Human antibody repertoires in normal physiology and in autoimmune disease

Abstract

The immune system has to balance the need for a broad protection from infectious agents against the risk of developing autoimmune disease. The elements of the immune system should provide clues about conditions under which physiological autoreactivity may develop into autoaggressive immunity, if analyzed against the background of the immune system in healthy individuals. A possible object for this analysis is the antibody repertoire, since antibodies have been implicated in the effector phase and etiology of several autoimmune diseases. Therefore, we studied utilization of the genes coding for the variable part of the immunoglobulin molecule in healthy individuals as well as in patients with autoimmune disease, using RNA in situ hybridization and a newly developed competitive quantitative DNA-based PCR. For the analysis of gene utilization and mutational frequencies, we used a VH-family specific PCR in combination with cloning and nucleotide sequence analysis. Our results demonstrate that in adults the VH gene family repertoire is remarkably stable in time, as well as very similar between individuals of similar genetic backgrounds. We also found differences in VH, D and JH expression between lymphocytes from the spleen and from peripheral blood. On the level of the single gene we found, in healthy individuals, an age-related differential utilization of D and JH genes and an impaired affinity maturation in VH6-containing rearrangements. The number of cells carrying mutations decreased with age, as did the number of mutations per gene.

To learn more about the possible role of B cell repertoires in the pathogenesis of autoimmune aggression, we studied two autoimmune diseases: IDDM (insulin dependent diabetes mellitus) as a model for a T cell mediated disease, and AITP (autoimmune idiopathic thrombocytopenic purpura) as a model for an antibody mediated disease. On VH gene family level, our results show an increase in VH6 gene utilization in the spleen of AITP patients. Interestingly, this change occurs in small resting lymphocytes rather than in naturally activated ones, indicating the absence of selection and actual utilization of the VH6 gene in these patients. This was confirmed by a sequence analysis of recombinations containing the VH6 gene, in which we found low mutational frequencies in functional VH6-containing recombinations both in AITP and in IDDM patients. Among non- functional rearrangements, however, we observed an increase in mutational frequencies in VH6-containing recombinations that appeared to be based in part on different mechanisms. In IDDM patients, the higher mutational frequencies were due both to an increase in the relative number of VH6 genes carrying mutations and to an actual increase in the number of mutations per gene, which suggests the presence of a high number of activated and differentiated B cells in the periphery. In AITP patients, however, only the number of clones that carried mutations was increased.

In conclusion, the VH gene family repertoire appears to be remarkably stable both in time and between individuals, indicating a strict, genetic control. Deviations found in patients with autoimmune disease indicate a modified selection which, however, does not affect the actual VH gene family repertoire of these patients. Therefore, these deviations are more likely a consequence than a cause of the autoimmune process. The age-related changes found in our studies do not correlate with the changes observed in our two models of autoimmune disease and are therefore not likely to be related to the reported increase in autoreactivity in elderly individuals.