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Shared VH1-46 gene usage by pemphigus vulgaris autoantibodies indicates common humoral immune responses among patients.

Cho MJ, Lo AS, Mao X, Nagler AR, Ellebrecht CT, Mukherjee EM, Hammers CM, Choi EJ, Sharma PM, Uduman M, Li H, Rux AH, Farber SA, Rubin CB, Kleinstein SH, Sachais BS, Posner MR, Cavacini LA, Payne AS - Nat Commun (2014)

Bottom Line: Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs.Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding.Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies (autoAbs) against desmoglein 3 (Dsg3). Here, we clone anti-Dsg3 antibodies (Abs) from four PV patients and identify pathogenic VH1-46 autoAbs from all four patients. Unexpectedly, VH1-46 autoAbs had relatively few replacement mutations. We reverted antibody somatic mutations to their germline sequences to determine the requirement of mutations for autoreactivity. Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs. Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding. Our data suggest that VH1-46 autoantibody gene usage is commonly found in PV because VH1-46 Abs require few to no mutations to acquire Dsg3 autoreactivity, which may favour their early selection. Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

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VH1-46 anti-Dsg3 mAbs have relatively few CDR replacement mutations. Frequency of replacement (R, black) and silent (S, white) mutations in the CDRs and FWRs are shown for (a) VH1-46 anti-Dsg3 mAbs and (b) select members of other anti-Dsg3 clonal lineages. “>N” = N/0. VH1-46 Abs have relatively low R:S ratios in the CDRs compared to the FWRs, represented by the proportion of the bar that is black. In contrast, VH1-69 anti-Dsg mAbs ((D31)2/29 and (D3)1d/2c), have more frequent R mutations and elevated R:S ratios in the CDRs compared to the FWRs, with statistical evidence of positive antigen-driven selection (shown in Supplementary Table 3).
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Figure 2: VH1-46 anti-Dsg3 mAbs have relatively few CDR replacement mutations. Frequency of replacement (R, black) and silent (S, white) mutations in the CDRs and FWRs are shown for (a) VH1-46 anti-Dsg3 mAbs and (b) select members of other anti-Dsg3 clonal lineages. “>N” = N/0. VH1-46 Abs have relatively low R:S ratios in the CDRs compared to the FWRs, represented by the proportion of the bar that is black. In contrast, VH1-69 anti-Dsg mAbs ((D31)2/29 and (D3)1d/2c), have more frequent R mutations and elevated R:S ratios in the CDRs compared to the FWRs, with statistical evidence of positive antigen-driven selection (shown in Supplementary Table 3).

Mentions: Analysis of variable region gene usage revealed that mAbs using VH1-46 were identified in all 4 patients (Table 1), including one patient with two clonally unrelated VH1-46 mAbs (patient 2). VK2-24 light chain gene usage was shared in two VH1-46 heterohybridoma mAbs isolated from patients 3 and 4, but no VK/VL gene usage was found in common among all 4 patients. Patient 1 additionally demonstrated three clonally expanded families using VH1-69 (7 unique members), VH3-07 (3 unique members), and VH4-04 (3 unique members). All mAbs had higher frequencies of mutations in their complementarity determining regions (CDRs) compared to the framework regions (FWRs) (Figure 2). This pattern is typical of somatically mutated antibody sequences due to the uneven distribution of somatic mutation hot- and cold-spots among the CDRs and FWRs and suggests that the mutations are physiologic rather than errors during PCR amplification, which would result in similar frequencies of mutations across the antibody sequence. Interestingly, VH1-46 mAbs had a relatively low number of replacement mutations in the CDRs (median 4, range 2-5) compared to non-VH1-46 mAbs (median 8, range 2-10) (p≤0.01 by Wilcoxon rank-sum test) (Supplementary Table 3). Correspondingly, many VH1-46 mAbs had low replacement to silent (R:S) mutation ratios in the complementarity determining regions (CDRs) relative to the framework regions (FWRs), with CDR values >2.9 typically being observed in Abs that have undergone positive antigen-driven selection21 (Figure 2a). Statistical analysis of somatic mutations using a Bayesian estimation (BASELINe) test22, 23, which compares the observed versus expected frequency of replacement mutations under the hypothesis of no selection, indicated that VH1-46 mAbs did not demonstrate statistically significant evidence of positive antigen-driven selection in the CDRs (Supplementary Table 3). In contrast, VH1-69 clonal lineage 1 demonstrated statistically significant evidence of positive antigen-driven selection in the CDRs. Additionally, several members in VH1-69 clonal lineage 2 showed a trend toward positive antigen-driven selection (0.05<p<0.1 highlighted in light gray), with increasing sigma values reflecting increasing selection pressures among members of the lineage.


Shared VH1-46 gene usage by pemphigus vulgaris autoantibodies indicates common humoral immune responses among patients.

Cho MJ, Lo AS, Mao X, Nagler AR, Ellebrecht CT, Mukherjee EM, Hammers CM, Choi EJ, Sharma PM, Uduman M, Li H, Rux AH, Farber SA, Rubin CB, Kleinstein SH, Sachais BS, Posner MR, Cavacini LA, Payne AS - Nat Commun (2014)

VH1-46 anti-Dsg3 mAbs have relatively few CDR replacement mutations. Frequency of replacement (R, black) and silent (S, white) mutations in the CDRs and FWRs are shown for (a) VH1-46 anti-Dsg3 mAbs and (b) select members of other anti-Dsg3 clonal lineages. “>N” = N/0. VH1-46 Abs have relatively low R:S ratios in the CDRs compared to the FWRs, represented by the proportion of the bar that is black. In contrast, VH1-69 anti-Dsg mAbs ((D31)2/29 and (D3)1d/2c), have more frequent R mutations and elevated R:S ratios in the CDRs compared to the FWRs, with statistical evidence of positive antigen-driven selection (shown in Supplementary Table 3).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4120239&req=5

Figure 2: VH1-46 anti-Dsg3 mAbs have relatively few CDR replacement mutations. Frequency of replacement (R, black) and silent (S, white) mutations in the CDRs and FWRs are shown for (a) VH1-46 anti-Dsg3 mAbs and (b) select members of other anti-Dsg3 clonal lineages. “>N” = N/0. VH1-46 Abs have relatively low R:S ratios in the CDRs compared to the FWRs, represented by the proportion of the bar that is black. In contrast, VH1-69 anti-Dsg mAbs ((D31)2/29 and (D3)1d/2c), have more frequent R mutations and elevated R:S ratios in the CDRs compared to the FWRs, with statistical evidence of positive antigen-driven selection (shown in Supplementary Table 3).
Mentions: Analysis of variable region gene usage revealed that mAbs using VH1-46 were identified in all 4 patients (Table 1), including one patient with two clonally unrelated VH1-46 mAbs (patient 2). VK2-24 light chain gene usage was shared in two VH1-46 heterohybridoma mAbs isolated from patients 3 and 4, but no VK/VL gene usage was found in common among all 4 patients. Patient 1 additionally demonstrated three clonally expanded families using VH1-69 (7 unique members), VH3-07 (3 unique members), and VH4-04 (3 unique members). All mAbs had higher frequencies of mutations in their complementarity determining regions (CDRs) compared to the framework regions (FWRs) (Figure 2). This pattern is typical of somatically mutated antibody sequences due to the uneven distribution of somatic mutation hot- and cold-spots among the CDRs and FWRs and suggests that the mutations are physiologic rather than errors during PCR amplification, which would result in similar frequencies of mutations across the antibody sequence. Interestingly, VH1-46 mAbs had a relatively low number of replacement mutations in the CDRs (median 4, range 2-5) compared to non-VH1-46 mAbs (median 8, range 2-10) (p≤0.01 by Wilcoxon rank-sum test) (Supplementary Table 3). Correspondingly, many VH1-46 mAbs had low replacement to silent (R:S) mutation ratios in the complementarity determining regions (CDRs) relative to the framework regions (FWRs), with CDR values >2.9 typically being observed in Abs that have undergone positive antigen-driven selection21 (Figure 2a). Statistical analysis of somatic mutations using a Bayesian estimation (BASELINe) test22, 23, which compares the observed versus expected frequency of replacement mutations under the hypothesis of no selection, indicated that VH1-46 mAbs did not demonstrate statistically significant evidence of positive antigen-driven selection in the CDRs (Supplementary Table 3). In contrast, VH1-69 clonal lineage 1 demonstrated statistically significant evidence of positive antigen-driven selection in the CDRs. Additionally, several members in VH1-69 clonal lineage 2 showed a trend toward positive antigen-driven selection (0.05<p<0.1 highlighted in light gray), with increasing sigma values reflecting increasing selection pressures among members of the lineage.

Bottom Line: Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs.Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding.Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies (autoAbs) against desmoglein 3 (Dsg3). Here, we clone anti-Dsg3 antibodies (Abs) from four PV patients and identify pathogenic VH1-46 autoAbs from all four patients. Unexpectedly, VH1-46 autoAbs had relatively few replacement mutations. We reverted antibody somatic mutations to their germline sequences to determine the requirement of mutations for autoreactivity. Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs. Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding. Our data suggest that VH1-46 autoantibody gene usage is commonly found in PV because VH1-46 Abs require few to no mutations to acquire Dsg3 autoreactivity, which may favour their early selection. Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

Show MeSH
Related in: MedlinePlus