Murine B Cell Development and Antibody Responses to Model Antigens Are Not Impaired in the Absence of the TNF Receptor GITR

The Glucocorticoid-Induced Tumor necrosis factor Receptor GITR, a member of the tumor necrosis factor receptor superfamily, has been shown to be important in modulating immune responses in the context of T cell immunity. B lymphocytes also express GITR, but a role of GITR in humoral immunity has not been fully explored. To address this question, we performed studies to determine the kinetics of GITR expression on naïve and stimulated B cells and the capacity of B cells to develop and mount antibody responses in GITR−/− mice. Results of our studies indicate that all mature B cells express GITR on the cell surface, albeit at different levels. Expression of GITR on naïve mature B cells is upregulated by BCR signaling, but is counteracted by helper T cell-related factors and other inflammatory signals in vitro. In line with these findings, expression of GITR on germinal center and memory B cells is lower than that on naïve B cells. However, the expression of GITR is strongly upregulated in plasma cells. Despite these differences in GITR expression, the absence of GITR has no effect on T cell-dependent and T cell-independent antibody responses to model antigens in GITR−/− mice, or on B cell activation and proliferation in vitro. GITR deficiency manifests only with a slight reduction of mature B cell numbers and increased turnover of naïve B cells, suggesting that GITR slightly contributes to mature B cell homeostasis. Overall, our data indicate that GITR does not play a significant role in B cell development and antibody responses to T-dependent and independent model antigens within the context of a GITR-deficient genetic background

The antigen-specific CD8+ T cell repertoire in unimmunized mice includes memory phenotype cells bearing markers of homeostatic expansion

Memory T cells exhibit superior responses to pathogens and tumors compared with their naive counterparts. Memory is typically generated via an immune response to a foreign antigen, but functional memory T cells can also be produced from naive cells by homeostatic mechanisms. Using a recently developed method, we studied CD8 T cells, which are specific for model (ovalbumin) and viral (HSV, vaccinia) antigens, in unimmunized mice and found a subpopulation bearing markers of memory cells. Based on their phenotypic markers and by their presence in germ-free mice, these preexisting memory-like CD44hi CD8 T cells are likely to arise via physiological homeostatic proliferation rather than a response to environmental microbes. These antigen-inexperienced memory phenotype CD8 T cells display several functions that distinguish them from their CD44lo counterparts, including a rapid initiation of proliferation after T cell stimulation and rapid IFN-γ production after exposure to proinflammatory cytokines. Collectively, these data indicate that the unprimed antigen-specific CD8 T cell repertoire contains antigen-inexperienced cells that display phenotypic and functional traits of memory cells

GITR-deficient mice mount relatively normal antibody responses to model antigens.

<p>(A) Total serum levels of IgM, IgG1, IgG2b, IgG2a and IgE in 4 month-old GITR^+/+ (129S1 strain, white bars, empty circles) and GITR^−/− mice (gray bars, filled circles). <i>n</i> = 7. (B) GITR^+/+ (129S1, white bars, empty circles) and GITR^−/− (grey bars, filled circles) mice (<i>n</i> = 5 per group) were immunized i.p. with 5 µg NP₂₈-FICOLL. Sera were collected before (day 0) and 4 and 7 days after immunization, and NP-specific IgM and IgG3 were measured by ELISA. (C) GITR^+/+ (129S1, white bars, empty circles) and GITR^−/− (grey bars, filled circles) mice (<i>n</i> = 5 per group) were immunized i.p. with 100 µg NP₃₆-CGG in Alum. Sera were collected at indicated time points and NP-specific IgM, IgG1, IgG2b and IgG2a were measured by ELISA. (D) Mice described in (C) were boosted with 5 µg/ml NP₃₆-CGG injected i.p. 178 days after primary immunization, and sera were collected at indicated times after boost. NP-specific IgG1, IgG2b and IgG2a were measured by ELISA (<i>n</i> = 4 per group). Bars and circles represent geometric means and individual mice, respectively. *<i>p</i><0.05; **<i>p</i>≤0.002; ***<i>p</i>≤0.0002.</p

BCR stimulation enhances GITR expression on B cells.

<p>B cells were purified from the spleen of BALB/c mice by negative selection and all B cell cultures were performed in the presence of 4 ng/ml of BAFF to sustain cell survival. (A) Representative GITR expression on B cells cultured for 48 h in medium alone (gray line) or in the presence of either 10 µg/ml F(ab′)₂ anti-IgM antibodies (dashed line) or 20 µg/ml LPS (intact black line). (B) B cells were cultured in either medium alone (dashed line) or with 10 µg/ml F(ab′)₂ anti-IgM antibodies (intact line). Expression of GITR, CD69 and CD86 were analyzed at 8, 24 and 48 h of culture. The bar graph represents average GITR mean fluorescent intensity (MFI) on B cells cultured with medium or anti-IgM antibodies for 48 h from 3 independent experiments. **<i>p</i>≤0.002, <i>n</i> = 3. (C) B cells were stimulated in culture for 24 h with 10 µg/ml F(ab′)₂ anti-IgM antibodies without (dashed line) or with 0.1 µM actinomycin D (ActD) to inhibit transcription, or 2 µM cycloheximide (Chx) to inhibit protein synthesis (intact black line). Gray line indicates GITR expression on cells cultured in medium alone. The diluent DMSO (0.2–0.4%) was present in all cultures. The bar graph represents average GITR MFI on B cells cultured with medium, anti-IgM antibodies, or anti-IgM antibodies with the indicated inhibitor from 3 independent experiments. Differences were not statistically significant, but were observed in all three independent experiments. (D) GITR (left) and CD86 (right) expression on B cells cultured for 48 h with 10 µg/ml F(ab′)₂ anti-IgM antibodies alone (dashed line) or in the presence of 0.2 µM of cyclosporin A (CsA, intact black line). Expression on B cells cultured in medium alone (gray line) is shown for comparison. The bar graph represents average GITR MFI on B cells cultured with medium, anti-IgM antibodies, or anti-IgM antibodies with CsA from 3 independent experiments. *<i>p</i>≤0.05, <i>n</i> = 3. All histograms represent live, B220⁺ lymphoid cells. Isotype control staining for GITR (light shaded histogram) is shown in (B) and (C) and it was similar on stimulated or nonstimulated cells (data not shown). Data are representative of 2–6 independent experiments.</p

Helper T cell factors inhibit GITR induction on BCR-stimulated B cells.

<p>B cells were purified from the spleen of wild-type BALB/c mice by negative selection and used for <i>in vitro</i> analyses. All B cell cultures were performed in the presence of 4 ng/ml of BAFF to sustain cell survival. (A) GITR expression on B cells cultured for 48 h in medium alone, with 2 µg/ml F(ab′)₂ anti-IgM antibodies (gray line, left panel), 15 µg/ml anti-CD40 antibodies (dashed line, left and right panels), a combination of anti-IgM and anti-CD40 antibodies (intact black line, left panel), or a combination of anti-CD40 antibodies and 0.2 µM CsA (intact black line, right panel). (B) GITR expression on B cells cultured for 48 h in medium alone (filled histogram), 2 µg/ml F(ab′)₂ anti-IgM antibodies (gray line), 50 ng/ml IL-4, 100 (not shown) and 1000 U/ml IFNγ, 100 (not shown) and 1000 U/ml IFNα (dashed line) or a combination of anti-IgM antibodies and cytokines (intact black line) as indicated. There was no difference between 100 and 1000 U/ml of IFNγ and IFNα (data not shown). Isotype control staining for GITR (light shaded histogram) is shown in (A) and (B) and it was similar on stimulated or nonstimulated cells (data not shown). (C) GITR expression on B cells cultured with 15 µg/ml anti-CD40 antibodies alone (dashed line) or in combination with 50 ng/ml IL-4, 1000 U/ml IFNγ, or 1000 U/ml IFNα (intact line), as indicated. Note that the isotype control antibody staining was included in all analyses but omitted in plots that display more than 3 samples for clarity.</p

GITR appears dispensable for entry of B cells in the active cell subsets.

<p>(A) GITR^+/+ (129S1, white bars) and GITR^−/− (black bars) mice (<i>n</i> = 5) were fed BrdU continuously for one week. Bars represent frequency of BrdU⁺ cells in naïve B220⁺ (PNA⁻), germinal center (GC), memory (MC), and plasma (PC) cell compartments gated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031632#pone-0031632-g001" target="_blank">Fig. 1</a>. Error bars represent SD. ***<i>p</i>≤0.003. Similar results were found in mice fed BrdU for two weeks (data not shown). (B) Schematic for the generation of GITR-deficient and sufficient mixed bone marrow chimeras. Bone marrow cells from CD45.2 GITR^−/− and GITR^+/+ 129S1 mice were mixed at a 1∶3 ratio with bone marrow cells isolated from CD45.1 C57BL/6 SJL mice. Cell mixtures were injected i.v. into lethally irradiated C57BL/6 mice, which were evaluated 8 weeks later. (C) Bone marrow, spleen and lymph nodes from mixed bone marrow chimeras (<i>n</i> = 5 per group) generated as described in (B) were analyzed by flow cytometry to determine the frequency of CD45.2⁺ cells in B cell subsets. B cell subsets were gated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031632#pone-0031632-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031632#pone-0031632-g004" target="_blank">4</a>. Open bars and symbols represent CD45.2⁺ GITR^+/+ B cells and filled bars and symbols represent CD45.2⁺ GITR^−/− B cells. The graphs represent means and SEM for each indicated B cell subset. (D) The relative size of the CD45.2⁺ B cell population in each spleen B cell compartment was measured relative to that of the transitional B cell population in each mouse described in (C). The bar graph represents the average fold change (+SEM) in the size of each CD45.2 B cell subset relative to the transitional B cell subset, which was set at 1. Open bars represent CD45.2⁺ GITR^+/+ B cells and filled bars represent CD45.2⁺ GITR^−/− B cells. *<i>p</i><0.05.</p

GITR expression and signaling do not alter B cell activation and proliferation.

<p>(A) B cells were purified from the spleen of wild-type BALB/c mice by negative selection and cultured for 48 h in medium alone (filled histogram) or in the presence of 1 µg/ml GITRL (gray line), 2 µg/ml F(ab′)₂ anti-IgM antibodies (dashed line) or a combination of these reagents (intact black line). All B cell cultures were performed in the presence of 4 ng/ml of BAFF to sustain cell survival. Histograms indicate CD69 (left) and CD86 (right) expression on live B220⁺ lymphocytes. Data are representative of 3 independent experiments. (B) Total spleen cells from GITR^+/+ (129S1 strain) and GITR^−/− mice were purified over a Ficoll gradient, labeled with CFSE and cultured for 4 days. Cultured cells were either left untreated or stimulated with 10 µg/ml F(ab′)₂ anti-IgM antibodies, 15 µg/ml anti-CD40 antibodies, a combination of anti-IgM and anti-CD40 antibodies, or a combination of anti-IgM antibodies and 50 ng/ml IL-4, as indicated at the top. All B cell cultures were performed in the presence of 4 ng/ml of BAFF to sustain cell survival. Cells from GITR-sufficient mice were treated as stated above in the presence or absence of 10 µg/ml anti-GITR agonistic antibodies (DTA-1 clone). Histograms represent CFSE dilution in live, B220⁺ B lymphocytes. Numbers represent frequency of dividing (CFSE^low) cells in the population. Data are representative of 2 independent experiments.</p

B cells express GITR starting at the transitional stage of development.

<p>(A) Bone marrow and spleen cells from wild-type (129S1) mice were analyzed <i>ex vivo</i> by flow cytometry for the expression of indicated markers. Live cells in dot plots were gated as indicated on top. Histograms depict GITR expression on B cells in subsets gated as shown in the dot plots in top panel. Expression of GITR on gated CD4⁺ T cells is shown as a positive control for GITR staining. Rat IgG2b was used as an isotype control for GITR staining. Data are representative of 3 independent experiments. Similar GITR expression was observed on B cells from BALB/c mice (data not shown). (B) GITR^+/+ (BALB/c) mice were immunized i.p. with 100 µg NP₃₉-CGG in Alum. At days 4, 7, and 15 post immunization, spleen cells were harvested from 2 immunized and 2 nonimmunized (naïve) mice at each point and analyzed for GITR expression on germinal center B cells reactive with NP. The dot plots show NP and PNA binding on B220⁺PNA^high gated B cells from one naive (left panel) and one immunized (right panel) mouse at day 7. There were no NP⁺ germinal center B cells at day 4, and reduced frequency at day 15 (data not shown). The histogram represents live, B220⁺IgD⁻CD3⁻CD11b⁻PNA^high germinal center B cells. GITR expression on NP⁺ germinal center B cells of one mouse at day 7 (black intact line) and one mouse at day 15 (gray intact line) following immunization are shown compared to those on total B220⁺PNA⁻ B cells (dark-shaded histogram and dashed black line) in respective animals and on lymphocytes of one GITR^−/− mouse (light-shaded histogram).</p