Computational approaches to study germline-encoded and expressed adaptive immune receptor repertoires

The somatic recombination of a couple variable (V), diversity (D), and joining (J) genes into millions of B and T cell receptors (BCRs and TCRs) is the key mechanism by which the adaptive immune system recognizes an endlessly diverse set of pathogens. The loci encoding V, D, and J genes are complex: nearly every individuals has a unique set, deletions and duplications are commonplace, and intergenic and interallelic variation is enormous. TCRs are made of either an alpha and beta chain or a gamma and delta chain. BCRs comprise two identical heavy chains and two identical light chains, the latter from either the kappa or lambda locus. My thesis is a study of TCR and BCR population diversity, response to antigenic stimuli, and the computational methods used to analyze them. Our lab developed a method called IgDiscover to determine personal genotypes from expressed TCR and BCR repertoire data. As a computational scientist, I applied our lab's method as well as population genetics methods, amplicon denoising approaches, and methods I developed during my thesis work to elucidate the genetics and functionality of TCRs and BCRs.

In paper I, we sequenced all four types of TCR repertoires (alpha, beta, delta, gamma) in 45 individuals across four diverse superpopulations (sub-Saharan African, European, East Asian, South Asian) and applied IgDiscover to determine each person's TCR V, D, and J genotype. We discovered 175 previously undescribed V and J alleles, with a majority containing coding variants. We reported large inter-individual and inter-population diversity and many instances of population specific alleles. Non-African specific Neanderthal novel variants of the TRGV4, TRAJ24, TRAJ26, and TRAV12-2 genes were discovered. The TRGV4 variant was particularly divergent from previously described alleles and exhibited decreased binding to the TRGV4 butyrophilin-like (BTNL) ligand.

In paper II, we longitudinally sequenced the immunoglobulin heavy (IGH) repertoires of the BCR and isolated monoclonal antibodies of two individuals first infected by SARS-CoV-2 then immunized with an mRNA spike vaccine. The sampling timepoints following infection and vaccination were timed to capture the peak of adaptive immune system activity, allowing us to trace numerous BCR lineages through time. We functionally characterized 31 SARS-CoV-2 neutralizing antibodies, with four neutralizing Omicron BA.1, despite the study occurring prior to the emergence of Omicron, demonstrating that affinity maturation can broaden the response. Combined IGH repertoire and monoclonal antibody lineage tracing revealed that vaccination expanded a broad range of affinity-matured infection- induced antibody lineages.

Paper III describes a novel method for identifying and filtering PCR chimeras in adaptive immune receptor repertoire sequencing (AIRR-seq) data. We analyzed 319 published libraries and demonstrated the universal presence of chimeras in AIRR-seq data with different frequencies depending on the type of AIRR dataset. Further, we created new AIRR-seq libraries to determine which PCR conditions produced more chimerism. Evaluation on simulated TCR and IGH data revealed that our tool performed better than existing non-domain-specific software making it a useful tool for the AIRR community.

List of scientific papers

I. Martin Corcoran, Mark Chernyshev*, Marco Mandolesi*, Sanjana Narang, Mateusz Kaduk, Kewei Ye, Christopher Sundling, Anna Färnert, Taras Kreslavsky, Carolina Bernhardsson, Maximilian Larena, Mattias Jakobsson, Gunilla B Karlsson Hedestam

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

Immunity, 2023, 56, 635-652 *Equal Contribution.

https://doi.org/10.1016/j.immuni.2023.01.026

II. Mark Chernyshev*, Mrunal Sakharkar*, Ruth I. Connor, Haley L. Dugan, Daniel J. Sheward, C. G. Rappazzo, Aron Stålmarck, Mattias N. E. Forsell, Peter F. Wright, Martin Corcoran, Ben Murrell, Laura M. Walker, Gunilla B. Karlsson Hedestam

"Vaccination of SARS-COV-2-infected individuals expands a broad range of clonally diverse affinity-matured B cell lineages"

Nature Communications, 2023, 14, article 2249 *Equal Contribution.

https://doi.org/10.1038/s41467-023-37972-1

III. Mark Chernyshev*, Aron Stålmarck*, Martin Corcoran, Gunilla B. Karlsson Hedestam, Ben Murrell

"Detection of PCR chimeras in adaptive immune receptor repertoire sequencing using hidden Markov models" * Equal Contribution. [Submitted]