Characterizing immune repertoires by high throughput sequencing: strategies and applications

Abstract

As the key cellular effectors of adaptive immunity, T and B lymphocytes utilize specialized receptors to recognize, respond to, and neutralize a diverse array of extrinsic threats. These receptors (immunoglobulins in B lymphocytes, T cell receptors in T lymphocytes) are incredibly variable, the products of specialized genetic diversification mechanisms that generate complex lymphocyte repertoires with extensive collections of antigen specificities. Recent advances in high throughput sequencing (HTS) technologies have transformed our ability to examine antigen receptor repertoires at single nucleotide, and more recently, single cell, resolution. Here we review current approaches to examining antigen receptor repertoires by HTS, and discuss inherent biological and technical challenges. We further describe emerging applications of this powerful methodology for exploring the adaptive immune system.

Keywords: T cell receptor diversity; antibody diversity; high throughput sequencing; immune profiling; immune repertoires; immunoglobulin diversity.

Figure 1

Diversification of antigen receptor repertoires. (A) BCRs and TCRs are similarly organized. Each receptor is composed of two distinct subunit chains (BCR: light chain and heavy chain, TCR: α chain and β chain). The antigen binding surface is formed by the variable region of each chain, which is encoded by recombined V, J, and D (BCR heavy and TCRβ) gene segments. (B) Antigen receptor diversification. A schematic of the BCR heavy locus is shown; with the exception of somatic hypermutation and class-switch recombination, analogous mechanisms proceed at the TCRβ locus (with differences in segment organization). Antigen receptor repertoire diversity is primarily established during lymphocyte development, during which V (orange), D (green), and J (yellow) gene segments are rearranged through the process of V(D)J recombination. Numbers of distinct V, D, and J segments are shown for each antigen receptor locus [2]. During the recombination process, nucleotides may be added or deleted at segment junctions (magenta), contributing to additional sequence diversity. Complementarity determining regions are indicated. BCR-specific secondary diversification may occur following antigen recognition. In somatic hypermutation processes, mutations (red) are introduced throughout the variable region such that modified BCRs may be selected through affinity maturation. In class-switch recombination, gene segments encoding constant regions (blue) are rearranged resulting in the production of antibodies with different isotypes and corresponding effector functions. Abbreviations: BCR, B cell receptor; TCR, T cell receptor; V, J, and D, Variable, Joining, and Diversity gene segments.

Figure 2

Library preparation strategies for antigen receptor HTS. The extraordinary diversity of antigen receptor sequences poses challenges for targeted amplification and library preparation; although not comprehensive, a general overview of select PCR priming strategies is presented here. A generic antigen receptor schematic representative of BCR heavy or TCRβ is shown. (A) PCR amplification and library preparation from genomic DNA. Multiplex PCR strategies, in which complex mixtures of primers complementary to many or all possible V segment sequences, can be used to amplify portions of variable region sequences from genomic DNA. Multiplex primers targeting portions of V segments (red) can be used in conjunction with J segment primers (navy blue) for amplification. Upon incorporation of HTS adaptors (light gray boxes), long read HTS can be used to capture a majority of variable region sequence. Alternatively, short read HTS can be used to sequence only the CDR3 region. (B) Reverse transcription PCR amplification and library preparation from mRNA. In antigen receptor transcript mRNA, the juxtaposition of constant region exons adjacent to the variable region offers a reverse priming site with minimal diversity. In invariant adaptor strategies, the complexities and potential biases of V segment multiplex PCR are bypassed by incorporating a defined adaptor sequence (dark gray box) upstream of the variable region by oligonucleotide ligation or template switch methods during reverse transcription. A single primer to the adaptor sequence (dark gray) can then be used with constant region primers (violet) to generate amplicons that contain complete variable region open reading frames (ORFs). These can be sequenced in entirety by long read paired-end HTS or sequenced with short read HTS for CDR3-targeted studies. Similar adaptor-mediated PCR strategies using multiplex J primers for CDR3 sequencing are also available (not shown). Alternatively, following reverse transcription without invariant adaptors, multiplex primers to V regions and J regions (as for genomic DNA amplification) or constant regions (not shown), can be used for CDR3-targeted short read HTS. Hashed lines, HTS reads. V, D, J, and constant segment colors as in Figure 1. Abbreviations: BCR, B cell receptor; TCR, T cell receptor; V, J, and D, Variable, Joining, and Diversity gene segments; HTS, high throughput sequencing; CDR3, complementarity determining region 3.