B cell repertoire expansion occurs in meningeal ectopic lymphoid tissue
Klaus Lehmann-Horn, Sheng-zhi Wang, Sharon A. Sagan, Scott S. Zamvil, H.-Christian von Büdingen
Klaus Lehmann-Horn, Sheng-zhi Wang, Sharon A. Sagan, Scott S. Zamvil, H.-Christian von Büdingen
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Research Article

B cell repertoire expansion occurs in meningeal ectopic lymphoid tissue

Abstract

Ectopic lymphoid tissues (ELT) can be found in multiple sclerosis (MS) and other organ-specific inflammatory conditions. Whether ELT in the meninges of central nervous system (CNS) autoimmune disease exhibit local germinal center (GC) activity remains unknown. In an experimental autoimmune encephalomyelitis model of CNS autoimmunity, we found activation-induced cytidine deaminase, a GC-defining enzyme, in meningeal ELT (mELT) densely populated by B and T cells. To determine GC activity in mELT, we excised meningeal lymphoid aggregates using laser capture microscopy and evaluated B cell repertoires in mELT and secondary lymphoid organs by next-generation immune repertoire sequencing. We found immunoglobulin heavy chain variable region sequences that were unique to mELT and had accumulated functionally relevant somatic mutations, together indicating localized antigen-driven affinity maturation. Our results suggest that B cells in mELT actively participate in CNS autoimmunity, which may be relevant to mELT in MS and ELT in other chronic inflammatory conditions.

Authors

Klaus Lehmann-Horn, Sheng-zhi Wang, Sharon A. Sagan, Scott S. Zamvil, H.-Christian von Büdingen

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Figure 8

Somatic IgG-VH mutations occurring in mELT cause significant alterations in the binding affinity of the corresponding rmAb to the antigen MOG.

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Somatic IgG-VH mutations occurring in mELT cause significant alterations...
(A) Amino acid alignment of the CDR1–CDR3 regions of 9 mutated IgG-VH sequences found in mELT exclusively with the germline sequence (knockin Ig-VH derived from the 8.18-c5 hybridoma). Six mutated sequences are from mouse A12 and 3 from mouse A20. AA position labeling according to IMGT. Dashes indicate consensus with the germline. (B and C) The 9 mutated Ig-VH sequences were coexpressed with the light chain of the 8.18-c5 hybridoma. Non-mutated rm8.18-c5 rmAb served as reference rmAb. Biolayer interferometry (Octet RED384) was performed to measure binding affinity to MOG. (B) Exemplary association and dissociation diagrams of one mutated rmAb (A12-H2) and the reference rmAb (rm8.18-c5) to MOG in a dilution series. Dark lines, raw data; red lines, fitted curves. Based on association (on) and dissociation (off) rates for each rmAb and the reference rmAb measured in the same experiment, KDs were determined. rmAb A12-H2 (top): Kon = 1.59E+05 (1/ms) (±9.10E+02), Koff = 1.59E-04 (1/s) (±4.24E-06), r2 > 0.99; KD = 9.97E-10 (M). rm8.18-c5 (bottom): Kon = 2.64E+05 (1/Ms) (±1.85E+03), Koff = 4.82E-04 (1/s) (±6.35E-06), r2 > 0.99, KD = 1.83E-9 (M). (C) The ratio KDrm8.18-c5/KDmut rmAb (designated relative binding affinity) is shown for all 9 mutated rmAb, for each mouse separately. For each pair of mutated rmAb and its reference rmAb, data points of 3–5 independent experiments and the mean are plotted. The data point in red represents the rmAb pair depicted in B. **P ≤ 0.01; ***P ≤ 0.001; unpaired 2-tailed t test with Welch’s correction (comparing the rmAb with the highest binding affinity with all others). VH, heavy chain variable region; mELT, meningeal ectopic lymphoid tissue; rmAb, recombinant monoclonal Ab; MOG, myelin oligodendrocyte glycoprotein; CDR, complementarity determining region; IMGT, International ImMunoGeneTics.