B-1 lymphocytes in mice and nonhuman primates

Abstract

B-1 cells comprise subpopulations of B lymphocytes in mice that display developmental, phenotypic, and functional characteristics that are distinct from those of conventional B cell populations (B-2 cells). Despite the known importance of murine B-1a (CD5(+) ) and B-1b (CD5(-) ) cells in the production of natural antibodies and rapid antigen-specific humoral responses to infection, evidence for B-1 cells in primates, including humans, is very limited. Identifying these cells in humans proves challenging given the limited number of cells that can be obtained from sites expected to harbor increased frequencies of these cells (i.e., peritoneal and pleural cavities) and the need to perform functional analyses on these cells, which, in the case of B-1b cells, must be carried out in vivo. My laboratory has used cynomolgus macaques and African green monkeys to bypass these limitations and to identify and extensively analyze primate B cell populations with the phenotypic and functional characteristics of mouse B-1a and B-1b cells. Our results reveal striking similarities between primate and murine B-1 cells, including a conserved functional role for primate B-1b-like cells in immunity to T cell-independent type 2 antigens.

Keywords: B-1a cells; B-1b cells; T cell-independent type 2 antigens; nonhuman primates.

Figure 1. B-1b cells reconstitute protective antibody responses to PPS in B-1b-cell deficient CD19Tg mice and B cell-deficient Rag-1^−/− mice

A) CD19^−/− mice are deficient in B-1a cells whereas CD19Tg mice are deficient in B-1b cells. B-1 (B220⁺CD11b⁺) and B-2 (B220⁺CD11b⁻) lymphocytes are indicated (left column) with histograms showing CD5 expression by peritoneal B-1 (B220⁺CD11b⁺-gated) cells (right column). Isotype-matched control antibody staining is indicated by a dotted line. B–C) Reconstituting hCD19Tg mice with peritoneal B-1b cells from CD19^−/− mice rescues responsiveness to PPS-3. Peritoneal B-1b or B-2 cells from CD19^−/− mice were isolated by FACS (B). FACS-purified peritoneal B cells or enriched spleen and lymph node B cells from CD19^−/− mice were transferred i.p. into hCD19Tg mice (10⁵ cells/mouse). Mice were immunized with PPS-3 3 weeks later with PPS-3-specific antibody titers determined by ELISA (C). D–E) Transfer of WT B-1b cells into Rag-1^−/− mice reconstitutes PPS3-specific IgM and IgG responses and provides protection against lethal S. pneumoniae infection. D) Purified WT peritoneal B-1a cells, B-1b cells, or unfractionated spleen or LN cells were transferred i.p. or i.v. into Rag-1^−/− mice (4 × 10⁵ B cells/mouse; ≥3 mice/group). Mice were immunized with 0.5 µg PPS-3 3 days later, with PPS-3-specific IgM (d7) and IgG3 (d14) antibody levels measured by ELISA. E) Rag-1^−/− mice reconstituted with B-1b cells were infected with 10² colony forming units of serotype 3 S. pneumoniae 14 days post-immunization. *Chi-square analysis indicated significant differences in survival. Adapted from Haas et al..

Figure 2. Heterogeneity in expression of surface markers on B-1b cells

A) Gating strategy to identify B-1a, B-1b, and B-2 cells in lymphocytes from peritoneal cavity lavages of wild type C57BL/6 mice. B) CD19, CD21/35, CD23, CD43, and B220 expression levels on peritoneal cavity B-1a, B-1b, and B-2 B cells in wild type and CD19^−/− mice on a C57BL/6 background.

Figure 3. B-1–like cells are present in higher primates

A) Sample gating strategy to identify B cells in NHP tissues. B cells were defined as either CD19⁺CD20⁺ cells present within the lymphocyte gate. CD11b expression (solid line) by B cells in AGM peritoneal cavity (P. cavity), omentum, spleen, and blood is shown in histograms in the lower panel. B) FSC, IgM, CD21, and CD19 expression by CD11b⁺ (solid line) and CD11b⁻ B cells (gray shading), gated as shown in (A). C) Constitutive active Stat3(p705) expression (solid line) by CD19⁺ spleen B and peritoneal CD11b⁻ and CD11b⁺ B cells in AGMs (top histograms) and mice (bottom histograms). Histograms with dashed lines indicate intracellular mAb isotype control staining. Adapted from Yammani and Haas.

Figure 4. Ag-specific B cell responses to TNP-Ficoll are similar between mice and non-human primates (NHP)

A) Ag-specific B cells (upper panels) and their CD11b expression levels (lower panels) in mice and AGM prior to and 5 days following TNP-Ficoll immunization. B220 and CD19 were used to identify splenic mouse B cells and CD20 and CD19 were used to identify primate blood B cells. Representative CD11b⁺ Ag-specific B cell frequencies before and following (d5) immunization are indicated. B) CD19, CD21, PD-1, and CD86 expression levels by gated Ag-specific B cells on d0 (thin line) and 5 days post immunization (thick line). Shaded histograms indicate expression by non-Ag-specific B cells in immune animals. Dotted lines indicate isotype control staining for Ag-specific B cells. Adapted from Yammani and Haas and Haas.

Figure 5. Ag-specific B-1 cells are significantly increased in NHP spleens 6 weeks following TNP-Ficoll immunization and display an IgM^hiIgD^loCD80⁺CD27⁺ phenotype

(A–I) Flow cytometric analysis of Ag-specific B cells in spleens of naive and immune cynomolgus macaques 6 weeks post TNP-Ficoll immunization. A) Representative flow cytometric analysis of Ag-specific splenic B cells (gated). B) CD11b expression on Ag-specific B cells from naive (shaded histogram) and immune (solid line) animals. C) Frequencies of CD11b⁺ and CD11b⁻ Ag-specific B cells among splenocytes. D) IgM and IgD expression by Ag-specific B cells, with representative IgM^hiIgD^lo population-gating shown. E) Frequencies of Ag-specific and non–Ag-specific B cells expressing an IgM^hiIgD^lo B cell phenotype. F) Representative CD11b expression by Ag-specific IgM^hiIgD^lo (solid line) and IgM^lo (shaded histogram) B cells in immune animals. G) CD19, CD20, CD80, and CD27 expression by CD11b⁺ (thick solid line) and CD11b⁻ (shaded histogram) Ag-specific B cells and non–Ag-specific B cells (thin line) in immune animals. H) CD27 expression by Ag-specific B cells in naive and immune spleen (left panel) and frequencies of Ag-specific B cells expressing CD27. N.D., “none detected”, indicates the frequency of Ag-specific B cells staining positive for CD27 was not significantly increased over the frequencies of these cells binding to isotype-matched control antibody (indicated by horizontal dashed line). I) Frequencies of Ki67⁺ B cells in immune animals. For all analyses, n = 3 cynomolgus macaques per group. Histograms with dashed horizontal lines depict isotype control staining by Ag-specific B cells from immune animals. Significant differences in mean (±SEM) frequencies are indicated, *p < 0.05. Adapted from Yammani and Haas.