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Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool.

Turner M, Gulbranson-Judge A, Quinn ME, Walters AE, MacLennan IC, Tybulewicz VL - J. Exp. Med. (1997)

Bottom Line: Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells.Normally only a proportion of immature B cells is recruited into the recirculating pool.Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom.

ABSTRACT
The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

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Positive selection of immature B cells. Flow cytometric analysis of cells  from radiation chimeras reconstituted with liver cells from Syk+/−/3-83 (Syk+/−),  Syk−/−/3-83 (Syk−/−), and Syk−/−/3-83/Bcl-2Tg (Syk−/− + Bcl-2) fetuses or from  an adult Syk+/−/3-83/Bcl-2Tg (Syk+/− + Bcl-2) mouse. In separate experiments chimeras made with Syk+/−/3-83/Bcl-2Tg fetal liver gave essentially the same results as  presented in this figure (data not shown). Chimeras made with Syk+/+/3-83 fetal  liver gave results indistinguishable from Syk+/−/3-83 chimeras (data not shown). (a)  The first column from the left shows the expression of B220 and the transgenic BCR  (3-83) on bone marrow cells; B220+3-83+ cells are boxed and their fraction as a percentage of all lymphoid cells is shown. The second column shows the expression of  IgMa and IgDa by B220+3-83+ cells with gating to show immature (IgM+IgD−),  transitional (IgM+IgDlow), and mature (IgM+IgDhigh) B cells; the percentage for each  gated population is expressed as a fraction of all lymphocytes. (b) The first column  shows the gating on immature (B220low3-83low) cells that was used to generate the  histograms of CD43 expression on B220+3-83+ bone marrow cells shown in the second column; percentages represent the fraction of gated immature B220low3-83low  cells that are CD43−. (c) Expression of B220 and 3-83 in the spleen; B220+3-83+  cells are boxed and their fraction as a percentage of all lymphoid cells is shown. (d) Mean numbers ± SEM of B220+3-83+ cells in the bone marrow (per  femur) and spleen are shown. In the marrow, these have been subdivided into immature (IgM+IgD−), transitional (IgM+IgDlow) and mature  (IgM+IgDhigh) B cells using the gating shown in a. Data in (a–c) are representative of at least six mice of each genotype. Absolute cell numbers in the marrow were determined using eight Syk+/−/3-83 mice, six Syk−/−/3-83 mice, three Syk+/−/3-83/Bcl-2Tg mice, and four Syk−/−/3-83/Bcl-2Tg mice;  data for the spleen cell numbers was determined using four Syk+/−/3-83 mice, four Syk−/−/3-83 mice, three Syk+/−/3-83/Bcl-2Tg mice, and two  Syk−/−/3-83/Bcl-2Tg mice.
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Figure 3: Positive selection of immature B cells. Flow cytometric analysis of cells from radiation chimeras reconstituted with liver cells from Syk+/−/3-83 (Syk+/−), Syk−/−/3-83 (Syk−/−), and Syk−/−/3-83/Bcl-2Tg (Syk−/− + Bcl-2) fetuses or from an adult Syk+/−/3-83/Bcl-2Tg (Syk+/− + Bcl-2) mouse. In separate experiments chimeras made with Syk+/−/3-83/Bcl-2Tg fetal liver gave essentially the same results as presented in this figure (data not shown). Chimeras made with Syk+/+/3-83 fetal liver gave results indistinguishable from Syk+/−/3-83 chimeras (data not shown). (a) The first column from the left shows the expression of B220 and the transgenic BCR (3-83) on bone marrow cells; B220+3-83+ cells are boxed and their fraction as a percentage of all lymphoid cells is shown. The second column shows the expression of IgMa and IgDa by B220+3-83+ cells with gating to show immature (IgM+IgD−), transitional (IgM+IgDlow), and mature (IgM+IgDhigh) B cells; the percentage for each gated population is expressed as a fraction of all lymphocytes. (b) The first column shows the gating on immature (B220low3-83low) cells that was used to generate the histograms of CD43 expression on B220+3-83+ bone marrow cells shown in the second column; percentages represent the fraction of gated immature B220low3-83low cells that are CD43−. (c) Expression of B220 and 3-83 in the spleen; B220+3-83+ cells are boxed and their fraction as a percentage of all lymphoid cells is shown. (d) Mean numbers ± SEM of B220+3-83+ cells in the bone marrow (per femur) and spleen are shown. In the marrow, these have been subdivided into immature (IgM+IgD−), transitional (IgM+IgDlow) and mature (IgM+IgDhigh) B cells using the gating shown in a. Data in (a–c) are representative of at least six mice of each genotype. Absolute cell numbers in the marrow were determined using eight Syk+/−/3-83 mice, six Syk−/−/3-83 mice, three Syk+/−/3-83/Bcl-2Tg mice, and four Syk−/−/3-83/Bcl-2Tg mice; data for the spleen cell numbers was determined using four Syk+/−/3-83 mice, four Syk−/−/3-83 mice, three Syk+/−/3-83/Bcl-2Tg mice, and two Syk−/−/3-83/Bcl-2Tg mice.

Mentions: It is possible that the block in maturation of Syk−/− immature B cells was not complete, but simply inefficient, and the failure to observe any Syk−/− recirculating cells was due to the reduced number of immature B cells produced in the marrow of the Syk−/− chimeras (23). To attempt to overcome this, we made further chimeras with Syk−/− and control fetal livers containing the 3-83μδ (3-83) BCR transgene; this encodes IgM and IgD specific for H-2Kk and H-2Kb, but not H-2Kd; the background of both the donor and host in all these chimeras was H-2d (29). Although 3-83–expressing B cells were abundant in the bone marrow of the Syk-deficient chimeras, all these cells expressed IgM with little or no IgD and thus do not represent recirculating cells, which are typically IgM+IgDhi (Fig. 3, a and d). In Syk−/− chimeras made without a BCR transgene, most B lineage cells in the marrow are pro-B cells, and thus it was possible that the 3-83–expressing cells in the Syk−/−/3-83 chimeras would still be phenotypically pro-B cells, despite the expression of a rearranged BCR (23). However, flow cytometric analysis demonstrated that most of the 3-83–expressing cells in these chimeras do not express CD43, a marker of pro-B cells that is turned off as the cells mature into pre-B/immature B cells (Fig. 3 b; reference 30). Thus, the Syk−/−/3-83 chimeras contained large numbers of Syk−/− cells with an immature B cell phenotype. The mechanism whereby the expression of the 3-83 BCR partially rescues the pro-B→ pre-B cell developmental block is unclear. Despite the abundance of these immature cells in the bone marrow, very few Syk-deficient cells could be detected in the spleen and lymph nodes of the chimeras and again these were immature in phenotype (IgM+IgD−/low; Fig. 3, c and d, and data not shown). Furthermore, immunohistology demonstrated that in contrast to wild-type 3-83+ B cells, the few Syk-deficient cells were again confined to the outer T zone and red pulp of the spleen (Fig. 2, c and d).


Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool.

Turner M, Gulbranson-Judge A, Quinn ME, Walters AE, MacLennan IC, Tybulewicz VL - J. Exp. Med. (1997)

Positive selection of immature B cells. Flow cytometric analysis of cells  from radiation chimeras reconstituted with liver cells from Syk+/−/3-83 (Syk+/−),  Syk−/−/3-83 (Syk−/−), and Syk−/−/3-83/Bcl-2Tg (Syk−/− + Bcl-2) fetuses or from  an adult Syk+/−/3-83/Bcl-2Tg (Syk+/− + Bcl-2) mouse. In separate experiments chimeras made with Syk+/−/3-83/Bcl-2Tg fetal liver gave essentially the same results as  presented in this figure (data not shown). Chimeras made with Syk+/+/3-83 fetal  liver gave results indistinguishable from Syk+/−/3-83 chimeras (data not shown). (a)  The first column from the left shows the expression of B220 and the transgenic BCR  (3-83) on bone marrow cells; B220+3-83+ cells are boxed and their fraction as a percentage of all lymphoid cells is shown. The second column shows the expression of  IgMa and IgDa by B220+3-83+ cells with gating to show immature (IgM+IgD−),  transitional (IgM+IgDlow), and mature (IgM+IgDhigh) B cells; the percentage for each  gated population is expressed as a fraction of all lymphocytes. (b) The first column  shows the gating on immature (B220low3-83low) cells that was used to generate the  histograms of CD43 expression on B220+3-83+ bone marrow cells shown in the second column; percentages represent the fraction of gated immature B220low3-83low  cells that are CD43−. (c) Expression of B220 and 3-83 in the spleen; B220+3-83+  cells are boxed and their fraction as a percentage of all lymphoid cells is shown. (d) Mean numbers ± SEM of B220+3-83+ cells in the bone marrow (per  femur) and spleen are shown. In the marrow, these have been subdivided into immature (IgM+IgD−), transitional (IgM+IgDlow) and mature  (IgM+IgDhigh) B cells using the gating shown in a. Data in (a–c) are representative of at least six mice of each genotype. Absolute cell numbers in the marrow were determined using eight Syk+/−/3-83 mice, six Syk−/−/3-83 mice, three Syk+/−/3-83/Bcl-2Tg mice, and four Syk−/−/3-83/Bcl-2Tg mice;  data for the spleen cell numbers was determined using four Syk+/−/3-83 mice, four Syk−/−/3-83 mice, three Syk+/−/3-83/Bcl-2Tg mice, and two  Syk−/−/3-83/Bcl-2Tg mice.
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Figure 3: Positive selection of immature B cells. Flow cytometric analysis of cells from radiation chimeras reconstituted with liver cells from Syk+/−/3-83 (Syk+/−), Syk−/−/3-83 (Syk−/−), and Syk−/−/3-83/Bcl-2Tg (Syk−/− + Bcl-2) fetuses or from an adult Syk+/−/3-83/Bcl-2Tg (Syk+/− + Bcl-2) mouse. In separate experiments chimeras made with Syk+/−/3-83/Bcl-2Tg fetal liver gave essentially the same results as presented in this figure (data not shown). Chimeras made with Syk+/+/3-83 fetal liver gave results indistinguishable from Syk+/−/3-83 chimeras (data not shown). (a) The first column from the left shows the expression of B220 and the transgenic BCR (3-83) on bone marrow cells; B220+3-83+ cells are boxed and their fraction as a percentage of all lymphoid cells is shown. The second column shows the expression of IgMa and IgDa by B220+3-83+ cells with gating to show immature (IgM+IgD−), transitional (IgM+IgDlow), and mature (IgM+IgDhigh) B cells; the percentage for each gated population is expressed as a fraction of all lymphocytes. (b) The first column shows the gating on immature (B220low3-83low) cells that was used to generate the histograms of CD43 expression on B220+3-83+ bone marrow cells shown in the second column; percentages represent the fraction of gated immature B220low3-83low cells that are CD43−. (c) Expression of B220 and 3-83 in the spleen; B220+3-83+ cells are boxed and their fraction as a percentage of all lymphoid cells is shown. (d) Mean numbers ± SEM of B220+3-83+ cells in the bone marrow (per femur) and spleen are shown. In the marrow, these have been subdivided into immature (IgM+IgD−), transitional (IgM+IgDlow) and mature (IgM+IgDhigh) B cells using the gating shown in a. Data in (a–c) are representative of at least six mice of each genotype. Absolute cell numbers in the marrow were determined using eight Syk+/−/3-83 mice, six Syk−/−/3-83 mice, three Syk+/−/3-83/Bcl-2Tg mice, and four Syk−/−/3-83/Bcl-2Tg mice; data for the spleen cell numbers was determined using four Syk+/−/3-83 mice, four Syk−/−/3-83 mice, three Syk+/−/3-83/Bcl-2Tg mice, and two Syk−/−/3-83/Bcl-2Tg mice.
Mentions: It is possible that the block in maturation of Syk−/− immature B cells was not complete, but simply inefficient, and the failure to observe any Syk−/− recirculating cells was due to the reduced number of immature B cells produced in the marrow of the Syk−/− chimeras (23). To attempt to overcome this, we made further chimeras with Syk−/− and control fetal livers containing the 3-83μδ (3-83) BCR transgene; this encodes IgM and IgD specific for H-2Kk and H-2Kb, but not H-2Kd; the background of both the donor and host in all these chimeras was H-2d (29). Although 3-83–expressing B cells were abundant in the bone marrow of the Syk-deficient chimeras, all these cells expressed IgM with little or no IgD and thus do not represent recirculating cells, which are typically IgM+IgDhi (Fig. 3, a and d). In Syk−/− chimeras made without a BCR transgene, most B lineage cells in the marrow are pro-B cells, and thus it was possible that the 3-83–expressing cells in the Syk−/−/3-83 chimeras would still be phenotypically pro-B cells, despite the expression of a rearranged BCR (23). However, flow cytometric analysis demonstrated that most of the 3-83–expressing cells in these chimeras do not express CD43, a marker of pro-B cells that is turned off as the cells mature into pre-B/immature B cells (Fig. 3 b; reference 30). Thus, the Syk−/−/3-83 chimeras contained large numbers of Syk−/− cells with an immature B cell phenotype. The mechanism whereby the expression of the 3-83 BCR partially rescues the pro-B→ pre-B cell developmental block is unclear. Despite the abundance of these immature cells in the bone marrow, very few Syk-deficient cells could be detected in the spleen and lymph nodes of the chimeras and again these were immature in phenotype (IgM+IgD−/low; Fig. 3, c and d, and data not shown). Furthermore, immunohistology demonstrated that in contrast to wild-type 3-83+ B cells, the few Syk-deficient cells were again confined to the outer T zone and red pulp of the spleen (Fig. 2, c and d).

Bottom Line: Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells.Normally only a proportion of immature B cells is recruited into the recirculating pool.Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom.

ABSTRACT
The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

Show MeSH
Related in: MedlinePlus