Scarcity of autoreactive human blood IgA+ memory B cells

Julie Prigent, Valérie Lorin, Ayrin Kök, Thierry Hieu, Salomé Bourgeau, Hugo Mouquet, Julie Prigent, Valérie Lorin, Ayrin Kök, Thierry Hieu, Salomé Bourgeau, Hugo Mouquet

Abstract

Class-switched memory B cells are key components of the "reactive" humoral immunity, which ensures a fast and massive secretion of high-affinity antigen-specific antibodies upon antigenic challenge. In humans, IgA class-switched (IgA+ ) memory B cells and IgA antibodies are abundant in the blood. Although circulating IgA+ memory B cells and their corresponding secreted immunoglobulins likely possess major protective and/or regulatory immune roles, little is known about their specificity and function. Here, we show that IgA+ and IgG+ memory B-cell antibodies cloned from the same healthy humans share common immunoglobulin gene features. IgA and IgG memory antibodies have comparable lack of reactivity to vaccines, common mucosa-tropic viruses and commensal bacteria. However, the IgA+ memory B-cell compartment contains fewer polyreactive clones and importantly, only rare self-reactive clones compared to IgG+ memory B cells. Self-reactivity of IgAs is acquired following B-cell affinity maturation but not antibody class switching. Together, our data suggest the existence of different regulatory mechanisms for removing autoreactive clones from the IgG+ and IgA+ memory B-cell repertoires, and/or different maturation pathways potentially reflecting the distinct nature and localization of the cognate antigens recognized by individual B-cell populations.

Keywords: Autoreactivity; IgA Antibodies; Immunoglobulin genes; Memory B cells; Polyreactivity.

© 2016 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figures

Figure 1
Figure 1
Immunoglobulin gene repertoire of IgA+ memory B‐cell antibodies. Single CD19+CD27+IgA+ and CD19+CD27+IgG+ B cells from PBMCs of healthy donors were FACS sorted and their heavy‐ and light‐chain variable domains (IgH and IgL) amplified and sequenced. All immunoglobulin gene characteristics were determined by analyzing IgH and IgL sequences of 297 single IgA+ B cells isolated from four individuals (n = 58 for hd1, n = 56 for hd2, n = 84 for hd3, and n = 99 for hd4) and 70 single IgG+ B cells from two of them (n = 31 for hd2 and n = 39 for hd4) using IgBLAST; (http://www.ncbi.nlm.nih.gov/igblast) and IMGT® (http://www.imgt.org) online tools. (A) Box plot showing the frequency of IgA antibodies expressing IgA1 (α1) or IgA2 (α2) subclass as determined using Blast® alignment tool (NIH) and Fcα1 and Fcα2 gene sequences as reference (IMGT®). (B) Heat maps comparing the VH and JH gene usages between IgA+ and IgG+ memory B‐cell antibodies. Color gradient is proportional to the frequency for gene usages with darker colors indicating high frequencies while white corresponds to a frequency of 0. Groups were compared using 2 × 5 Fisher's Exact test. ns, not significant. (C) Circos plots generated from the immunoglobulin gene analysis using “circlize” (v0.3.1) R package compare the frequency of VH(DH)JH rearrangements between IgA+ and IgG+ memory B‐cell antibodies. Heat bar shows the results of the 2 × 5 Fisher's Exact test used to compare both groups. (D) Bar graphs comparing the CDRH3 aminoacid length and number of positive charges in the CDRH3 between IgA+ and IgG+ memory B‐cell antibodies. Groups were compared using 2 × 5 Fisher's Exact test. ns, not significant. (E) Comparison of the number of mutations in VH genes (VH mut.) between IgA+ and IgG+ memory B‐cell antibodies, and between IgA1 and IgA2 subclasses. The average number of mutations is indicated below each dot plot. Groups were compared using Student's t‐test with Welch's correction. ns, not significant. (F) Bar graphs comparing the frequency of Igκ− and/or Igλ−expressing antibodies between IgA+ and IgG+ memory B‐cell compartments. Groups were compared using 2 × 5 Fisher's Exact test. ns, not significant. (G) Same as in (B) but for Vκ and Jκ gene usages. (H) Same as in (B) but for Vλ and Jλ gene usages (I) Same as in (E) but for IgA Vκ and Vλ genes.
Figure 2
Figure 2
Reactivity of IgA+ memory B‐cell antibodies against vaccines, viral pathogens, and commensal bacteria. The reactivity of recombinant IgA+ and IgG+ memory antibodies against selected vaccines, mucosa‐tropic viruses, and commensal bacteria was determined by ELISA. In total, 251 recombinant IgA (from four healthy individuals; n = 59 for hd1, n = 47 for hd2, n = 67 for hd3, and n = 78 for hd4), and 61 IgG monoclonal antibodies (from hd2 and hd4 donors, n = 28 and n = 33, respectively) were produced and tested. Antibodies were tested in triplicate and in two independent experiments. Averaged data from one representative experiment are shown. (A) Flu, influenza virus; DTP, diphtheria‐tetanus‐polio; MMR, measles‐mumps‐rubella; HBV, hepatitis B virus. (B) Adeno, adenovirus type 5; Rota, rotavirus; Rhino, rhinovirus type 1A; HIV‐1. (C) Selected commensal bacteria. Dotted black lines indicate the reactivity of the polyreactive control antibody ED38 38. Dotted green line indicates the average reactivity of the negative control antibody mGO53 8 against the tested antigens. Polyreactive antibodies reacting non‐specifically with the different antigens tested are shown in red.
Figure 3
Figure 3
Polyreactivity of IgA+ memory B‐cell antibodies. (A) The reactivity of recombinant IgA+ and IgG+ memory antibodies against KLH, dsDNA, insulin and LPS was measured by ELISA. In total, 251 recombinant IgA (from four healthy individuals; n = 59 for hd1, n = 47 for hd2, n = 67 for hd3, and n = 78 for hd4), and 61 IgG monoclonal antibodies (from hd2 and hd4 donors, n = 28 and n = 33, respectively) were tested and analyzed. Duplicate values were obtained from at least three independent experiments. Representative data from one experiment are shown as mean values. Dotted green and black lines represent the negative control antibody mGO53 8 and positive control antibody ED38 38, respectively. Horizontal lines show cut‐off OD405 nm for positive reactivity. Pie charts summarize the frequency of polyreactive (black) and non‐polyreactive (white) IgA+ and IgG+ memory B‐cell clones. The number of tested antibodies is indicated in the pie chart center. Groups were compared using 2 × 2 Fisher's Exact test. ns, not significant. (B) Bar graph comparing the frequency of polyreactive antibodies (as indicated above bars) between IgA+ memory B cells, and other B‐cell compartments: mature naïve B cells (MN) 8, IgG+ memory B cells from donor hd2‐hd4, and from historical data 15, 17. Each symbol represents a donor. Groups were compared using 2 × 5 Fisher's Exact test.*p < 0.05; **p < 0.01; ns, nonsignificant. The number of antibodies tested in each group is indicated below each bar. (C) Bar graph comparing the frequency of polyreactive (black) and non‐polyreactive (white) IgA+ memory B cells according to IgA sub‐class (IgA1 or IgA2), and light chains used (Igκ or Igλ). Groups were compared using 2 × 5 Fisher's Exact test.ns, nonsignificant. (D) Bar graphs comparing the frequency of polyreactive and nonpolyreactive IgA+ memory B cells according to CDRH3 aminoacid length and number of positive charges in the CDRH3. Groups were compared using 2 × 5 Fisher's Exact test. ns, nonsignificant. (E) Heat maps comparing the frequency of VH and JH gene usages between polyreactive and nonpolyreactive IgA+ memory B cells. (F) Circos plots generated from the immunoglobulin gene analysis using “circlize” (v0.3.1) R package compare the frequency of VH(DH)JH rearrangements between polyreactive and nonpolyreactive IgA+ memory B cells. Heat bar shows the results of the 2 × 5 Fisher's Exact test used to compare both groups. (G) Dot plots comparing the number of mutations in VH genes between polyreactive and non‐polyreactive IgA+ memory B cells. Groups were compared using Student's t‐test with Welch's correction. ns, not significant.
Figure 4
Figure 4
Self‐reactivity of IgA+ memory B‐cell antibodies. The reactivity of the recombinant IgA+ and IgG+ memory antibodies against HEp‐2 cell antigens was evaluated by ELISA and indirect immunofluorescence assay (IFA). In total, 251 recombinant IgA (from four healthy individuals; n = 59 for hd1, n = 47 for hd2, n = 67 for hd3, and n = 78 for hd4), and 61 IgG monoclonal antibodies (from hd2 and hd4 donors, n = 28 and n = 33, respectively) were tested and analyzed. (A) Dot plots comparing the frequency of HEp‐2 reactive antibodies by ELISA (as indicated above dots) between IgA+ and IgG+ memory B cells. The data shown correspond to the mean values of triplicate obtained in two independent experiments. (B) and (C) show representative HEp‐2 cell IFA staining patterns of antibodies cloned from IgA+ (B), and IgG+ (C) memory B cells, respectively, tested in two experiments (Magnification ×40). White scale bar indicates 12 μm. (D) Bar graph comparing the frequency of combined ELISA/IFA HEp‐2 reactive antibodies (as indicated above bars) between IgA+ memory B cells, and other B‐cell compartments: mature naïve B cells (MN) 8, IgG+ memory B cells from donor hd2‐hd4 (blue dots), and from historical data (green dots) 15, 17. Each symbol represents a donor. Groups were compared using 2 × 5 Fisher's Exact test.*p < 0.05; **p < 0.01; ***p < 0.001; ns, nonsignificant. The number of antibodies tested in each group is indicated below each bar. (E) Pie charts comparing the frequency (as indicated below and on charts) of non HEp‐2 reactive (white) and HEp‐2 reactive IgA+ and IgG+ memory B‐cell antibodies according to the different IFA reactivity patterns observed: cytoplasmic (light grey), nuclear plus cytoplamic (dark gray), and nuclear (dark). The number of tested antibodies is indicated in the pie chart center. Groups were compared using 2 × 5 Fisher's Exact test.
Figure 5
Figure 5
Poly‐ and self‐reactivity of class‐switched IgA+ and germline‐encoded antibodies. The reactivity of native IgA+ memory (n = 14) and reverted (n = 9) antibodies, which were selected from the four donors, were tested by ELISA against polyreactive ligands, and by ELISA and IFA against HEp‐2 cell antigens. (A) Heat map comparing the poly‐ and HEp‐2 reactivity of recombinant IgA+ memory antibodies expressed with an IgG1 or with their original IgA Fc portion. The data shown correspond to the mean values of triplicate obtained in two independent experiments. Color intensity is proportional to the reactivity level with darker colors indicating high binding while light colors show moderate binding (white = no binding). (B) ELISA graphs comparing the reactivity of mutated and reverted IgA antibodies against KLH, dsDNA, insulin, and LPS. Dotted lines represent the positive control antibody ED38 38. Horizontal lines show cut‐off OD405 nm for positive reactivity. Green line shows the negative control antibody mGO53 8. One representative experiment is shown. Heat maps summarizing the reactivity of the mutated and reverted IgA antibodies to the various antigens tested expressed as a mean of triplicate values obtained from two experiments. Color gradient is proportional to the reactivity level with darker colors indicating high binding while light colors show moderate binding (white = no binding). Pie charts summarize the frequency of polyreactive (black) and non‐polyreactive (white) antibodies. The number of tested antibodies is indicated in the pie chart center. Groups were compared using 2 × 2 Fisher's Exact test. (C) Dot plot comparing the HEp‐2 reactivity detected by ELISA of mutated (Mut.) versus reverted to germline (GL) IgA antibodies. Pie charts summarize the frequency of HEp‐2 (black) and non‐HEp‐2 (white) reactive antibodies. The number of tested antibodies is indicated in the pie chart center. The data shown correspond to the mean values of triplicate obtained in two independent experiments. Groups were compared using 2 × 2 Fisher's Exact test. (D) HEp‐2 cell IFA staining patterns obtained with the original mutated IgA antibodies and their germline counterparts (Reverted), tested in two experiments (magnification ×40). White scale bar indicates 12 μm. Pie charts summarize the frequency of HEp‐2 (black) and non‐HEp‐2 (white) reactive antibodies. The number of tested antibodies is indicated in the pie chart center. Groups were compared using 2 × 2 Fisher's Exact test.

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