Adaptive immune receptor genotyping using the corecount program.

We present a new Rep-Seq analysis tool called corecount, for analyzing genotypic variation in immunoglobulin (IG) and T cell receptor (TCR) genes. corecount is highly efficient at identifying V alleles, including those that are infrequently used in expressed repertoires and those that contain 3' end variation that are otherwise refractory to reliable identification during germline inference from expressed libraries. Furthermore, corecount facilitates accurate D and J gene genotyping. The output is highly reproducible and facilitates the comparison of genotypes from multiple individuals, such as those from clinical cohorts. Here, we applied corecount to the genotypic analysis of IgM libraries from 16 individuals. To demonstrate the accuracy of corecount, we Sanger sequenced all the heavy chain IG alleles (65 IGHV, 27 IGHD and 7 IGHJ) from one individual from whom we also produced two independent IgM Rep-seq datasets. Genomic analysis revealed that 5 known IGHV and 2 IGHJ sequences are truncated in current reference databases. This dataset of genomically validated alleles and IgM libraries from the same individual provides a useful resource for benchmarking other bioinformatic programs that involve V, D and J assignments and germline inference, and may facilitate the development of AIRR-Seq analysis tools that can take benefit from the availability of more comprehensive reference databases.

Analysis of IGH allele content in a sample group of rheumatoid arthritis patients demonstrates unrevealed population heterogeneity.

Immunoglobulin heavy chain (IGH) germline gene variations influence the B cell receptor repertoire, with resulting biological consequences such as shaping our response to infections and altering disease susceptibilities. However, the lack of information on polymorphism frequencies in the IGH loci at the population level makes association studies challenging. Here, we genotyped a pilot group of 30 individuals with rheumatoid arthritis (RA) to examine IGH allele content and frequencies in this group. Eight novel IGHV alleles and one novel IGHJ allele were identified in the study. 15 cases were haplotypable using heterozygous IGHJ6 or IGHD anchors. One variant, IGHV4-34*01_S0742, was found in three out of 30 cases and included a single nucleotide change resulting in a non-canonical recombination signal sequence (RSS) heptamer. This variant allele, shown by haplotype analysis to be non-expressed, was also found in three out of 30 healthy controls and matched a single nucleotide polymorphism (SNP) described in the 1000 Genomes Project (1KGP) collection with frequencies that varied between population groups. Our finding of previously unreported alleles in a relatively small group of individuals with RA illustrates the need for baseline information about IG allelic frequencies in targeted study groups in preparation for future analysis of these genes in disease association studies.

Archaic humans have contributed to large-scale variation in modern human T cell receptor genes.

Human T cell receptors (TCRs) are critical for mediating immune responses to pathogens and tumors and regulating self-antigen recognition. Yet, variations in the genes encoding TCRs remain insufficiently defined. Detailed analysis of expressed TCR alpha, beta, gamma, and delta genes in 45 donors from four human populations-African, East Asian, South Asian, and European-revealed 175 additional TCR variable and junctional alleles. Most of these contained coding changes and were present at widely differing frequencies in the populations, a finding confirmed using DNA samples from the 1000 Genomes Project. Importantly, we identified three Neanderthal-derived, introgressed TCR regions including a highly divergent TRGV4 variant, which mediated altered butyrophilin-like molecule 3 (BTNL3) ligand reactivity and was frequent in all modern Eurasian population groups. Our results demonstrate remarkable variation in TCR genes in both individuals and populations, providing a strong incentive for including allelic variation in studies of TCR function in human biology.

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies.

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies.

Rhesus and cynomolgus macaque immunoglobulin heavy-chain genotyping yields comprehensive databases of germline VDJ alleles.

Definition of the specific germline immunoglobulin (Ig) alleles present in an individual is a critical first step to delineate the ontogeny and evolution of antigen-specific antibody responses. Rhesus and cynomolgus macaques are important animal models for pre-clinical studies, with four main sub-groups being used: Indian- and Chinese-origin rhesus macaques and Mauritian and Indonesian cynomolgus macaques. We applied the (Ig) gene inference tool IgDiscover and performed extensive Sanger sequencing-based genomic validation to define germline VDJ alleles in these 4 sub-groups, comprising 45 macaques in total. There was allelic overlap between Chinese- and Indian-origin rhesus macaques and also between the two macaque species, which is consistent with substantial admixture. The island-restricted Mauritian cynomolgus population displayed the lowest number of alleles of the sub-groups, yet maintained high individual allelic diversity. These comprehensive databases of germline IGH alleles for rhesus and cynomolgus macaques provide a resource toward the study of B cell responses in these important pre-clinical models.

Extensive dissemination and intraclonal maturation of HIV Env vaccine-induced B cell responses.

Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages.