Stereotypic persistent B cell receptor clonotypes in Alzheimer’s Disease

The growing evidence suggest that the effect of Alzheimer’s Disease (AD) is not solely restricted to the brain. Numerous groups investigated the relation between the central nervous system and peripheral system, whereupon the peripheral system is closely involved in accordance with the progression of disease and systemic immunity. In our previous report, we identified shared B cell receptor (BCR) sequences among 10 AD patients which showed similar class-switching orientation, identical isotypes and high somatic hypermutation rate. For further investigation, we recruited 44 patients with AD at baseline and 37 patients for second follow-up to identify the shared BCR sequences among patients and persistent BCR within an individual patient. We were able to annotate 3983 unique AD specific BCR clonotypes and one of the clonotype showed binding affinity against human Aβ42 peptide. Our finding can provide evidence of common BCRs in AD patients exposed to antigenic stimulus which are related to AD pathogenesis. For a period of 75 weeks, peripheral blood (PB) samples were collected from 44 Alzheimer patients with a median age of 75 years (standard deviation; 7.04) and sex ratio of 34% and 66% (male vs female). Except for seven patients (Patient ID 3, 5, 8, 19, 28, 30, and 37), PB samples were collected twice with interval between 14 and 53 weeks. The duration for the first and second PB sampling was 61 and 34 weeks, respectively. From these 81 PB samples, cDNA was prepared and subjected to the amplification of a gene fragment encoding variable domain (VH) and N-terminal part of constant domain 1 (CH1) of B cell receptor (BCR) heavy chain using a specific primer set (Extended Table 2), which were analyzed in next generation sequencing with a median read of 245,033 (standard deviation of 184,462, Extended Table 3). For the analysis of BCR repertoire, we first removed naïve BCR sequences, which we define as those with IgM or ID isotype containing less than 1 somatic hypermutation (SHM). Then we grouped the remaining sequences into clonotypes, which was defined as a collection of sequences sharing the same CDR3 sequence at the amino acid level as well as encoded by identical IGHV and IGHJ genes15. Among these BCR clonotypes, AD patient-specific clonotypes were selected by excluding clonotypes found in a control group (CG) established in our prior studies, which was constituted with 55 vaccinees (285 PB samples obtained chronologically) injected three times with COVID-19 vaccine14.Control group specific non-naïve BCR clonotypes were also obtained following the same procedures. Before further analysis, we tried to remove any possibility of aerosol molecular contamination during NGS library preparation and index switching during sequencing15. For this, we excluded all the stereotypic clonotypes in which each patient or control did not show unique BCR sequences at the nucleotide level. After all these selection processes we could select 3,983 and 83,821 BCR clonotypes specific to AD patient group or control group, respectively