In order to investigate characteristics of the humoral immune response in the CSF of a patient with a typical course of NB, we sought to resurrect the antigen specificities of antibodies from CSF-derived cePC by recombinant reconstruction of their secreted IgG. Instead of analyzing the clonally diverse CSF Ig itself, we dissected the antigen-driven immune responses in the CSF down to the clonal level, which made it possible to discern the antigen specificity of individual expanded CSF plasma cell clones. We validated our system by recombinant expression of a well-characterized MAb (8-18c5), demonstrating that our methodology preserves both the specificity and the affinity of the antibodies secreted by the “original” antibody-producing cells.
By means of assembly of Ig H-chain sequences with identical CDR3 regions, we were able to identify three expanded plasma cell clones in the CSF of a patient with NB (RA17, RA20, and RA77). The VH sequences (n = 4) contained in the RA17 group had a number of nucleotide mutations, suggesting that these sequences were the result of somatic hypermutation during affinity maturation. No mutations were found in the RA20 and RA77 groups of sequences. However, a comparison with germ line V-region segments revealed mutations in the H- and L-chain V-region sequences in all groups (Table ), thus providing additional evidence that the antibodies had undergone affinity maturation.
Of the recombinant human MAbs derived from the groups containing identical H-chain CDR3 regions, one (RA77) specifically reacted with B. burgdorferi
, the causative infectious agent of NB, as determined by ELISA and immunofluorescence staining of B. burgdorferi
with RA77. No cross-reactivity of RA77 with T. pallidum
spirochetes or E. coli
could be detected, indicating that RA77 is specific for B. burgdorferi
. This finding of a B. burgdorferi
-specific antibody produced by a cePC is consistent with previous studies showing that OCB in the CSF of NB patients contain antibodies directed against B. burgdorferi
), particularly in view of recent data from our laboratory supporting the conclusion that cePC are in fact the producers of OCB (unpublished data). To date, the only other inflammatory disease for which a link between CNS-derived plasma cells and the causative agent has been demonstrated is subacute sclerosing panencephalitis. Burgoon et al. obtained measles virus-specific antibodies from individual plasma cells excised from postmortem brain tissue of a patient with subacute sclerosing panencephalitis by laser capture microdissection (6
). Our findings, together with those of Burgoon et al., suggest that such an experimental setup may be helpful in identifying disease-relevant target antigens in neuroinflammatory diseases of unknown origin.
To further analyze the exact antigen specificity of the B. burgdorferi
-reactive RA77 MAb, we performed immunoprecipitation and subsequent MALDI-TOF analyses. In this way, we were able to identify the cognate antigen of RA77, namely, the B. burgdorferi
flagellum protein p41. While we found a high-affinity interaction between antibody RA77 and native B. burgdorferi
lysate, the affinity to recombinant p41 protein was significantly lower. This finding suggests that the interaction between RA77 and native p41 may depend not only on the amino acid sequence and resulting conformation but also on potential posttranslational modifications. Such modifications have been demonstrated in flagellum proteins of other spirochetes (3
). Another explanation for the lower affinity of antibody RA77 for the recombinant p41 protein than for the native protein may be partial misfolding of the recombinant protein. Nevertheless, the high-affinity interaction between RA77 and native B. burgdorferi
lysate suggests that RA77 is the result of an affinity-matured antibody response.
Because the patient whose cePC we used in this study suffered from facial nerve palsy, a common clinical presentation of NB, we investigated the possible specificity of RA17, RA20, and RA77 for facial nerve tissue. Interestingly, one of these antibodies, RA17, displayed reactivity with this tissue. To further evaluate the tissue components that RA17 is specific for, we performed double-immunofluorescence staining with a myelin-specific antibody (8-18c5), which strongly suggested myelin reactivity of RA17. Although the results did not overlap completely, costaining with the 8-18c5 antibody and RA17 indicated reactivity with identical structures, namely myelin. However, the myelin reactivity of RA17 was not limited to facial nerve tissue but also applied to CNS white matter myelin. Double staining with RA17 and a neurofilament-specific antibody indirectly confirmed this notion, as RA17 stained structures enfolding neurons. Immunofluorescence staining with RA17 of a number of different human control tissues (colon, kidney, and lung) yielded negative results, suggesting CNS-restricted reactivity. This autoimmune reactivity is consistent with previous studies hypothesizing that the immune responses taking place in NB may also have autoimmune features (11
). The origin of autoimmunity in NB could be either cross-reactivity of Borrelia
-specific antibodies with epitopes shared with human tissues (“molecular mimicry”), such as that observed in Lyme arthritis (9
), or an immune response triggered by the emergence of novel epitopes during infection-mediated tissue destruction (19
). Because no reactivity of RA17 with B. burgdorferi
was observed, in the case of this MAb the latter hypothesis may be favored. While we could determine the specificity for the cePC RA17 and RA77, the antigens that originally drove the B-cell response into clonal expansion and final-stage maturation into plasma cells within the CNS compartment remain undetermined for RA20. Nevertheless, our data suggest that autoimmunity is a feature represented within the cePC repertoire analyzed here. Future work should provide further insight into the functional role of such autoreactive antibodies in NB.
In conclusion, by recombinant reconstruction of the antigen specificities of antibodies secreted by individual expanded CSF plasma cell clones, we were able to dissect complex repertoires of the adaptive immune system in response to CNS infection with B. burgdorferi. We provide evidence that the antigen-driven immune response in NB not only results in high-affinity pathogen-specific antibodies but can also lead to the generation of humoral autoimmunity apparently independent of mechanisms such as “molecular mimicry.”