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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 mAbs require few to no somatic mutations to bind Dsg3 (a) F706 germline-reverted (GL) and (b) F779 GL Abs do not bind Dsg3. (c) 3.2 GL Ab binds Dsg3 by ELISA with reduced affinity (specificity of binding confirmed by surface plasmon resonance, Table 3). (d) PVE4-8 GL and (e) 4.2 GL Abs retain binding to Dsg3 by ELISA. Black/gray hatched lines indicate GL1/2 Abs, respectively. (f-j) Germline-reverted non-VH1-46 mAbs do not demonstrate binding to Dsg3 by ELISA. GL LC/HC recombinant Abs were generated for those mAbs that were insoluble in their fully GL state. (k) Indirect immunofluorescence (IIF) staining of human skin confirms Dsg3 autoreactivity of PVE4-8 GL and 4.2 GL Abs in native skin tissue. IIF binding of 3.2 GL was not detectable, presumably due to low affinity. Scale bar, 20 μM. Error bars indicate s.e.m. Data are representative of 3-5 independent experiments.
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Figure 3: VH1-46 mAbs require few to no somatic mutations to bind Dsg3 (a) F706 germline-reverted (GL) and (b) F779 GL Abs do not bind Dsg3. (c) 3.2 GL Ab binds Dsg3 by ELISA with reduced affinity (specificity of binding confirmed by surface plasmon resonance, Table 3). (d) PVE4-8 GL and (e) 4.2 GL Abs retain binding to Dsg3 by ELISA. Black/gray hatched lines indicate GL1/2 Abs, respectively. (f-j) Germline-reverted non-VH1-46 mAbs do not demonstrate binding to Dsg3 by ELISA. GL LC/HC recombinant Abs were generated for those mAbs that were insoluble in their fully GL state. (k) Indirect immunofluorescence (IIF) staining of human skin confirms Dsg3 autoreactivity of PVE4-8 GL and 4.2 GL Abs in native skin tissue. IIF binding of 3.2 GL was not detectable, presumably due to low affinity. Scale bar, 20 μM. Error bars indicate s.e.m. Data are representative of 3-5 independent experiments.

Mentions: The relative paucity of replacement mutations in the CDRs of VH1-46 mAbs and the lack of evidence of positive selection for those mutations prompted us to investigate whether the VH1-46 mAbs identified from PV patients required somatic mutations to bind Dsg3. We reverted somatically mutated (SM) residues in the V(D)J gene segments to their corresponding germline (GL) sequences and tested for their ability to bind Dsg3 by ELISA and indirect IF staining of human skin. Germline predictions were identified by analysis of variable regions with 3 sequence alignment programs, SODA, Vbase2, and IMGT/V-QUEST. Some mAbs had more than one GL sequence prediction due to different D gene assignments, and hence multiple GL versions were evaluated (Supplementary Table 1). F706GL and F779GL1/GL2 mAbs were unable to bind Dsg3 in the absence of somatic mutations (Figure 3a,b). 3.2GL demonstrated weak binding to Dsg3 by ELISA (Figure 3c), with no detectable IF staining of human skin (Figure 3k). PVE4-8GL and 4.2GL1/GL2maintained binding to Dsg3 by ELISA (Figure 3d,e) confirmed by indirect IF staining of human skin (Figure 3k). None of the VH1-46 SM/GL mAbs were polyreactive, as measured by Hep2 immunofluorescence and ELISA (Supplementary Figure 1). We also reverted somatic mutations in non-VH1-46 anti-Dsg3 mAbs. (D3)4/30GL (VH3-30) was insoluble and was unable to be tested. (D3)1d/2c (VH1-69), (D31)2/29 (VH1-69), (D31)3c/9 (VH3-07), (D31)12b/6 (VH4-04), and VH5a (VH5-a) all required somatic mutations to bind Dsg3 (Figure 3f-j). Mutation reversion in only the light chain or only the heavy chain indicated that the heavy chain is more important for determining binding to Dsg3, as indicated for F706, F779, (D31)2/29, 4.2, (D31)12b/6, and VH5a (Figure 3 and Supplementary Figure 2).


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 mAbs require few to no somatic mutations to bind Dsg3 (a) F706 germline-reverted (GL) and (b) F779 GL Abs do not bind Dsg3. (c) 3.2 GL Ab binds Dsg3 by ELISA with reduced affinity (specificity of binding confirmed by surface plasmon resonance, Table 3). (d) PVE4-8 GL and (e) 4.2 GL Abs retain binding to Dsg3 by ELISA. Black/gray hatched lines indicate GL1/2 Abs, respectively. (f-j) Germline-reverted non-VH1-46 mAbs do not demonstrate binding to Dsg3 by ELISA. GL LC/HC recombinant Abs were generated for those mAbs that were insoluble in their fully GL state. (k) Indirect immunofluorescence (IIF) staining of human skin confirms Dsg3 autoreactivity of PVE4-8 GL and 4.2 GL Abs in native skin tissue. IIF binding of 3.2 GL was not detectable, presumably due to low affinity. Scale bar, 20 μM. Error bars indicate s.e.m. Data are representative of 3-5 independent experiments.
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Related In: Results  -  Collection

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Figure 3: VH1-46 mAbs require few to no somatic mutations to bind Dsg3 (a) F706 germline-reverted (GL) and (b) F779 GL Abs do not bind Dsg3. (c) 3.2 GL Ab binds Dsg3 by ELISA with reduced affinity (specificity of binding confirmed by surface plasmon resonance, Table 3). (d) PVE4-8 GL and (e) 4.2 GL Abs retain binding to Dsg3 by ELISA. Black/gray hatched lines indicate GL1/2 Abs, respectively. (f-j) Germline-reverted non-VH1-46 mAbs do not demonstrate binding to Dsg3 by ELISA. GL LC/HC recombinant Abs were generated for those mAbs that were insoluble in their fully GL state. (k) Indirect immunofluorescence (IIF) staining of human skin confirms Dsg3 autoreactivity of PVE4-8 GL and 4.2 GL Abs in native skin tissue. IIF binding of 3.2 GL was not detectable, presumably due to low affinity. Scale bar, 20 μM. Error bars indicate s.e.m. Data are representative of 3-5 independent experiments.
Mentions: The relative paucity of replacement mutations in the CDRs of VH1-46 mAbs and the lack of evidence of positive selection for those mutations prompted us to investigate whether the VH1-46 mAbs identified from PV patients required somatic mutations to bind Dsg3. We reverted somatically mutated (SM) residues in the V(D)J gene segments to their corresponding germline (GL) sequences and tested for their ability to bind Dsg3 by ELISA and indirect IF staining of human skin. Germline predictions were identified by analysis of variable regions with 3 sequence alignment programs, SODA, Vbase2, and IMGT/V-QUEST. Some mAbs had more than one GL sequence prediction due to different D gene assignments, and hence multiple GL versions were evaluated (Supplementary Table 1). F706GL and F779GL1/GL2 mAbs were unable to bind Dsg3 in the absence of somatic mutations (Figure 3a,b). 3.2GL demonstrated weak binding to Dsg3 by ELISA (Figure 3c), with no detectable IF staining of human skin (Figure 3k). PVE4-8GL and 4.2GL1/GL2maintained binding to Dsg3 by ELISA (Figure 3d,e) confirmed by indirect IF staining of human skin (Figure 3k). None of the VH1-46 SM/GL mAbs were polyreactive, as measured by Hep2 immunofluorescence and ELISA (Supplementary Figure 1). We also reverted somatic mutations in non-VH1-46 anti-Dsg3 mAbs. (D3)4/30GL (VH3-30) was insoluble and was unable to be tested. (D3)1d/2c (VH1-69), (D31)2/29 (VH1-69), (D31)3c/9 (VH3-07), (D31)12b/6 (VH4-04), and VH5a (VH5-a) all required somatic mutations to bind Dsg3 (Figure 3f-j). Mutation reversion in only the light chain or only the heavy chain indicated that the heavy chain is more important for determining binding to Dsg3, as indicated for F706, F779, (D31)2/29, 4.2, (D31)12b/6, and VH5a (Figure 3 and Supplementary Figure 2).

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