In spite of selection against B cells bearing autoreactive antigen receptors during ontogeny in the bone marrow, substantial frequencies of autoreactive B cells in the periphery are observed. There are two hypotheses, not mutually exclusive, to account for this fact. One such hypothesis holds that such selection is intrinsically leaky, and that autoreactivity in the periphery is due largely to original autoreactivity that has slipped through the selective filter. The other hypothesis does not depend on leakiness of central B cell tolerance, but instead relies on the generation of autoreactivity by somatic mutations incorporated after primary B cell development.
These two hypotheses make different predictions regarding the relationships one will find between the genetic signatures of autoreactive Ig in the bone marrow and those in the periphery. If peripheral autoreactivity is due primarily to the escape of original autoreactivity from central tolerance, we expect to find that autoreactive peripheral B cells will be similar to pre-selection and autoreactive B cells in the bone marrow. The main distinguishing feature of such cells is a long CDR3. We find no statistically significant difference between CDR3 lengths in autoreactive and non-autoreactive Ig, though CDR3 is much longer in NP sequences than in any other class, confirming the observation by others that selection during primary ontogeny favors shorter CDR3.
No such correspondence is necessary under the second hypothesis. What one does expect to see under the second hypothesis are differences in somatic mutation, and we do indeed find that the mutation frequency is about 50% higher in autoreactivity-associated genes than in non-autoreactive genes. This result is consistent with many previous studies of the role of somatic mutation in autoimmunity [17, 18, 19, 20, 21, 22]. Beyond the simple observation of differential mutation frequencies, however, we do see additional genetic biases that distinguish the autoreactives from the normals.
We observe, for example, differential usage in both VH and JH gene segments, with an increase in VH1 family usage in genes from both the general autoimmune (A) and rheumatoid arthritis (RA) datasets, as well as a corresponding reduction in family VH4 usage. Family VH4 is typically second-highest in usage frequency and has been reported to provide some of the most frequently used segments in the adult Ig repertoire, particularly V4-34 [23, 24, 25, 26].
If peripheral autoreactivity were due largely to the leakiness of central tolerance, we would expect gene segment usage in autoantibodies to trend toward the pre-selection repertoire represented by the non-productively rearranged genes. This is not what we find. In fact, the VH segment use in the A and RA genes is not intermediate between the NP and normal genes, it is less like that of the NP genes than the normals are. VH1, for example, has a higher frequency in the normal genes than in the NP genes, and higher frequency still in the A and AR genes, while VH4 is lower in normals than in NP and lower still in A and AR.
The selective enhancement of usage for VH1 and VH5 segments in the A and RA genes may be due partially to the presence of charged residues in the framework region of these gene segments. Studies of autoimmune anti-erythrocyte human protein antibodies and non-autoimmune anti-staphylococcal antibodies show that framework region 1 may play a critical role in effective binding to these antigens [27, 28]. The VH1 and VH5 family genes contain a lysine-lysine amino acid motif in framework 1, which would produce a positively charged region in the folded protein. No other VH segment contains a contiguous pair of positively charged residues. We did also observe an enhancement of VH5 segment usage in the RA genes. Thus, it may be possible that part of the framework region 1 of the VH1 family genes promotes binding to some self-antigens.
We did observe, however, significant differences in the proportions of N-nucleotides among CDR3 nucleotides between either autoreactive set and the normals. This result is consistent with those from studies showing that TdT-deficient mice crossed with autoimmune-prone mice, which make B- and T-cells with nearly no N-nucleotides, have lower incidence of autoimmune disease and longer life spans than non-TdT deficient autoimmune-prone controls [29, 30].
These analyses suggest to us that the premutation genetic signatures observed in the autoreactive sets are indicative of autoreactive potential, and mark those genes that are more likely to give rise to autoreactivity under somatic mutation. B-cell central tolerance can only counter-select V(D)J rearrangements that are already autoreactive, not those with a tendency to become autoreactive. Germline evolution, however, can and does select gene segments for their potential repertoire under somatic mutation [31, 32].
The idea is that germline VH, DH, and JH gene segments have been shaped by evolution to avoid becoming autoreactive too easily under somatic hypermutation. When much of CDR3 is encoded by N-nucleotides rather than by these time-tested gene segments, autoreactivity may be much closer in genotype space than it would otherwise be.