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Violation of an evolutionarily conserved immunoglobulin diversity gene sequence preference promotes production of dsDNA-specific IgG antibodies.

Silva-Sanchez A, Liu CR, Vale AM, Khass M, Kapoor P, Elgavish A, Ivanov II, Ippolito GC, Schelonka RL, Schoeb TR, Burrows PD, Schroeder HW - PLoS ONE (2015)

Bottom Line: This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3.For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion.Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

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Related in: MedlinePlus

Divergence in the absolute numbers of B lineage subpopulations from the bone marrow, spleen, and peritoneal cavity of homo- and heterozygous ΔD-DFL and ΔD-iD mice relative to their littermate controls.Percent loss or gain in homo- and heterozygous ΔD-DFL(top) and ΔD-iD (bottom) mice relative to wild type littermate controls in the average absolute number of cells in bone marrow fractions B (CD19+ CD43+ HSA+ BP-1-), C (CD19+ CD43+ HSA+ BP-1+), D (CD19+ CD43- IgM- IgD-), E (CD19+ CD43- IgM+ IgD-), and F (CD19+ CD43- IgMlo IgDhi); and in splenic transitional T1 (CD19+ AA4.1+ sIgMhi CD23-), T2 (CD19+ AA4.1+ sIgMhi CD23+), marginal zone (MZ, CD19+ CD21hi CD23lo), and mature (M, CD19+ CD21lo CD23hi) B-cell subsets. The distribution of B cell numbers by subset in homozygous ΔD-DFL and ΔD-iD mice has been previously published [31,32] and is included for clarity. The standard error of the mean of each B lineage subpopulation for the littermate controls averaged approximately 11% of the absolute number of cells in each subpopulation. For ΔD-DFL and ΔD-iD, the standard error of the mean is shown as an error bar. ‘*’, p ≤ 0.05 and ‘****’, p < 0.0001.
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pone.0118171.g008: Divergence in the absolute numbers of B lineage subpopulations from the bone marrow, spleen, and peritoneal cavity of homo- and heterozygous ΔD-DFL and ΔD-iD mice relative to their littermate controls.Percent loss or gain in homo- and heterozygous ΔD-DFL(top) and ΔD-iD (bottom) mice relative to wild type littermate controls in the average absolute number of cells in bone marrow fractions B (CD19+ CD43+ HSA+ BP-1-), C (CD19+ CD43+ HSA+ BP-1+), D (CD19+ CD43- IgM- IgD-), E (CD19+ CD43- IgM+ IgD-), and F (CD19+ CD43- IgMlo IgDhi); and in splenic transitional T1 (CD19+ AA4.1+ sIgMhi CD23-), T2 (CD19+ AA4.1+ sIgMhi CD23+), marginal zone (MZ, CD19+ CD21hi CD23lo), and mature (M, CD19+ CD21lo CD23hi) B-cell subsets. The distribution of B cell numbers by subset in homozygous ΔD-DFL and ΔD-iD mice has been previously published [31,32] and is included for clarity. The standard error of the mean of each B lineage subpopulation for the littermate controls averaged approximately 11% of the absolute number of cells in each subpopulation. For ΔD-DFL and ΔD-iD, the standard error of the mean is shown as an error bar. ‘*’, p ≤ 0.05 and ‘****’, p < 0.0001.

Mentions: Homozygous ΔD-iD and ΔD-DFL mice both exhibit B cell lymphopenia in Hardy fractions D and E in the bone marrow [31,32]. Homozygous ΔD-iD mice also exhibit B cell lymphopenia in the follicular B cell subset in the spleen and recirculating, mature Hardy Fraction F in the bone marrow (Fig. 8) [31,32]. To assess the effect of heterozygosity on B cell numbers, we re-examined previously published [36] and unpublished studies of heterozygous D-altered BALB/c mice (Fig. 8). By focusing on the percent increase, or decrease, in absolute B cell numbers when compared to wild type, this analysis emphasizes relative differences in B cell numbers at specific B cell developmental stages. With the exception of the follicular B cell subset that exhibited a 25% reduction in B cell numbers (p<0.05), the absolute numbers of B cells in bone marrow and splenic subsets in the heterozygous ΔD-DFL/WT mice were statistically indistinguishable from those in WT/WT littermate controls. Unlike their ΔD-DFL counterparts, heterozygous ΔD-iD/WT mice exhibited reductions in the absolute number of Hardy Fraction D and E. However, among the mature splenic and bone marrow B cell fractions, heterozygous ΔD-iD/WT mice also exhibited normalization of B cell numbers.


Violation of an evolutionarily conserved immunoglobulin diversity gene sequence preference promotes production of dsDNA-specific IgG antibodies.

Silva-Sanchez A, Liu CR, Vale AM, Khass M, Kapoor P, Elgavish A, Ivanov II, Ippolito GC, Schelonka RL, Schoeb TR, Burrows PD, Schroeder HW - PLoS ONE (2015)

Divergence in the absolute numbers of B lineage subpopulations from the bone marrow, spleen, and peritoneal cavity of homo- and heterozygous ΔD-DFL and ΔD-iD mice relative to their littermate controls.Percent loss or gain in homo- and heterozygous ΔD-DFL(top) and ΔD-iD (bottom) mice relative to wild type littermate controls in the average absolute number of cells in bone marrow fractions B (CD19+ CD43+ HSA+ BP-1-), C (CD19+ CD43+ HSA+ BP-1+), D (CD19+ CD43- IgM- IgD-), E (CD19+ CD43- IgM+ IgD-), and F (CD19+ CD43- IgMlo IgDhi); and in splenic transitional T1 (CD19+ AA4.1+ sIgMhi CD23-), T2 (CD19+ AA4.1+ sIgMhi CD23+), marginal zone (MZ, CD19+ CD21hi CD23lo), and mature (M, CD19+ CD21lo CD23hi) B-cell subsets. The distribution of B cell numbers by subset in homozygous ΔD-DFL and ΔD-iD mice has been previously published [31,32] and is included for clarity. The standard error of the mean of each B lineage subpopulation for the littermate controls averaged approximately 11% of the absolute number of cells in each subpopulation. For ΔD-DFL and ΔD-iD, the standard error of the mean is shown as an error bar. ‘*’, p ≤ 0.05 and ‘****’, p < 0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4338297&req=5

pone.0118171.g008: Divergence in the absolute numbers of B lineage subpopulations from the bone marrow, spleen, and peritoneal cavity of homo- and heterozygous ΔD-DFL and ΔD-iD mice relative to their littermate controls.Percent loss or gain in homo- and heterozygous ΔD-DFL(top) and ΔD-iD (bottom) mice relative to wild type littermate controls in the average absolute number of cells in bone marrow fractions B (CD19+ CD43+ HSA+ BP-1-), C (CD19+ CD43+ HSA+ BP-1+), D (CD19+ CD43- IgM- IgD-), E (CD19+ CD43- IgM+ IgD-), and F (CD19+ CD43- IgMlo IgDhi); and in splenic transitional T1 (CD19+ AA4.1+ sIgMhi CD23-), T2 (CD19+ AA4.1+ sIgMhi CD23+), marginal zone (MZ, CD19+ CD21hi CD23lo), and mature (M, CD19+ CD21lo CD23hi) B-cell subsets. The distribution of B cell numbers by subset in homozygous ΔD-DFL and ΔD-iD mice has been previously published [31,32] and is included for clarity. The standard error of the mean of each B lineage subpopulation for the littermate controls averaged approximately 11% of the absolute number of cells in each subpopulation. For ΔD-DFL and ΔD-iD, the standard error of the mean is shown as an error bar. ‘*’, p ≤ 0.05 and ‘****’, p < 0.0001.
Mentions: Homozygous ΔD-iD and ΔD-DFL mice both exhibit B cell lymphopenia in Hardy fractions D and E in the bone marrow [31,32]. Homozygous ΔD-iD mice also exhibit B cell lymphopenia in the follicular B cell subset in the spleen and recirculating, mature Hardy Fraction F in the bone marrow (Fig. 8) [31,32]. To assess the effect of heterozygosity on B cell numbers, we re-examined previously published [36] and unpublished studies of heterozygous D-altered BALB/c mice (Fig. 8). By focusing on the percent increase, or decrease, in absolute B cell numbers when compared to wild type, this analysis emphasizes relative differences in B cell numbers at specific B cell developmental stages. With the exception of the follicular B cell subset that exhibited a 25% reduction in B cell numbers (p<0.05), the absolute numbers of B cells in bone marrow and splenic subsets in the heterozygous ΔD-DFL/WT mice were statistically indistinguishable from those in WT/WT littermate controls. Unlike their ΔD-DFL counterparts, heterozygous ΔD-iD/WT mice exhibited reductions in the absolute number of Hardy Fraction D and E. However, among the mature splenic and bone marrow B cell fractions, heterozygous ΔD-iD/WT mice also exhibited normalization of B cell numbers.

Bottom Line: This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3.For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion.Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

Show MeSH
Related in: MedlinePlus