Analyzing infection-driven immune perturbations by quantitative IR-Seq

Immune repertoire sequencing (IR-Seq) rapidly emerged with the advent of high-throughput sequencing as a means of characterizing the adaptive immune system. Early generations of IR-Seq were plagued by sequencing errors and low diversity coverage. We developed Molecular Identifier Clustering-based IR-Seq (MIDCIRS) to quantitatively and comprehensively measure the immune repertoire from a small amount of blood. We used naive B cells to formulate a general framework for IR-Seq experimental validation and quality control and showed that MIDCIRS can be applied to as few as 1,000 naive B cells with excellent diversity coverage. Using MIDCIRS, we studied the antibody repertoire response to acute malaria infection in young children. We found that the infant antibody repertoire is surprisingly competent at introducing somatic hypermutations (SHM) and diversifying B cell clonal lineages in response to malaria infection. Detailed analysis of memory B cell-containing lineages in malaria-experienced toddlers revealed that memory B cells further mutate upon malaria rechallenge. IgM-expressing memory B cells largely retain IgM expression upon rechallenge, but a subset class switch to IgG and IgA. Accurate antibody repertoire analysis requires not only accurate sequencing data, but also correct reference germline allele sequences. Mismatches between the reference sequence and an individual’s true germline sequence would be mistakenly counted as SHMs, inflating the SHM load and skewing the repertoire analysis. We developed a simple yet effect method for predicting novel germline allele sequences from antibody repertoire data and validating them via targeted sequencing of non-rearranged genomic DNA. HIV infection has a profound impact on the CD4⁺ T cell compartment, which can have a devastating effect on the adaptive immune system as a whole. Paradoxically, while peripheral CD4⁺ T cell counts drop with disease severity, T[subscript FH] cells show an inverse relationship. We found that these expanded T[subscript FH] cells exhibit a functionally restricted phenotype, which could contribute to ineffective antibody responses both to HIV and unrelated vaccines. Using MIDCIRS, we found that these expanded T[subscript FH] cells are enriched with HIV-specific sequences and show signs of antigen-driven convergent evolution, suggesting that HIV-specific T cells are selected and recruited into the T[subscript FH] compartment during infection.