Elevated Detection of Dual Antibody B Cells Identifies Lupus Patients With B Cell-Reactive VH4-34 Autoantibodies

Abstract

About 5% of B cells in healthy mice and humans are allelically or isotypically included and hence co-express two different antibodies. In mice, dual antibody B cells (B2R) expand with systemic autoimmunity, co-express autoreactive and non-autoreactive antibodies, and participate in immune responses, but this phenomenon is strain dependent. This study was developed with two goals: 1) to establish the contribution of TLR and IFN receptor signaling to the development of germinal center B cells that express two antibodies in MRL/lpr mice; and 2) to determine whether B2R B cells are increased and particularly activated in a subset of adult patients diagnosed with systemic lupus erythematosus (SLE). Results from the MRL/lpr studies indicate that the enhanced differentiation of dual-κ B cells into germinal center B cells is due to a heightened response to TLR7 and TLR9 signaling, further fueled by an increased response to type II IFN. To understand the clinical and translational implications of our observations in mouse B2R B cells, cohorts of SLE patients and healthy controls were recruited and evaluated for expression of dual BCRs. Results from flow cytometry and microscopy revealed supraphysiological frequencies of κ+λ+ B2R cells in one fourth of the SLE patients. Abnormal numbers of κ+λ+ B cells correlated with higher frequencies of activated naïve B cells and age-associated B cells, and a lower proportion of "B cells that are naïve IgD+" (BND). However, results from single cell V(D)J sequencing demonstrated that these high κ+λ+ SLE patients harbored normal frequencies of κ+λ+ and other B2R B cells. and we further show that their B cells were instead decorated by κ and λ VH4-34 autoantibodies. Thus, our findings indicate that elevated flow cytometric detection of isotypically-included B cells can identify patients with high titers of B cell-reactive VH4-34 autoantibodies and abnormal distribution of B cell subsets relevant to autoimmunity.

Keywords: B cell; SLE; VH4-34; antibodies; autoimmunity; lupus; single cell RNA-seq.

Conflict of interest statement

JT receives royalties from Alexion Pharmaceuticals, Inc. and is a consultant for Q32 Bio, Inc., a company developing complement inhibitors. He also holds stock and will receive royalty income from Q32 Bio, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Peterson, Boackle, Taitano, Sang, Lang, Kelly, Rahkola, Miranda, Sheridan, Thurman, Rao, Torres and Pelanda.

Figures

Figure 1

IFNα and IFNγ synergize with TLR7 and TLR9 to promote the differentiation of single-κ and dual-κ MRL/lpr B cells into GC B cells. (A) Schematic of the in vivo treatment of 6 week old MRL/lpr mice with the TLR9 ligand CpG. (B) Flow cytometric analysis and gating strategy of splenic B cells from MRL/lpr mice injected with either PBS or CpG. The cells were serially gated to measure the frequency of PNAhighCD38low GC B cells within the single-κ and dual-κ B cell populations. (C, D) Frequencies of dual-κ B cells and PNAhighCD38low GC B cells within the single-κ and dual-κ cell populations, in MRL/lpr mice treated with either PBS or CpG. N=4, analyzed in two independent experiments. Data is shown as mean+SD. Statistical analysis was performed with a one-tailed unpaired t-test. (E) Flow cytometric analysis of B cells enriched from the spleen of MRL/lpr mice (8-15 weeks of age) and cultured for 60 hours in media alone or with R848, R848+IFNα, or R848+IFNγ. The cells were serially gated to measure BCL6+GL7high GC B cells within the single-κ and dual-κ B cell populations. (F) Frequencies of BCL6+GL7high GC B cells within the single-κ and dual-κ B cell populations of MRL/lpr B cells cultured as described in (E). N=6, analyzed over one experiment (data from a similar experiment are in Supplementary Figures S1D–F). Data are shown as mean+SD. Statistical analysis was performed with a one-tailed unpaired t-test. *p ≤ 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Figure 2

Detection of κ+λ+ B cells in SLE patients and healthy controls. (A) Representative flow cytometric analysis and gating strategy of CD19+ B cells from the blood of healthy controls and SLE patients. The top two plots to the right demonstrate isotype control staining for Igκ and Igλ. (B) Igκ and Igλ expression on CD19+ B cells from representative healthy controls (HC) and SLE-L patients (top), and from all SLE-H patients with high κ+λ+ cells (bottom). Data were collected over a total of 18 independent analyses. (C) Frequency of κ+λ+ cells within the blood CD19+ B cell population in each healthy control and SLE patient. N=18 in each group. (D) Frequency of κ+λ+ cells within the blood CD20+ B cell population in healthy controls (N=5) and ALPS patients (N=10). Data were collected over 3 independent analyses. (E) Representative Image-Stream analysis of PBMCs stained for CD19 (red), Igλ (green), and Igκ (blue). The top three rows show example of CD19+ B cells that express both κ and λ; the cell shown in the third row was defined as bright for both κ and λ (κhighλhigh). Bottom two rows are examples of single λ+ or κ+ cells. Brightfield, individual fluorescence, and merge fluorescence are shown for each cell. (F) Frequency of all κ+λ+ cells and of bright (κhighλhigh) cells analyzed by Image-Stream among CD19+ cells from the blood of N=4 healthy controls and N=4 SLE patients with high κ+λ+ cell frequency. The analysis was performed on an average of 2263 ± 740 CD19+ cells (range 1470-3074) per sample. Data are from one experiment. In all graphs, each symbol is an individual, lines represent mean ± SD, and statistical analysis was performed with a Mann-Whitney U test. *p < 0.05; **p < 0.01; ns, not significant.

Figure 3

Distribution of κ+λ+ B cells among human B cell subsets. (A) Representative flow cytometric analysis and gating strategy of human CD19+ B cell subsets in PBMCs. All B cell subsets, except for the CD38highCD27high (PBs&PCs) subset representing plasmablasts (PBs) and plasma cells (PCs), were analyzed for surface (s) Igκ and Igλ expression, and the frequency of κ+λ+ cells was measured only on cells positive for sIgκ and/or sIgλ, as shown for the CD38vsCD24 plot. For the CD38highCD27high (PBs&PCs) subset, Igκ and Igλ were measured after cell fixation and permeabilization and, therefore, include intracellular (IC) expression. (B) Frequency of κ+λ+ cells within each B cell subset from healthy controls (N=18), and from SLE patients with low (SLE-L, N=12) and high (SLE-H, N=6) frequency of κ+λ+ cells. Data were collected over a total of 18 independent analyses. Each symbol is a subject, and bars represent mean+SD. Differences between groups were analyzed by a two-tailed Mann-Whitney U test. Differences between the HC group and each of the SLE groups are reported on top of each SLE bar. Differences between the transitional B cell subset and the other B cell subsets only in the SLE-H group, are depicted with horizontal lines on top of graph, and only if significant. *p ≤ 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Figure 4

Distribution of B cell subsets in SLE patients and healthy controls. (A) Distribution of CD19+ B cells among B cell subsets in blood of healthy controls (HC, N=18) and SLE patients with low κ+λ+ (SLE-L, N=12) or high κ+λ+ (SLE-H, N=6) cells. Mature naïve B cells are graphed separately to visualize better group differences within the smaller B cell subsets. The B cell subsets were gated as shown in Figure 3A. PBs&PCs(1) were gated as CD38highCD24low, while PBs&PCs(2) were gated as CD38highCD27high. Data were collected over a total of 18 independent analyses. Each symbol is a subject, and bars represent mean+SD. Statistical analysis was performed with a Mann-Whitney U test. Statistical significance for differences between each SLE group and the healthy control group are shown on top of each SLE group bar. Differences between the SLE-L and SLE-H groups are shown with horizontal lines on top of the SLE bars and only if significant. (B, C) Scatter plot analyses of the percentage of ABCs (y-axis) relative to the percentage (on x-axis) of either BND (B) or DN (C) cells within the CD19+ B cell population of SLE patients (left graphs) and healthy controls (right graphs). Cells were analyzed as shown in Figure 3A and data were collected over the course of 18 independent analyses. Data were analyzed by Simple linear regression. *p ≤ 0.05; **p < 0.01; ***p < 0.0001; ns, not significant.

Figure 5

Single cell V(D)J-seq analysis of human B cells. (A) Average (mean+SD) Ig VH usage among all (switched and unswitched) B cell clonotypes from three healthy controls (HC) and three SLE-H patients. VH usage was obtained via 10X Genomics V(D)J-seq se analysis over two independent runs. The number of clonotypes per sample was between 887 and 5,418. Statistical analysis was performed with two-tailed unpaired t-tests. (B) Percentage of unswitched (IgM/D) or switched (Igsw) B cell clonotypes bearing one heavy chain and a κ and a λ light chains. Symbols represent individual subjects and bars are mean+SD. Statistical analysis was performed with two-tailed Mann-Whitney U tests. (C) Scatter plot analysis of the percentage of Ig switched (IgM–IgD–) B cells measured by 10X (y-axis) or flow cytometry (x-axis) in the same SLE-H and healthy control subjects. Data were analyzed by Simple linear regression. (D, E) Amino acid (AA) length of the heavy chain CDR3 sequence (D) and Jκ usage (E) in IgM/D (unswitched) κ+ and κ+λ+ clonotypes from healthy controls and SLE-H patients. Statistical analysis was performed with two-tailed unpaired t-tests. *p ≤ 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant (differences were not significant when lacking asterisks).

Figure 6

Analysis of VH4-34 B cells and antibodies. (A) Frequency of VH4-34-bearing clonotypes among all unswitched (IgM/D) and switched (Igsw) clones obtained by 10X Genomics V(D)J sequencing in three healthy controls (HC) and three SLE-H subjects. (B) Relative titers of VH4-34 IgM (left) and VH4-34 IgG (right) in sera of healthy control (HC, N=4), SLE-L (N=11) and SLE-H (N=6) subjects. Titers are shown on logarithmic scale and relative to one of the HC samples. (C) Representative flow cytometric analyses of 9G4 (anti-VH4-34) staining vs CD19 (top) and IgM (bottom) on CD19+ gated B cells from healthy and SLE subjects. (D) Frequency of 9G4+ B cells measured by flow cytometry in healthy (N=6), SLE-L (N=6) and SLE-H (N=3) subjects over 4 independent experiments. (E, F) Scatter plot analyses of the percentage of 9G4+ B cells (y-axis) relative to either (on x-axis) VH4-34 IgM sera titers (E) or the frequency of κ+λ+ B cells (F) in SLE patients. N=9, 6 SLE-L and 3 SLE-H. Data were analyzed by simple linear regression. (G) Schematic modeling the decoration of B cells by VH4-34, κ or λ, antibodies. (H) Flow cytometric analyses of CD19 vs 9G4 (top), and Igκ vs Igλ (bottom), on CD19+ gated B cells from healthy control HC-18 that were incubated for 30 min at 4°C with undiluted sera from the same healthy subject or from three SLE-H individuals, as indicated. Data are from one of two independent experiments (data from second experiment are in Supplementary Figure S5D). (I) Frequency of κ+λ+ cells measured by flow cytometry within the CD19+ B cell populations of HC PBMCs incubated with sera from either HC or SLE-H subjects. N=3 for HC cells+HC sera; N=6 for HC cells+SLE-H sera; analyzed over 2 independent experiments. In all bar graphs, each symbol is an individual, bars represent mean+SD, and statistical analyses were performed with Mann-Whitney U tests. *p ≤ 0.05; **p < 0.01; ***p < 0.001; ns, not significant.