.. _receptor-model: 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 (:term:`IR`) and recognize the same :term:`epitopes`. In single-cell RNA-sequencing (scRNA-seq) data, T or B cells sharing identical complementarity-determining regions 3 (:term:`CDR3`) nucleotide sequences of both :term:`VJ` and :term:`VDJ` chains (e.g. both α and β :term:`TCR` chains) make up a clonotype. Scirpy provides an option to additionally require clonotypes to have the same :term:`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 (:cite:`Brady2010-gh`), scRNA-seq datasets can feature a considerable number of cells with more than one pair of :term:`VJ` and :term:`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 (:cite:`Fischer2019`, :cite:`Zhang2018-ip`), or select the CDR3 sequence with the highest expression level (:cite:`Afik2017-sg`). 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 :term:`Dual IR` population (:cite:`Schuldt2019`, :cite:`Ji2010-bn`, :cite:`Vettermann2010`). 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 :cite:`Stubbington2016-kh`, where immune cells are allowed to have a primary and a secondary pair of :term:`VJ` and :term:`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 :term:`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 :term:`clonotypes` based on *identical* :term:`CDR3` *nucleotide sequences* and into :term:`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 (:cite:`Fischer2019`, :cite:`Glanville2017-ay`, :cite:`TCRdist`). 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.