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Silencing of B cell receptor signals in human naive B cells.

Feldhahn N, Schwering I, Lee S, Wartenberg M, Klein F, Wang H, Zhou G, Wang SM, Rowley JD, Hescheler J, Krönke M, Rajewsky K, Küppers R, Müschen M - J. Exp. Med. (2002)

Bottom Line: In a functional assay, we show that down-regulation of inhibitory IgSF receptors and increased responsiveness to BCR stimulation in memory as compared with naive B cells at least partly results from interleukin (IL)-4 receptor signaling.Thus, LIRB1 and IL-4 may represent components of two nonoverlapping gene expression programs in naive and memory B cells, respectively: in naive B cells, a large group of inhibitory IgSF receptors can elevate the BCR signaling threshold to prevent these cells from premature activation and clonal expansion before GC-dependent affinity maturation.In memory B cells, facilitated responsiveness upon reencounter of the immunizing antigen may result from amplification of BCR signals at virtually all levels of signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, 50931 Köln, Germany.

ABSTRACT
To identify changes in the regulation of B cell receptor (BCR) signals during the development of human B cells, we generated genome-wide gene expression profiles using the serial analysis of gene expression (SAGE) technique for CD34(+) hematopoietic stem cells (HSCs), pre-B cells, naive, germinal center (GC), and memory B cells. Comparing these SAGE profiles, genes encoding positive regulators of BCR signaling were expressed at consistently lower levels in naive B cells than in all other B cell subsets. Conversely, a large group of inhibitory signaling molecules, mostly belonging to the immunoglobulin superfamily (IgSF), were specifically or predominantly expressed in naive B cells. The quantitative differences observed by SAGE were corroborated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. In a functional assay, we show that down-regulation of inhibitory IgSF receptors and increased responsiveness to BCR stimulation in memory as compared with naive B cells at least partly results from interleukin (IL)-4 receptor signaling. Conversely, activation or impairment of the inhibitory IgSF receptor LIRB1 affected BCR-dependent Ca(2+) mobilization only in naive but not memory B cells. Thus, LIRB1 and IL-4 may represent components of two nonoverlapping gene expression programs in naive and memory B cells, respectively: in naive B cells, a large group of inhibitory IgSF receptors can elevate the BCR signaling threshold to prevent these cells from premature activation and clonal expansion before GC-dependent affinity maturation. In memory B cells, facilitated responsiveness upon reencounter of the immunizing antigen may result from amplification of BCR signals at virtually all levels of signal transduction.

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Genotype and phenotype of purified B cell subsets. Bone marrow pre-B cells (CD10+ CD19+), peripheral blood naive B cells (CD19+ CD27−), tonsillar GC B cells (CD20+ CD77+), and peripheral blood memory B cells (CD19+ CD27+) were purified as described in Materials and Methods. The mRNA expression of Cμ, Cγ1, Cκ, and VpreB was analyzed by semiquantitative RT-PCR analysis (A). The identity of the purified subsets was further verified by flow cytometry (B): FACS® plots for preB cells (CD10+ CD19+), naive (CD20+ CD27−), GC (CD20+ CD77+), and memory B cells (CD20+ CD27+; from top to bottom) are given.
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fig1: Genotype and phenotype of purified B cell subsets. Bone marrow pre-B cells (CD10+ CD19+), peripheral blood naive B cells (CD19+ CD27−), tonsillar GC B cells (CD20+ CD77+), and peripheral blood memory B cells (CD19+ CD27+) were purified as described in Materials and Methods. The mRNA expression of Cμ, Cγ1, Cκ, and VpreB was analyzed by semiquantitative RT-PCR analysis (A). The identity of the purified subsets was further verified by flow cytometry (B): FACS® plots for preB cells (CD10+ CD19+), naive (CD20+ CD27−), GC (CD20+ CD77+), and memory B cells (CD20+ CD27+; from top to bottom) are given.

Mentions: CD34+ HSCs (reference 7) and CD10+ CD19+ pre-B cells (Fig. 1 B) were purified from human bone marrow, CD19+CD27− naive and CD19+CD27+ memory B cells were purified from peripheral blood and CD38+CD77+ GC B cells from tonsils and analyzed by flow cytometry (Fig. 1 B). The identity of the purified subsets was further supported by the analysis of their genotype (Table I). As opposed to memory B cells, Ig VH1-gene rearrangements amplified from pre-B cells and naive B cells were virtually devoid of somatic mutations. Consistent with ongoing selection for the expression of a functional Ig heavy chain in bone marrow pre-B cells, nonproductive VH1-gene rearrangements (either due to loss of reading frame or rearrangement of a pseudogene of the VH1 family) were overrepresented in the isolated pre-B cell population (Table I). Prior to the SAGE analysis, specific fragments of the Cμ-, Cγ1-, Cκ, and VpreB genes were amplified from pre-B cells, naive, and memory B cells by RT-PCR, which was normalized for β2-microglobulin (Fig. 1 A). As expected, expression of VpreB is confined to pre-B cells, which, in turn, lack expression of Igκ light chains and IgG1 heavy chains. Cγ1 transcripts were detected in memory but not pre-GC B cells (Fig. 1 A). The identity of the purified B cell subsets was also confirmed retrospectively by the expression pattern of a set of subset-specific markers within the SAGE-libraries (Table II).


Silencing of B cell receptor signals in human naive B cells.

Feldhahn N, Schwering I, Lee S, Wartenberg M, Klein F, Wang H, Zhou G, Wang SM, Rowley JD, Hescheler J, Krönke M, Rajewsky K, Küppers R, Müschen M - J. Exp. Med. (2002)

Genotype and phenotype of purified B cell subsets. Bone marrow pre-B cells (CD10+ CD19+), peripheral blood naive B cells (CD19+ CD27−), tonsillar GC B cells (CD20+ CD77+), and peripheral blood memory B cells (CD19+ CD27+) were purified as described in Materials and Methods. The mRNA expression of Cμ, Cγ1, Cκ, and VpreB was analyzed by semiquantitative RT-PCR analysis (A). The identity of the purified subsets was further verified by flow cytometry (B): FACS® plots for preB cells (CD10+ CD19+), naive (CD20+ CD27−), GC (CD20+ CD77+), and memory B cells (CD20+ CD27+; from top to bottom) are given.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193982&req=5

fig1: Genotype and phenotype of purified B cell subsets. Bone marrow pre-B cells (CD10+ CD19+), peripheral blood naive B cells (CD19+ CD27−), tonsillar GC B cells (CD20+ CD77+), and peripheral blood memory B cells (CD19+ CD27+) were purified as described in Materials and Methods. The mRNA expression of Cμ, Cγ1, Cκ, and VpreB was analyzed by semiquantitative RT-PCR analysis (A). The identity of the purified subsets was further verified by flow cytometry (B): FACS® plots for preB cells (CD10+ CD19+), naive (CD20+ CD27−), GC (CD20+ CD77+), and memory B cells (CD20+ CD27+; from top to bottom) are given.
Mentions: CD34+ HSCs (reference 7) and CD10+ CD19+ pre-B cells (Fig. 1 B) were purified from human bone marrow, CD19+CD27− naive and CD19+CD27+ memory B cells were purified from peripheral blood and CD38+CD77+ GC B cells from tonsils and analyzed by flow cytometry (Fig. 1 B). The identity of the purified subsets was further supported by the analysis of their genotype (Table I). As opposed to memory B cells, Ig VH1-gene rearrangements amplified from pre-B cells and naive B cells were virtually devoid of somatic mutations. Consistent with ongoing selection for the expression of a functional Ig heavy chain in bone marrow pre-B cells, nonproductive VH1-gene rearrangements (either due to loss of reading frame or rearrangement of a pseudogene of the VH1 family) were overrepresented in the isolated pre-B cell population (Table I). Prior to the SAGE analysis, specific fragments of the Cμ-, Cγ1-, Cκ, and VpreB genes were amplified from pre-B cells, naive, and memory B cells by RT-PCR, which was normalized for β2-microglobulin (Fig. 1 A). As expected, expression of VpreB is confined to pre-B cells, which, in turn, lack expression of Igκ light chains and IgG1 heavy chains. Cγ1 transcripts were detected in memory but not pre-GC B cells (Fig. 1 A). The identity of the purified B cell subsets was also confirmed retrospectively by the expression pattern of a set of subset-specific markers within the SAGE-libraries (Table II).

Bottom Line: In a functional assay, we show that down-regulation of inhibitory IgSF receptors and increased responsiveness to BCR stimulation in memory as compared with naive B cells at least partly results from interleukin (IL)-4 receptor signaling.Thus, LIRB1 and IL-4 may represent components of two nonoverlapping gene expression programs in naive and memory B cells, respectively: in naive B cells, a large group of inhibitory IgSF receptors can elevate the BCR signaling threshold to prevent these cells from premature activation and clonal expansion before GC-dependent affinity maturation.In memory B cells, facilitated responsiveness upon reencounter of the immunizing antigen may result from amplification of BCR signals at virtually all levels of signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, 50931 Köln, Germany.

ABSTRACT
To identify changes in the regulation of B cell receptor (BCR) signals during the development of human B cells, we generated genome-wide gene expression profiles using the serial analysis of gene expression (SAGE) technique for CD34(+) hematopoietic stem cells (HSCs), pre-B cells, naive, germinal center (GC), and memory B cells. Comparing these SAGE profiles, genes encoding positive regulators of BCR signaling were expressed at consistently lower levels in naive B cells than in all other B cell subsets. Conversely, a large group of inhibitory signaling molecules, mostly belonging to the immunoglobulin superfamily (IgSF), were specifically or predominantly expressed in naive B cells. The quantitative differences observed by SAGE were corroborated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. In a functional assay, we show that down-regulation of inhibitory IgSF receptors and increased responsiveness to BCR stimulation in memory as compared with naive B cells at least partly results from interleukin (IL)-4 receptor signaling. Conversely, activation or impairment of the inhibitory IgSF receptor LIRB1 affected BCR-dependent Ca(2+) mobilization only in naive but not memory B cells. Thus, LIRB1 and IL-4 may represent components of two nonoverlapping gene expression programs in naive and memory B cells, respectively: in naive B cells, a large group of inhibitory IgSF receptors can elevate the BCR signaling threshold to prevent these cells from premature activation and clonal expansion before GC-dependent affinity maturation. In memory B cells, facilitated responsiveness upon reencounter of the immunizing antigen may result from amplification of BCR signals at virtually all levels of signal transduction.

Show MeSH
Related in: MedlinePlus