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TLR7 influences germinal center selection in murine SLE.

Boneparth A, Huang W, Bethunaickan R, Woods M, Sahu R, Arora S, Akerman M, Lesser M, Davidson A - PLoS ONE (2015)

Bottom Line: Deletion of 3H9 B cells occurred in the bone marrow and the remaining 3H9 follicular B cells manifested a decrease in surface IgM.We were unable to demonstrate a consistent effect of Tlr7 dose or Yaa on somatic mutations.Our data show that TLR7 excess influences the selection, expansion and diversification of B cells in the germinal center, independent of other genes in the Yaa locus.

View Article: PubMed Central - PubMed

Affiliation: Center for Autoimmunity and Musculoskeletal Diseases, Feinstein Institute for Medical Research, Manhasset, New York, 11030, United States of America.

ABSTRACT
TLR7 enhances germinal center maturation and migration of B cells to the dark zone where proliferation and somatic hypermutation occur. Our goal was to determine how Tlr7 dose influences selection of the autoreactive B cell repertoire in NZW/BXSB. Yaa mice bearing the site-directed heavy chain transgene 3H9 that encodes for the TLR7 regulated anti-CL response. To create a physiologic setting in which autoreactive B cells compete for survival with non-autoreactive B cells, we generated bone marrow chimeras in which disease onset occurred with similar kinetics and the transferred 3H9+ female non-Yaa, male Yaa or male TLR7(-/Yaa) cells could be easily identified by positivity for GFP. Deletion of 3H9 B cells occurred in the bone marrow and the remaining 3H9 follicular B cells manifested a decrease in surface IgM. Although there were differences in the naïve repertoire between the chimeras it was not possible to distinguish a clear pattern of selection against lupus related autoreactivity in TLR7(-/Yaa) or female chimeras. By contrast, preferential expansion of 3H9+ B cells occurred in the germinal centers of male Yaa chimeras. In addition, although all chimeras preferentially selected 3H9/Vκ5 encoded B cells into the germinal center and plasma cell compartments, 3H9 male Yaa chimeras had a more diverse repertoire and positively selected the 3H9/Vκ5-48/Jκ4 pair that confers high affinity anti-cardiolipin activity. We were unable to demonstrate a consistent effect of Tlr7 dose or Yaa on somatic mutations. Our data show that TLR7 excess influences the selection, expansion and diversification of B cells in the germinal center, independent of other genes in the Yaa locus.

No MeSH data available.


Related in: MedlinePlus

Binding characteristics of Vκ5 encoded light chains vary with Jκ usage.Serial dilutions of each heavy and light chain pair were tested for the reactivity with dsDNA (A), CL (B) or histones (C).Distribution of Jκ regions among Vκ5–45 (D) and Vκ5–48 (E) encoded light chains as a percentage of total Vκ5 encoded light chains is shown. Comparisons are with F 3H9 to F chimeras for Vκ5–45 and with M3H9 chimeras for Vκ5–48. * p < 0.05, † p < 0.01.
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pone.0119925.g007: Binding characteristics of Vκ5 encoded light chains vary with Jκ usage.Serial dilutions of each heavy and light chain pair were tested for the reactivity with dsDNA (A), CL (B) or histones (C).Distribution of Jκ regions among Vκ5–45 (D) and Vκ5–48 (E) encoded light chains as a percentage of total Vκ5 encoded light chains is shown. Comparisons are with F 3H9 to F chimeras for Vκ5–45 and with M3H9 chimeras for Vκ5–48. * p < 0.05, † p < 0.01.

Mentions: Proteinuria and survival data were analyzed using Kaplan-Meier curves and log rank test. Comparisons shown in Figs. 1A, 2 and 3A were performed using Mann-Whitney test. p values ≤ 0.05 were considered significant. Comparisons in Fig. 4 were performed using pair-wise Mann-Whitney test with a Bonferroni adjustment to account for multiple testing. In order to preserve an overall 5% significance level, a pairwise comparison was considered statistically significant if p < 0.025. For Fig. 5 the Kruskal-Wallis test was used to compare the M and F wt with their respective experimental groups. Upon finding a significant difference, Bonferroni adjusted multiple pairwise comparisons of the WT group to each of the other two gender-matched groups, were carried out using Mann-Whitney tests. A p value of <0.025 was considered significant. For all male vs. female comparisons the Mann-Whitney test was used. Statistical analysis of the Vκ repertoire data shown in Fig. 6 was performed as previously described [31]. Owing to the typically small sample sizes of single cells, the usual Pearson χ2 statistic is not valid for determining whether differences exist in the distributions of L chains across B cell subsets. Instead, the Fisher exact test is applicable. However, the computing time required for running the exact test on large, sparse tables is prohibitive and, moreover, the power of the test is low. Accordingly, identifying L chains of interest without using formal inferential methods (i.e., without using p values or confidence intervals) is needed. This exploratory approach is frequently used in identifying interesting genes in gene expression microarray studies [32–34]. To this end, we used a novel method for “screening” the r × c table for cell frequencies that appear to be inconsistent with the hypothesis that the B cell subset and L chain distributions are independent. This method uses a graphical approach (scree plots) to finding the entries in the table that depart most from the hypothesis. For additional details on the statistical methodology please refer to Lesser et al [35]. Comparisons in Fig. 7 were performed using chi-square analysis. Mutation frequencies in each subset were compared using the Mann-Whitney test.


TLR7 influences germinal center selection in murine SLE.

Boneparth A, Huang W, Bethunaickan R, Woods M, Sahu R, Arora S, Akerman M, Lesser M, Davidson A - PLoS ONE (2015)

Binding characteristics of Vκ5 encoded light chains vary with Jκ usage.Serial dilutions of each heavy and light chain pair were tested for the reactivity with dsDNA (A), CL (B) or histones (C).Distribution of Jκ regions among Vκ5–45 (D) and Vκ5–48 (E) encoded light chains as a percentage of total Vκ5 encoded light chains is shown. Comparisons are with F 3H9 to F chimeras for Vκ5–45 and with M3H9 chimeras for Vκ5–48. * p < 0.05, † p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368537&req=5

pone.0119925.g007: Binding characteristics of Vκ5 encoded light chains vary with Jκ usage.Serial dilutions of each heavy and light chain pair were tested for the reactivity with dsDNA (A), CL (B) or histones (C).Distribution of Jκ regions among Vκ5–45 (D) and Vκ5–48 (E) encoded light chains as a percentage of total Vκ5 encoded light chains is shown. Comparisons are with F 3H9 to F chimeras for Vκ5–45 and with M3H9 chimeras for Vκ5–48. * p < 0.05, † p < 0.01.
Mentions: Proteinuria and survival data were analyzed using Kaplan-Meier curves and log rank test. Comparisons shown in Figs. 1A, 2 and 3A were performed using Mann-Whitney test. p values ≤ 0.05 were considered significant. Comparisons in Fig. 4 were performed using pair-wise Mann-Whitney test with a Bonferroni adjustment to account for multiple testing. In order to preserve an overall 5% significance level, a pairwise comparison was considered statistically significant if p < 0.025. For Fig. 5 the Kruskal-Wallis test was used to compare the M and F wt with their respective experimental groups. Upon finding a significant difference, Bonferroni adjusted multiple pairwise comparisons of the WT group to each of the other two gender-matched groups, were carried out using Mann-Whitney tests. A p value of <0.025 was considered significant. For all male vs. female comparisons the Mann-Whitney test was used. Statistical analysis of the Vκ repertoire data shown in Fig. 6 was performed as previously described [31]. Owing to the typically small sample sizes of single cells, the usual Pearson χ2 statistic is not valid for determining whether differences exist in the distributions of L chains across B cell subsets. Instead, the Fisher exact test is applicable. However, the computing time required for running the exact test on large, sparse tables is prohibitive and, moreover, the power of the test is low. Accordingly, identifying L chains of interest without using formal inferential methods (i.e., without using p values or confidence intervals) is needed. This exploratory approach is frequently used in identifying interesting genes in gene expression microarray studies [32–34]. To this end, we used a novel method for “screening” the r × c table for cell frequencies that appear to be inconsistent with the hypothesis that the B cell subset and L chain distributions are independent. This method uses a graphical approach (scree plots) to finding the entries in the table that depart most from the hypothesis. For additional details on the statistical methodology please refer to Lesser et al [35]. Comparisons in Fig. 7 were performed using chi-square analysis. Mutation frequencies in each subset were compared using the Mann-Whitney test.

Bottom Line: Deletion of 3H9 B cells occurred in the bone marrow and the remaining 3H9 follicular B cells manifested a decrease in surface IgM.We were unable to demonstrate a consistent effect of Tlr7 dose or Yaa on somatic mutations.Our data show that TLR7 excess influences the selection, expansion and diversification of B cells in the germinal center, independent of other genes in the Yaa locus.

View Article: PubMed Central - PubMed

Affiliation: Center for Autoimmunity and Musculoskeletal Diseases, Feinstein Institute for Medical Research, Manhasset, New York, 11030, United States of America.

ABSTRACT
TLR7 enhances germinal center maturation and migration of B cells to the dark zone where proliferation and somatic hypermutation occur. Our goal was to determine how Tlr7 dose influences selection of the autoreactive B cell repertoire in NZW/BXSB. Yaa mice bearing the site-directed heavy chain transgene 3H9 that encodes for the TLR7 regulated anti-CL response. To create a physiologic setting in which autoreactive B cells compete for survival with non-autoreactive B cells, we generated bone marrow chimeras in which disease onset occurred with similar kinetics and the transferred 3H9+ female non-Yaa, male Yaa or male TLR7(-/Yaa) cells could be easily identified by positivity for GFP. Deletion of 3H9 B cells occurred in the bone marrow and the remaining 3H9 follicular B cells manifested a decrease in surface IgM. Although there were differences in the naïve repertoire between the chimeras it was not possible to distinguish a clear pattern of selection against lupus related autoreactivity in TLR7(-/Yaa) or female chimeras. By contrast, preferential expansion of 3H9+ B cells occurred in the germinal centers of male Yaa chimeras. In addition, although all chimeras preferentially selected 3H9/Vκ5 encoded B cells into the germinal center and plasma cell compartments, 3H9 male Yaa chimeras had a more diverse repertoire and positively selected the 3H9/Vκ5-48/Jκ4 pair that confers high affinity anti-cardiolipin activity. We were unable to demonstrate a consistent effect of Tlr7 dose or Yaa on somatic mutations. Our data show that TLR7 excess influences the selection, expansion and diversification of B cells in the germinal center, independent of other genes in the Yaa locus.

No MeSH data available.


Related in: MedlinePlus