B Cell Requirement for Robust Regulatory T Cell Responses to Friend Retrovirus Infection

Abstract

Regulatory T cells (Tregs) are immunosuppressive cells of the immune system that control autoimmune reactivity. Tregs also respond during immune reactions to infectious agents in order to limit immunopathological damage from potent effectors such as CD8⁺ cytolytic T lymphocytes. We have used the Friend virus (FV) model of retroviral infection in mice to investigate how viral infections induce Tregs. During acute FV infection, there is significant activation and expansion of thymus-derived (natural) Tregs that suppress virus-specific CD8⁺ T cell responses. Unlike conventional T cells, the responding Tregs are not virus specific, so the mechanisms that induce their expansion are of great interest. We now show that B cells provide essential signals for Treg expansion during FV infection. Treg responses are greatly diminished in B cell-deficient mice but can be restored by adoptive transfers of B cells at the time of infection. The feeble Treg responses in B cell-deficient mice are associated with enhanced virus-specific CD8⁺ T cell responses and accelerated virus control during the first 2 weeks of infection. In vitro experiments demonstrated that B cells promote Treg activation and proliferation through a glucocorticoid-induced receptor superfamily member 18 (GITR) ligand-dependent mechanism. Thus, B cells play paradoxically opposing roles during FV infection. They provide proliferative signals to immunsosuppressive Tregs, which slows early virus control, and they also produce virus-specific antibodies, which are essential for long-term virus control.IMPORTANCE When infectious agents invade a host, numerous immunological mechanisms are deployed to limit their replication, neutralize their spread, and destroy the host cells harboring the infection. Since immune responses also have a strong capacity to damage host cells and tissues, their magnitude, potency, and duration are under regulatory control. Regulatory T cells are an important component of this control, and the mechanisms that induce them to respond and exert immunosuppressive regulation are of great interest. In the current report, we show that B cells, the cells responsible for making pathogen-specific antibodies, are also involved in promoting the expansion of regulatory T cells during a retroviral infection. In vitro studies demonstrated that they do so via stimulation of the Tregs through interactions between cell surface molecules: GITR interactions with its ligand (GITRL) on B cells and GITR on regulatory T cells. These findings point the way toward therapeutics to better treat infections and autoimmune diseases.

Keywords: B cells; regulatory T cells; retroviruses; tumor necrosis factor receptors.

FIG 1

Fewer Tregs in B cell-deficient mice. Spleens were harvested from naive C57BL/10 mice (A to E) or B cell-deficient μMT mice (B to E), and CD4, CD8, CD19, Ki-67, and FOXP3 expression was analyzed by flow cytometry. (A) Representative gating strategy for CD4⁺ T cells (top) or CD8⁺ T cells (bottom). (B) Quantification of the percentage of FOXP3⁺ cells within the CD4⁺ T cell gate. (C) Quantification of the percentage of Ki-67⁺ cells within the Treg (CD4⁺ FOXP3⁺) gate. (D) Quantification of the percentage of CD8⁺ cells within the non-B cell lymphocyte gate (CD19⁻ lymphocytes). (E) Quantification of the percentage of Ki-67⁺ cells within the CD8⁺ T cell gate. Each dot in panels B to E represents an individual mouse pooled from experiments performed with 4 to 5 mice per group. Lines indicate the means of data for the group. In panels B to E, statistical significance was determined with a Student’s t test; ns, = P > 0.05.

FIG 2

Impaired Treg induction during FV infection of B cell-deficient mice. (A to E) C57BL/10 (B10) and μMT mice were infected with 20,000 SFFU of FV i.v. or left naïve, spleens were harvested at 1 week (B to E), 2 weeks (A to E), or 3 weeks (B and C) postinfection, and CD4, FOXP3, and CD43 expression was analyzed by flow cytometry. (A) Representative gating strategy for Tregs (CD4⁺ FOXP3⁺) in B10 mice (left) or μMT mice (right) at 2 wpi. (B to E) Quantification of the percentage of Tregs among CD4⁺ T cells (B), the total number of Tregs (C), the percentage of CD43⁺ Tregs (D), and the total number of CD43⁺ Tregs per spleen (E). In panels B to E, each dot represents an average of 5 to 12 mice from pooled experiments and error bars represent ± standard errors of the means (SEM). ***, P < 0.001; ****, P < 0.0001; ns, P > 0.05 (comparing B10 to μMT mice at the indicated time point, as determined by one-way ANOVA with Tukey's posttest for multiple comparisons).

FIG 3

B cells induce Treg proliferation and activation in vitro. (A to E) Untreated Tregs were harvested by FACS analysis of GFP⁺ cells from FOXP3-GFP mice and labeled with CellTrace Violet. B cells were harvested from naive or FV-infected mice at 1 wpi with CD19⁺ beads and stained with CellTrace Far Red. Tregs were cultured in the absence of B cells or with B cells at a B cell/Treg ratio of either 10:1 (A to D) or 1:1 (E) in the presence of 10 µg/ml anti-CD3 and 10 ng/ml IL-2. As a control, unstimulated (Unstim) Tregs were cultured in the absence of anti-CD3 and IL-2. For panel E, naive B cells were either left untreated or treated with 10 µg/ml anti-GITRL for 1 h prior to coculture. After 2.5 days of coculture, cells were harvested and CD4⁺ CellTrace Far Red⁻ Tregs were analyzed for FOXP3, Ki-67, CD43, and CellTrace violet expression by flow cytometry. (A) Representative dot plots of Ki-67 and CellTrace Violet expression on Tregs (CD4⁺ CellTrace Far Red⁻). The gate shows proliferating cells (Ki-67⁺ CellTrace Violet⁻). (B to E) Quantification of proliferation (% Ki-67⁺ CellTrace Violet⁻) (B and E), CD43 expression (C), and FOXP3 (D) expression among Tregs (CD4⁺ CellTrace Far Red⁻). In panels B to E, bars represent means ± SEM of data from 5 to 12 mice in pooled experiments. **, P > 0.01; ****, P > 0.0001 (as determined by one-way ANOVA with Tukey's posttest for multiple comparisons).

FIG 4

Enhanced anti-FV CD8⁺ T cell responses in B cell-deficient mice. (A to F) C57BL/10 (B10) mice and μMT mice were infected with 20,000 SFFU of FV i.v. or left naïve, spleens were harvested at 2 weeks (A to F) or 3 weeks (B to F) postinfection, and CD8, GrB, CD11a, and D^b-GagL dextramer binding was analyzed by flow cytometry. (A) Representative gating strategy for CD8⁺ T cells at 2 wpi. (B to F) Quantification of the percentage of dextramer⁺ (B), percentage of dextramer⁺ GrB⁺ (C), absolute number of dextramer⁺ GrB⁺ (D), percentage of CD11a⁺ GrB⁺ (E), and absolute number of CD11a⁺ GrB⁺ (F) cells among splenic CD8⁺ T cells. In panels B to F, each dot represents an average of 5 to 12 mice from pooled experiments and error bars show ± SEM. *, P < 0.05; **, P < 0.01 (comparing B10 mice to μMT mice at the indicated time point, as determined by one-way ANOVA with Tukey's posttest for multiple comparisons).

FIG 5

More-rapid control of FV in B cell-deficient mice. (A to D) C57BL/10 (B10) mice and μMT mice were infected with 20,000 SFFU of FV i.v. or left naïve, and spleens were harvested at 1, 2, or 3 weeks postinfection for infectious center assays. (A and B) Flow cytometry analysis of Ter119, CD11b, and FV glycogag (mAb34⁺) expression (see panels C and D). (A) Total FV infectious centers per spleen. (B) FV infectious centers per gram of spleen. (C) Percent Ter119⁺ (erythroid progenitor) cells within the total splenocyte gate. (D) Percent glycogag⁺ (% Infected, mAb34⁺) CD11b⁺ monocytes. Each dot represents an average of 5 to 12 mice from pooled experiments, and error bars show ± SEM. **, P < 0.01; ****, P < 0.0001 (comparing B10 mice to μMT mice at the indicated time point, as determined by one-way ANOVA with Tukey's posttest for multiple comparisons).

FIG 6

Induction of Tregs following adoptive transfer of B cells. Donor B cells were purified from naive B10 mice, and 5 × 10⁷ B cells/mouse were injected i.v. into μMT mice 1 day before infection with 20,000 SFFU of FV i.v. or left naive. B10 mice or uMT mice without B cell adoptive transfer were used as controls. At 2 weeks postinfection, spleens were harvested and the absolute number of CD4⁺ FOXP3⁺ cells per spleen was analyzed by flow cytometry. Each dot represents an individual mouse pooled from experiments performed with 2 to 4 mice per group. Bars indicate means. ***, P < 0.001 (as determined by one-way ANOVA with Tukey's posttest for multiple comparisons).