albeit without affecting anti-NP–specific Ab levels (E. Der and T.N. Jorgensen, unpublished observations). Thus, Gr1⁺ cells from nonautoimmune B6 mice may exert some, but not all, of the immunosuppressive functions we observe in (NZB × NZW)F1 mice.

Interestingly, for reasons yet unknown, Gr1-expressing cells seem to have a limited protective effect in females of (NZB × NZW)F1 mice (Ref. 28 and this study). In fact, upon aging or during active disease, female Gr1⁺ cells from lupus-prone mice and SLE patients become stimulatory as described by us and others (28, 48–50, J. Dimo, unpublished observations), suggesting that disease-related inflammatory factors present in lupus-prone females can influence the immunosuppressive function of immature myeloid Gr1⁺ cells. Identification of such factor(s) clearly represents an attractive new target for therapeutic use in SLE and other diseases with dysregulated Ab production.

In summary, Gr1-expressing cells from lupus-prone male (NZB × NZW)F1 mice suppress T and B cell responses, resulting in reduced GC formation and less PC differentiation. Discovering the mechanism through which Gr1⁺CD11b⁺ cells suppress in the context of lupus could open new doors for immunotherapy of lupus patients and new targets for therapeutic agents.

We thank Jennifer Powers for invaluable help with all flow cytometry–based cell sorts and Dr. Eric Pearlman for purified RB6-8C5 anti-Gr1 Ab.

The authors have no financial conflicts of interest.