Although in vitro studies suggest a role for HP-1γ in the immune system, it has not been determined if it contributes to immunity in vivo. The cbx-3 (gene encoding HP-1γ) mutant mouse was generated by gene-trapping technology as described previously (10, 28). We found that cbx-3^−/− mice died perinatally. Because haploinsufficiency of genes involved in epigenetic modifications has been shown to alter cellular functions (29), we asked if haploinsufficiency of cbx-3 was sufficient to affect the immune system. First we assessed if cbx-3 deficiency influenced progenitor lymphoid development. A survey of the bone marrow (BM) and thymus showed that progenitor B and T cells developed normally in cbx-3^+/− mice compared to littermate controls (Figures 1A,B). Mature B- and T-cell-development also remained normal in cbx-3^+/− mice (Figures 1C,D). Thus, HP-1γ is not required for progenitor or mature B- and T-cell-development.

To determine the physiological function of HP-1γ in the adaptive immune response in mice, we immunized littermate control and cbx-3^+/− mice with the T-dependent Ag 4-hydroxy-3-nitrophenylacetyl hapten conjugated to chicken gamma globulin (NP-CGG) in alum. On day 14 after immunization, the expected frequency of spleen B220⁺CD38^lo/−FAS⁺ GC as well as switched B220⁺CD38^lo∕−IgG1+ B cells was obtained from littermate controls, demonstrating that the GC reaction occurred normally (Figure 2). In contrast, the GC response was impaired in cbx-3^+/− mice. On day 14, the percent of spleen GC B cells in cbx-3^+/− mice decreased by 2.3-fold compared to control mice (p = 0.0002, Figures 2A,B,F). Correspondingly, there was a twofold reduction in the percent of switched IgG1+ spleen B cells in cbx-3^+/− mice compared to control mice (p = 0.0007, Figures 2A,C). Similarly, the frequency of IgG1+ GC B cells in cbx-3^+/− mice was reduced by 2.8-fold compared to wild-type littermate mice (Figures 2D,E, p < 0.0001). The spleen architecture of wt littermate and mutant mice remained intact; and more peanut agglutinin (PNA) positive GCs were detected in wt littermate mice than in mutant mice on day 14 after immunization with NP (Figure 2F). These results demonstrate that HP-1γ positively regulates the GC reaction and production of IgG1+ B cells, and haploinsufficiency of cbx-3 is sufficient to impair these processes. The defect cannot be compensated for by the presence of wild-type HP-1α and HP-1β in cbx-3^+/− mice suggesting that HP-1γ has a non-redundant function in immunity in vivo.

To determine if the diminished GC reaction in cbx-3^+/− mice results in defective serum anti-NP Ab response, we measured anti-NP activity in sera obtained from cbx-3^+/− and wt littermate mice on day 14 after NP immunization. Serum high- and low-affinity responses to NP can be measured by enzyme-linked immunosorbent assay (ELISA) using NP4 and NP25 Ags, respectively. On day 14 after immunization, serum IgG anti-NP25 Ab activity increased in littermate control and mutant mice compared to unimmunized animals, and the level was similar between the two groups. By contrast, the amount of serum anti-NP4 antibodies in littermate control mice was 4.75-fold higher than cbx-3^+/− mice (p = 0.006, Figure 3A). Accordingly, the ratio of NP4/NP25 Ab titer was threefold lower in cbx-3^+/− mice compared to littermate control mice (p = 0.02, Figure 3B). The low-affinity response was not affected by cbx-3 haploinsufficiency. Both littermate control and mutant mice produced low amounts of serum IgM Abs against NP, and the majority of IgM antibodies were of low-affinity (Figures 3C,D). There was no difference in the production of total pre-immune serum IgG₁ and IgM between wt littermate control and mutant mice (Figure 3E). Thus cbx-3^+/− mice could not mount high-affinity Ab response to NP.

To rule out the possibility that reduced GC and Ab responses resulted from defects in proliferation or class switching after Ag encounter, we carried out in vitro proliferation/switch assays. Spleen B cells from cbx-3^+/− mice proliferated and switched as well as littermate control B cells when activated through the B-cell receptor, Toll-like receptor (TLR) 4, or CD40 plus IL-4 (Figures 4A,B). Therefore, HP-1γ deficiency results specifically in impaired high-affinity, not total NP Ab response. HP-1γ does not control the IgM response to NP. Because HP-1γ deficiency does not perturb proliferation or switching, the defect in high-affinity Ab response observed in cbx-3^+/− mice implies that HP-1γ may regulate Ab affinity maturation.

T follicular helper cells play a crucial role in affinity maturation in part by selecting B cells to enter the GC, regulating GC positive selection, and directing B-cell differentiation to plasma cells and memory B cells. Hence, the high-affinity Ab response defect seen in cbx-3^+/− mice may arise from inefficient T_FH support. Fluorescence-activated cell sorting (FACS) analysis showed that as the immune response proceeded to day 14, the frequency of TCRβ⁺CD4⁺CXCR5^hiPD-1^hi T_FH cells decreased by 1.7-fold in cbx-3^+/− mice compared to wt littermate control mice (p < 0.0001, Figures 5A,B). Moreover, we did not detect any differences in Bcl6, Prdm1, or Aicda expression between wt littermate control and mutant mice suggesting that GC and plasma cell differentiation was not affected by HP-1γ deficiency (data not shown). Thus, HP-1γ governs Ab affinity maturation perhaps by controlling the size of the T_FH-cell compartment during an immune response to T-dependent Ags.

To determine if T_FH cells were directly responsible for the GC phenotype observed, we generated cbx-3^+/+/cbx-3^+/− mixed BM chimeras. Recombinase activating gene 2 and common γ chain double knock out (B6-Rag2^−/−cγ^−/−) mice were reconstituted with a 1:1 mix of either CD45.1 cbx-3^+/+/CD45.2 cbx-3^+/+ or CD45.1 cbx-3^+/+/CD45.2 cbx-3^+/− BM. Eight weeks after reconstitution chimeric mice were immunized with NP-CGG in alum. On day 14 after immunization, mice were analyzed to determine the frequency of GC B cells, switched IgG1+ B cells and T_FH cells derived from CD45.2 (control cbx-3^+/+ or cbx-3^+/−) donor BM in each mouse (Figure 6A). FACS analysis showed that CD45.2 cbx-3^+/+ and CD45.2 cbx-3^+/− chimeric mice had similar percentage of GC and IgG1+ B cells as well as T_FH cells (Figure 6B). Therefore, the GC defect observed in cbx-3^+/− is not intrinsic to B or T_FH cells.

Recently, Kim and colleagues showed that CD122⁺Ly49⁺CD3⁺ CD8⁺ regulatory T (T_reg) cells served to inhibit the expansion of T_FH population during an immune response to foreign Ags as well as to self-Ags (26, 27). Thus it is plausible that reduction in the T_FH compartment in cbx-3^+/− mice may be due to an increase in CD8⁺ T_reg cells within the CD8⁺ T-cell compartment. On days 7 and 14 after immunization, compared to wt littermate mice, cbx-3^+/− mice had 2.3- and 1.8-fold higher frequency of spleen CD8⁺ T_reg cells, respectively (p < 0.0001, Figures 7A,B). Correspondingly, the number of spleen CD8⁺ T_reg cells in cbx-3^+/− mice increased by 1.8- and 1.75-fold on days 7 and 14, respectively (p < 0.0001, Figure 7C). Next, western blots were carried out to assess the expression status of HP-1γ in mutant CD8⁺ T cells. To our surprise, CD8⁺ and CD4⁺ T cells as well as B cells from cbx-3^+/− mice expressed much less HP-1γ than control cells despite the presence of one wild-type allele (Figure 7D). To ensure that the expansion of CD8⁺ T_reg cells in cbx-3^+/− mice was intrinsic to the CD8⁺ T-cell population, mixed BM chimeras were generated as described in Figure 6A. Mice were allowed to reconstitute for 8 weeks. On day 14 after immunization, reconstituted mice were analyzed to assess the frequency of T_reg cells derived from CD45.2 (control cbx-3^+/+ or cbx-3^+/−) donor BM in each mouse. As shown in Figure 7E, CD45.2 cbx-3^+/− chimeric mice had 4.6-fold more CD8⁺ T_reg cells than CD45.2 cbx-3^+/+ control mice. Thus, the CD8⁺ T_reg population expanded in cbx-3^+/− mice. These results suggest that HP-1γ limits the size of CD8⁺ T_reg cells during an immune response, and the effects are intrinsic to these cells.

To investigate if CD8⁺ T_reg cells from cbx-3^+/− mice directly controlled the Ab response, adoptive transfers into B6-Rag2^−/−cγ^−/−recipients were performed. Group 1 (control) recipients received B cells, CD4⁺ and CD8⁺ T cells from wt littermate mice; group 2 (experimental) received B cells and CD4⁺ T cells from wt littermate mice, and CD8⁺ T cells from cbx-3^+/− mice; group 3 (control) received B cells and CD8⁺ T cells from wt littermate mice, and CD4⁺ T cells from cbx-3^+/− mice (Figure 8A). On day 10 after immunization with NP-CGG in alum, recipients were analyzed to assess the status of the GC response. The percent of GC B cells from group 2 was 2.2-fold lower than groups 1 and 3 (p = 0.04 and p = 0.02, respectively, Figure 8B). Additionally, group 2 recipients had 1.7- and 1.9-fold less T_FH cells than groups 1 and 3, respectively (Figure 8C). By contrast, the frequency of CD8⁺ T_reg cells from group 2 was three- and fourfold higher than groups 1 and 3, respectively (Figures 8D,E). Thus, HP-1γ-deficient CD8⁺ T cells alone have the propensity to reduce the GC response. The results suggest that HP-1γ positively regulates GC and high-affinity Ab responses to T-dependent Ags by curtailing the ability of CD8⁺ T cells to inhibit an immune response.

cbx-3 mutant mice were generated, as described in Ref. (10, 28). Mice were backcrossed to C57BL/6 for 12 generations. B6-Rag2^−/−Cγ^−/− and B6.SJL mice were purchased from Taconic. All mice were maintained in specific pathogen-free conditions. All mouse protocols were approved by the BIDMC Institutional Animal Care and Use Committee.

Fluorescence-activated cell sorting was performed on the BD 5-laser LSR II. Analysis was carried with FlowJo software (Tree Star, Inc.). All fluorochrome-conjugated antibodies were purchased from Biolegend or BD Biosciences. The following antibodies were used: ckit-APC (1:200); CD25-PE (1:200); IgM-FITC (1:500); CD8-Pacific blue (1:200); CD8-APC-Cy7 (1:300); CD8-PE-Cy7 (1:200); Ly-49-FITC (1:100); CD44-Pacific blue (1:200); IgD-PE (1:500); CD21-APC (1:200); CD23-PE (1:150); CD19-PE-Cy7 (1:300); B220-Pacific blue (1:300); CD38-APC (1:200); IgG1-FITC (1:50); FAS-PE (1:200); CD4-FITC (1:200); CD4-PE (1:150); TCRβ-Brilliant-Violet 412 (1:200); PD1-PE-Cy7 (1:100); CXCR5-Biotin (1:100); SA-PerCP (1:100); CD45.1-FITC (1:150); CD45.2-PE-Cy7 (1:100); CD45.2-Pacific blue (1:200); CD3-APC (1:200); CD122-Pacific blue (1:200).

Adult mice (7–8-week-old) were immunized with 50 μg of the T-dependent Ag 4-hydroxy-3-nitrophenylacetyl hapten conjugated to chicken gamma globulin (NP-CGG, BioSearch Technologies) per mouse in alum (Thermo Scientific) (ratio 1:1). Immune sera were obtained at days 7 and 14 after immunization. FACS analysis was performed on the same days.

Immunohistochemistry was performed using 4 μm thick formalin-fixed, paraffin-embedded tissue sections. Briefly, slides were soaked in xylene, passed through graded alcohols, and put in distilled water. Slides were then pre-treated with 1.0-mM EDTA, pH 8.0, or 1.0 mM citrate (Zymed) in a steam pressure cooker (Decloaking Chamber, BioCare Medical) as per manufacturer’s instructions, followed by washing in distilled water. All subsequent steps were performed at room temperature in a hydrated chamber. Slides were pre-treated with Peroxidase Block (DAKO) for 5 min to quench endogenous peroxidase activity, followed by Serum free Protein Block (DAKO) for 20 min. Biotinylated PNA (Vector Laboratories) was applied for 1 h (all diluted in DAKO diluents). Slides were washed in 50 mM Tris–Cl, pH 7.4. Slides were washed again, and detected with anti-streptavidin–HRP Envision + kit (DAKO) as per manufacturer’s instructions. After further washing, immunoperoxidase staining was developed using a DAB chromogen (DAKO) and counterstained with hematoxylin. Images were acquired with the Nikon Eclipse E600 and SPOT Insight four camera and software.

Antibody response to NP was determined by ELISA using NP(4)-BSA or NP(25)-BSA (BioSearch Technologies) from days 7 and 14 immune sera. ELISA was performed as described (11).

MACS-purified (Miltenyi Biotec) CD43⁻ or CD19⁺ B cells were activated in vitro at a density of 1–3 × 10⁶ cells/ml with 2 μg/ml of anti-CD40 clone HM40-3 (eBiosciences) plus 25 ng/ml of recombinant mouse IL-4 (R&D Systems), 10 μg/ml of goat F(ab′)₂ anti-mouse IgM (Jackson Immunoresearch), LPS (20 μg/ml), or LPS + IL-4 (Sigma).

MACS-purified CD43⁻ B cells labeled with CFSE were activated with indicated stimuli as above for 3 or 4 days. Data were analyzed using the proliferation platform of the FlowJo software (Tree Star Inc.).

Bone marrow cells from femurs and tibias of 4-week-old B6.SJL mice (CD45.1) were mixed with either 4-week-old cbx3^+/− or littermate control mice (CD45.2) at a 1:1 ratio. Mixed BM cells (2 × 10⁶) were injected i.v. into 7-week-old Rag2^−/−Cγ^−/−recipients. Eight weeks after BM reconstitution, recipients were immunized with NP-CGG in alum (ratio 1:1). Analysis was carried out 14 days after immunization.

Purified CD8⁺CD44⁻ (1 × 10⁶) cells were lysed with radio-immunoprecipitation assay (RIPA) buffer (Boston BioProducts) containing protease inhibitor cocktail (Roche) on ice for 30 min. Cells were centrifuged at 14,000 rpm for 15 min at 4°C. Protein concentration was determined by Bio-Rad Protein Assay Kit (Bio-Rad). Ten micrograms of protein extracts were denatured at 95°C for 10 min, separated by SDS-PAGE, and transferred onto PVDF membranes (EMD Millipore). Membranes were probed with antibodies against HP-1γ (Cell Signaling Technology) or β-actin (Sigma Aldrich). Proteins of interest were detected with HRP-conjugated secondary antibodies and visualized with the Pierce ECL Western blotting substrate (Thermo Scientific).

B, CD8⁺, and CD4⁺ cells were prepared from spleen and lymph nodes of 7-week-old cbx3^+/− and wt littermate mice as described (18). 2 × 10⁶ wild-type B cells, 1 × 10⁶ wild-type or mutant CD8⁺, and 1 × 10⁶ wild-type or mutant CD4⁺ cells were injected i.v. into 7-week-old Rag^−/−Cγ^−/−recipients. The following day, recipients were immunized with NP-CGG in alum (ratio 1:1). Analysis was carried out 10 days after immunization.

P values were calculated using one-way ANOVA and graphs were plotted with Prism 6 (GraphPad Software).