Immune receptor (IR) model

What is a clonotype?

A clonotype designates a collection of T or B cells that descend from a common, antecedent cell, and therefore, bear the same adaptive immune receptors (IR) and recognize the same epitopes.

In single-cell RNA-sequencing (scRNA-seq) data, T or B cells sharing identical complementarity-determining regions 3 (CDR3) nucleotide sequences of both VJ and VDJ chains (e.g. both α and β TCR chains) make up a clonotype. Scirpy provides an option to additionally require clonotypes to have the same V-gene, enforcing the CDR 1 and 2 regions to be the same.

Dual TCRs and allelically included B cells

Contrary to what would be expected based on the previously described mechanism of allelic exclusion ([BSB10]), scRNA-seq datasets can feature a considerable number of cells with more than one pair of VJ and VDJ chains. Since cells with more than one productive CDR3 sequence for each chain did not fit into our understanding of adaptive immune cell biology, most TCR analysis tools ignore these cells ([FWST19], [ZYZ+18]), or select the CDR3 sequence with the highest expression level ([AYB+17]). While in some cases these cells might represent artifacts (e.g. doublets of a CD8+ and a CD4+ T cell engaged in an immunological synapse), there is an increasing amount of evidence in support of a bone fide Dual IR population ([SB19], [JPG10], [VS10]).

Scirpy allows investigating the composition and phenotypes of both single- and dual-IR cells by leveraging a immune cell model similar to the one proposed in [SLonnbergP+16], where immune cells are allowed to have a primary and a secondary pair of VJ and VDJ chains. For each cell, the primary pair consists of the VJ- and VDJ-chain with the highest read count. Likewise, the secondary pair is the pair of VJ/VDJ-chains with the second highest expression level. Based on the assumption that each cell has only two copies of the underlying chromosome set, if more than two variants of a chain are recovered for the same cell, the excess IR chains are ignored by Scirpy and the corresponding cells flagged as Multichain-cell. This filtering strategy leaves the choice of discarding or including multichain cells in downstream analyses.

Clonotype definition

Scirpy implements a network-based clonotype definition that enables clustering cells into clonotypes based on identical CDR3 nucleotide sequences and into clonotype clusters based on

  • identical CDR3 amino acid sequences, or

  • similar CDR3 amino acid sequences based on pairwise sequence alignment.

The latter approach is inspired by studies showing that similar TCR sequences also share epitope targets ([FWST19], [GHN+17], [DFGH+17]). Based on these approaches, Scirpy constructs a global, epitope-focused cell similarity network. While convergence of the nucleotide-based clonotype definition to the amino acid based one hints at selection pressure, sequence alignment based networks offer the opportunity to identify cells that might recognize the same epitopes.