Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production

Abstract

To identify genes and mechanisms involved in humoral immunity, we did a mouse genetic screen for mutations that do not affect the first wave of antibody to immunization but disrupt response maturation and persistence. The first two mutants identified had loss-of-function mutations in the gene encoding a previously obscure member of a family of Rho-Rac GTP-exchange factors, DOCK8. DOCK8-mutant B cells were unable to form marginal zone B cells or to persist in germinal centers and undergo affinity maturation. Dock8 mutations disrupted accumulation of the integrin ligand ICAM-1 in the B cell immunological synapse but did not alter other aspects of B cell antigen receptor signaling. Humoral immunodeficiency due to Dock8 mutation provides evidence that organization of the immunological synapse is critical for signaling the survival of B cell subsets required for long-lasting immunity.

Figure 1. Discovery of two independent, non-complementing ENU mutant mouse strains with humoral immunodeficiency characterized by failure to sustain a primary immune response and failure of maturation of the antibody response

(a) Representative screening assay results. IgG1 antibody to CGG and ARS, IgM antibody to NP, and IgG2c antibody to B. pertussis measured by ELISA optical density (OD) in serum collected 14 days post-primary immunization (d14) and 6 days after booster immunization (d44). To detect low responders among large numbers of mice, serum was tested at a fixed dilution yielding sub-saturating antibody titers in wild-type animals (CGG 1/2000, 1/20,000; BP 1/50; NP 1/50; ABA 1/2000). (b) Antibody titers to CGG and ARS followed longitudinally 14, 28 and 56 days after primary immunization, determined by titrating each serum sample against a standard reference serum whose titer was set as 1. Statistical comparison by Student’s t-test: ns, not significant; * P < 0.05; ** P < 0.005; *** P < 0.0005). In a,b, columns are arithmetic means, bars standard error of the mean (s.e.m), and dots individual mice. Data are representative of 3 independent experiments.

Figure 2. Analysis of splenic and peritoneal lymphocyte subsets

(a, b) Number of immature transitional 1-3 B cells (T1-T3, Trans), mature follicular B cells (Foll), marginal zone B cell precursors (MZP) and marginal zone B cells (MZ) in spleen of individual mice of the indicated genotypes. MZ B cell numbers were statistically significant (unpaired, two-tailed t test, (a) P = 0.006, (b) P = 0.038), while other B subsets were not significantly different. Data representative of 2 independent experiments. (c) Representative plots (from >5 independent experiments) showing % of B cells in the MZ B cell subset. (d) Immunohistochemistry of splenic MZ B cell marker CD1d and marginal sinus marker MAdCAM. (e) Percentage of mutant or wild-type CD45.2⁺ cells reconstituting the indicated B cell subsets in the spleen of individual mixed chimera mice. Differences in MZ contribution are representative of two separate cohorts of pri/pri mixed chimeras and two cohorts of cpm/cpm mixed chimeras, while no consistent difference in contribution to follicular or transitional cells was observed. (f) Numbers of B1, B1a, B1b and B2 subsets in the peritoneum of mice of the indicated genotypes. Representative of two independent experiments. (g) Numbers of splenic T cells in individual mice of the indicated genotypes. Naïve T cell numbers (CD44^lo) were statistically significantly different (ANOVA) for both CD4⁺ (P = 0.0003) and CD8⁺ T cells (P = 0.0001). Differences in CD8CD44^hi cells were statistically significantly different (P = 0.048), but not in CD4CD44^hi. (h) Percentage of mutant or wild-type CD45.2⁺ cells reconstituting the indicated splenic cell subsets of individual mixed chimeras. Representative of three independent experiments.

Figure 3. Independent Dock8 mutations in pri and cpm mouse strains

(a) Conserved DOCK8 domains and mutations. (b) Dock8 exon 18-21 cDNA amplification products from thymus (Thy) and spleen (Spl) of wild-type and cpm mice. Data are representative of 4 independent experiments. (c) Sequence of cDNA clones from cpm/cpm mice in (b), aligned to wild-type Dock8 cDNA sequence. Number of clones of each type shown on left. //, segment of normal exon 20 sequence omitted. (d) Sequence alignment of DOCK8 orthologues and paralogues in the region surrounding the pri mutation. Mutated serine in bold; dashes indicate identity to mouse DOCK8; secondary structure elements based on the structure of DOCK9 ; triangles show Cdc42 contact residues in DOCK9 .

Figure 4. DOCK8 mutation causes an intrinsic defect in germinal center B cells

(a) Immunohistochemistry of splenic PNA⁺ germinal centers and IgD⁺ follicular mantle 7 days after SRBC-immunization (scale bars: upper 500μm; lower 200μm). (b) Enumeration of GC B cells as percentage of B220⁺ in untreated mice (nil) or 5, 7 and 11 days after SRBC-immunization. Columns are arithmetic means, bars standard error of the mean (s.e.m), and dots individual mice (c) Contribution to different B cell subsets in 50:50 mixed bone marrow chimeras 7 or 11 days after SRBC immunization. Columns show percentage of CD45.2⁺ cells of the indicated genotypes in individual, numbered chimeric mice. Each bar represents an individual mouse. Numbers indicate total number of GC cells ×10⁶ per spleen (mean+/−SD) in each group of mice. Data are from 3 independent experiments.

Figure 5. DOCK8 mutant B cells undergo normal T-cell dependent activation, switching and initial differentiation into GC cells in vivo

HEL-specific pri/pri or wild-type (+/+) B cells from SW_HEL transgenic mice, allotypically tagged by CD45.1, were adoptively transferred into wild-type mice that were immunized with HEL^2X-SRBC and analyzed on the indicated days after transfer and immunization. (a) Representative flow cytometric analyses of spleen gated on HEL⁺ CD45.1⁺ B220⁺ SW_HEL B cells, showing percentage of GL7⁺ Fas⁺ cells (left) and IgG1⁺ switched and unswitched cells (right). Control mice given SW_HEL B cells but immunized with unconjugated SRBC lacking HEL (SRBC) are marked (*). (b) Enumeration of number of HEL⁺ CD45.1⁺ B220⁺ SW_HEL B cells on the indicated days after immunization, and the percentage of SW_HEL B cells that were GL7⁺ Fas⁺ or switched to IgG1 in individual animals. Statistical analysis (wild-type vs. pri/pri) by unpaired t-test with Welch’s correction. Unfilled circles represent wild-type, filled circles pri/pri and (*) control srbc mice. Early time-course experiment performed once.

Figure 6. Intrinsic failure of DOCK8 mutant germinal center B cells to persist or undergo affinity maturation

HEL-specific pri/pri or wild-type (+/+) B cells from SW_HEL transgenic mice, allotypically tagged by CD45.1, were injected into wild-type B6 mice and the recipients immunized with HEL^2X-SRBC. (a) Confocal immunofluorescence of spleen cryosections from recipient mice 5 or 10 days after immunization, stained for HEL, IgD and CD3. GCs (arrows) and extrafollicular plasma cell foci (PC), and T cell zones (T) are marked. (b) Representative flow cytometry plots and enumeration of CD45.1⁺ HEL-binding B cells and GC cells derived from the HEL⁺ CD45.1⁺ SW_HEL cells, of the indicated genotypes, 5-9 days after adoptive transfer. Control mice given SW_HEL B cells but immunized with unconjugated SRBC without HEL (srbc) are marked (*). Panels also show total number of GC B cells per spleen in individual mice and the percentage of GC B cells that were SW_HEL B cells (day 9, +/+ vs. pri/pri, P = 0.008) and the percentage and absolute numbers of HEL+ CD45.1+ SW_HEL FAS^hi HEL^lo GC cells and IgG1-switched cells. Unfilled circles represent wild-type, filled circles pri/pri and (*) control srbc mice. (c) Structure of HEL complexed with the SW_HEL antibody HyHEL10 showing the position of the two HEL residues mutated in HEL^2X and four of the D101 contact residues in the heavy chain CDR2. (d) Results of sequencing of heavy chain genes from individual flow-sorted SW_HEL GC B cells (both +/+ or pri/pri). Graphs show the percentage of cells containing the indicated number of mutations in the CDR2; the total number of cells sequenced is indicated in the center. The replacement to silent ratio (R/S) and percentage of cells with a Y58F replacement mutation are also shown. (e) Enumeration of high affinity variants on day 9 post-immunization by direct assay for binding to nanomolar concentrations of D101R mutant HEL^3X (independent experiment from b, d). Flow cytometry plots, gated on B220⁺ CD45.1⁺ SW_HEL B cells, are from concatenated data collected on 11-22 million spleen cells combined from four individual recipients of either +/+ or pri/pri SW_HEL B cells or from two recipients immunized with uncoupled SRBC (*). The mean % of spleen cells that are CD45.1⁺ SW_HEL B cells is shown in blue and the mean % of spleen cells switched to IgG1 is shown in red. Staining with nanomolar HEL^3X reveals mutated B cells with higher affinity BCRs (oval gate), and the mean % spleen cells in this gate shown in black. Panels on right show data for individual animals in each group. Statistical analysis by Student’s t-test with Welch’s correction indicated a significant difference between pri/pri and +/+ SW_HEL cells in percentage of high affinity cells among either CD45.1+ B220+ cells (P = 0.038) or among CD45.1+ B220+ IgG1+ cells (P = 0.031) that remained on day 9. Data are representative of four independent experiments, except (d) and (e) which were each performed once but independently of each other.

Figure 7. DOCK8 mutations disrupt the formation of the B cell immunological synapse but not other aspects of signaling through the B cell antigen receptor

(a) HEL-specific MD4 wild-type or mutant B cells were settled onto lipid bilayers containing mono-biotinylated HEL (green) and Alexa-532-conjugated ICAM-1 (red) and imaged after 10 min of interaction. Differential interference contrast (DIC), confocal fluorescence for HEL and ICAM-1, and interference reflection microscopy (IRM) images of representative cells are shown. Right, quantification of the area of B cell contact with the bilayer, the relative amounts of antigen accumulated at the contact site, and the percentages of B cells forming a peripheral ring of ICAM-1 (pSMAC). Results representative of 3 independent experiments. Columns are means and error bars are s.e.m. (b) Intracellular calcium in mixtures of pri/pri CD45.2 (red trace) and wild-type CD45.1 spleen cells stimulated with anti-IgM (arrow). (c) Expression of CD25 and CD86 on lymph node B cells 18 h after stimulation with 1 or 10 μg/ml anti-IgM. Unstimulated, grey histogram; wild-type, black line; pri/pri, red filled. Representative plots from 4 independent mice. (d) Division of LN B cells measured by dilution of CFSE in B220⁺ 7AAD⁻ cells after 4 d culture with the indicated stimuli. Mutant pri/pri or cpm/cpm CD45.2 cells (red histograms) were CFSE labeled and cultured with wild-type CD45.1 cells (black histograms) in the same wells to control for experimental variation. Representative of 6 out of 8 independent samples tested: in 2 samples there was a subtle decrease in CFSE dilution by pri/pri cells (Supplementary Fig. 9).