Btk regulates multiple stages in the development and survival of B-1 cells

Abstract

B-1 cells are important players in the first line of defense against pathogens. According to current models for the origin of B-1 cells, they either represent a separate lineage from conventional B-2 cells or differentiate from conventional B-2 cells via an intermediate, B-1(int), in response to positive selection by antigen. Here we show that Btk, a Tec family kinase that mediates B cell antigen receptor (BCR) signaling, is required at multiple stages of B-1 cell development. VH12 anti-phosphatidylcholine (PtC) IgH transgenic mice provide a model for the induced differentiation of B-1 cells. This transgene selects for PtC-reactive cells and induces them to adopt a B-1 phenotype. Both processes have been shown to depend on Btk. To determine whether this is secondary to a requirement for Btk in the development of mature B-2 cells, we crossed VH12 transgenic mice to mice expressing low levels of Btk. B-2 cell development occurs normally in Btk(lo) mice despite reduced responsiveness to BCR crosslinking. Analysis of VH12.Btk(lo) mice reveals that Btk regulates the B-1(int) to B-1 transition and/or the survival of splenic B-1 cells, in part via a mechanism independent of its role in BCR signaling. We also show that Btk mediates the survival of, and expression of IL-10 by, those B-1 cells that do develop and migrate to the peritoneum. Multiple roles for Btk in B-1 cell development and maintenance may explain the particular sensitivity of this population to mutations in components of Btk signaling pathways.

Figure 1. Btk regulates anti-PtC B cell expansion and differentiation from B-1^intto B-1

Splenocytes from 6-1, 6-1.Btk^lo, and 6-1.Btk-/- mice were stained with FITC-encapsulated liposomes, anti-CD5 PE or anti-CD43 PE, anti-B220 PerCP, and anti-CD23 biotin + streptavidin APC. A) The percentage of lymphocytes in each quadrant is indicated. B) Liposome-binding cells were gated and analyzed for expression of CD5, CD43, and CD23. B-2 cells (CD23+CD5- or CD23+CD43-) are in the upper left quadrant, B-1^int cells (CD23+CD5+ or CD23+CD43+) are in the upper right quadrant, and B-1 (CD23-CD5+ or CD23-CD43+) cells are in the lower right quadrant. Quadrants were defined using mice in which clear distinctions between populations were observed. The percentage of gated cells in each quadrant is indicated. Two individual 6-1.Btk^lo mice are shown in B) to reflect the range of variability seen in the differentiation state of anti-PtC B cells in these mice. Data in A) and B) are representative of 10 6-1, 10 6-1.Btk^lo and 5 6-1.Btk-/- mice. C) The number of splenocytes, B220+ cells, anti-PtC cells, and B-2, B-1^int, or B-1 within the anti-PtC population as defined by CD23 vs. CD5 or CD23 vs. CD43 are indicated. Each symbol (diamonds) represents an individual mouse. Bars represent the mean (n = 8-10). * = p < 0.05.

Figure 2. CD80 is upregulated in B-1^intand B-1 cells of 6-1.Btk^lomice

Splenocytes were stained with FITC-encapsulated liposomes, anti-CD80 PE, anti-CD5 PerCP, and anti-CD23 biotin + streptavidin APC. A. CD23+liposome- (left) and liposome+ (regardless of CD23 expression) (right) populations were gated and examined for CD80 expression. B. CD80- and CD80+ populations within the liposome-binding gate of 6-1 Btk^lo mice (lower right histogram in A) were examined for expression of CD23 and CD5. Results are representative of 5 mice per group.

Figure 3. Reduced B-1 cell numbers in 6-1.Lyn-/-Btk^lomice

Splenocytes from 6-1.Lyn-/- and 6-1.Lyn-/-Btk^lo mice were analyzed as in Figure 1. A) The frequency of liposome-binding cells is presented as mean +/- SEM. n = 10 for 6-1 and 6-1.Btk^lo mice, 6 for 6-1.Lyn-/- and 6-1.Lyn-/-Btk^lo mice. B) Data are representative of 6 mice per group. C) The total number of cells that are anti-PtC and B-2, B-1^int, or B-1 as defined by CD23 vs. CD5 or CD23 vs. CD43 is indicated. Data are presented as mean +/- SEM, n = 4 for 6-1.Lyn-/- and 5 for 6-1.Lyn-/-Btk^lo.

Figure 4. Btk regulates peritoneal B-1 cell survival

A) The frequency of liposome-binding cells among all peritoneal cells is indicated. Data are mean +/- SD, n = 3 for 6-1, 4 for 6-1.Btk^lo, and 2 for 6-1.Btk-/-. B) Peritoneal cells were stained with FITC-encapsulated liposomes, anti-CD5 PE, anti-B220 PerCP and anti-CD23 biotin + streptavidin APC. Liposome-binding (top) or B220+ (bottom) cells within the lymphoid gate were examined for CD23 and CD5 expression. Data are representative of 3 mice for 6-1 and 4 for 6-1.Btk^lo. C) B220+ cells were purified from the peritoneum of pools of 2-3 mice per group using magnetic beads or flow cytometry. Cells were plated in 96 well plates at 10⁶ per well. The number of live cells (as measured by trypan blue exclusion) remaining at the indicated time is indicated. Data represent the mean +/- SEM, n = 6 for 6-1 and 5 for 6-1.Btk^lo. D) B220+ cells were purified from the peritoneum of pools of 2-3 mice per group using magnetic beads. IL-10 mRNA levels were measured by quantitative real-time PCR. GAPDH was used as a loading control. The relative expression of IL-10 in 6-1.Btk^lo cells compared to 6-1 wt cells (100%) is shown as mean +/- SD, n=3. E) Peritoneal cells were cultured at 2 × 10⁶ per ml for 3 days. IL-10 levels in the culture supernatants were measured by ELISA. Each symbol represents the average of duplicate samples from an individual mouse (n = 3).

Figure 5. Increased number of CD23+ liposome- B cells in 6-1.Btk^lomice

A) Splenocytes were stained with FITC-encapsulated liposomes or anti-IgMa FITC and anti-CD23 biotin + streptavidin APC. Results are representative of 10 mice per group. B) Splenocytes were stained with anti-CD21-FITC, anti-IgM PE, anti-CD5 PerCP and anti-CD23-biotin + streptavidin APC. CD23+CD5- cells were examined for expression of CD21 and IgM. Regions used to define T2 and mature (M) cells and the percentage of gated cells in each region are indicated. Results are representative of 3 mice per group. C) The frequency of B220+IgM- (pro and pre B cells), B220^lo IgM+ (immature B cells), B220^hi IgM+ (recirculating B cells), B220+CD43+ (pro B cells) and B220+CD43- (pre, immature, and recirculating B cells) among bone marrow cells is indicated as mean+/- SD, n=3-5. There was no significant difference in the total number of bone marrow cells recovered. D) CD23+ liposome- cells as defined by the upper left quadrant in A) were purified by flow cytometry from pools of 4 6-1 and 2 6-1.Btk^lo spleens. RNA was prepared and Ig light chains amplified by 5’ RACE. 34 individual clones from 6-1 mice and 38 from 6-1.Btk^lo mice were sequenced. The percentage of clones representing each light chain family is shown.

Figure 6. Model for the role of Btk in the focusing of VH12-expressing B cells toward anti-PtC B-1 cells

Circles represent VH12-expressing cells. Black circles express light chains other than Vk4/5, stippled circles represent Vk4/5 expressing cells that do not bind PtC, and empty circles depict anti-PtC cells expressing Vk4/5. In normal mice, the light chain repertoire is focused towards a limited number of light chains including Vk4/5 due to failure of many light chains to pair with VH12 and the inability of some light chains that do pair with VH12 to support the development of mature B cells (Tatu et al., 1999). Development of B cells beyond the T2 stage is considerably impaired in the complete absence of Btk signaling (xid and Btk-/-) (Desiderio, 1997, Satterthwaite and Witte, 2000, Khan, 2001, Loder et al., 1999) regardless of their specificity. PtC-specific cells that do develop in xid and Btk-/- mice fail to differentiate beyond B2 (Clarke and Arnold, 1998). Btk^lo mice can support differentiation of anti-PtC cells through the B-1^int stage, but only limited numbers of B-1 cells are present in the spleen. Some B-1 cells do develop and localize to the peritoneum, but they survive poorly and produce reduced levels of the survival factor IL-10. The current studies do not rule out an additional role for Btk in the newly described B-1 progenitor population present in fetal and adult bone marrow (Montecino-Rodriguez et al., 2006).