Paired analysis of TCRα and TCRβ chains at the single-cell level in mice

Abstract

Characterizing the TCRα and TCRβ chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRβ chain utilization is very limited for pathogen-and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ chains with single-cell resolution. Using CD8 + CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease. IntroductionRecent advances have allowed us to analyze the development and persistence of virus-specific CD8 + T cell-mediated immunity from naive CTL precursors (CTLps) in peripheral lymphoid tissue, through the antigen-driven phase in lymph nodes and spleen, to the CTL effectors in a site of virus-induced pathology, and then, ultimately, to the persistence and recall of immune memory (1-3). However, unless we use lymphocytes from TCR-transgenic mice, our capacity to follow the fate and persistence of defined clonotypes is very limited. Several approaches have been used to estimate the extent of TCR diversity and to track clonally expanded T cell populations throughout the course of antigen-specific CTL responses (4), but none has given the complete picture. A commonly used protocol is to double-stain CD8 + T cells with mAbs specific for TCR variable (V) region β (TRBV) and tetramers specific for peptide + class I MHC glycoprotein (pMHCI) epitopes (5-9). Such low-resolution analysis provides no insight into the extent of clonal diversity within a particular TRBV-specific population and offers little scope for determining the spectrum of TCRα usage, as there are few mAb reagents. Another approach, known as immunoscope or spectratyping, uses gel electrophoresis of total mRNA from TRBV-specific populations (RT-PCR product) to determine profiles of complementarity-determining region 3β (CDR3β) length (4, 10). Combining spectratyping with cloning and sequencing allows for more definitive identification of CDR3β transcripts, but the approach is compromised by the possibility of bias durin...

Functional TCR Retrieval from Single Antigen-Specific Human T Cells Reveals Multiple Novel Epitopes

“…In contrast with established primer sets for the amplification of TCR CDR3 regions from single T cells (10,13,14,45), our primers amplify full-length variable regions. In the elegant study by Ozawa and colleagues (9), they were able to generate matching TCR a/b cDNAs from approximately 20% of processed single cells using the 5 0 -RACE technique.…”

“…Several groups observed dual TCR expression (9)(10)(11)(12), raising the need for functional analysis. Not only are we able to study dual TCR expression, using this method we also can delineate the different (productively rearranged) TCR-a chains of a single T cell.…”

“…One approach is the molecular analysis of TCR diversity by analyzing the TCR-b chain CDR3 repertoire using CDR3 spectratyping techniques (7). During the last years, several PCR-based methods have been developed, allowing TCR repertoire analyses at the single-cell level (8)(9)(10)(11)(12) as well as the in vitro reconstitution of full-length a/b TCRs (13-16) from single cells. Likewise, next-generation sequencing methods have evolved that generate millions of short sequence reads, which enable high-throughput profiling of TCR repertoires but have not addressed single T cells, thus precluding analysis of paired TCR-a/b chains (17)(18)(19).…”

Heightened self-reactivity associated with selective survival, but not expansion, of naïve virus-specific CD8 + T cells in aged mice

“…The frequency and TCR usage of naïve and immune CD8 + T cells specific for a range of influenza A virus (IAV) epitopes is well characterized for B6 mice (17)(18)(19)(20), providing a convenient experimental system for investigating the characteristics and drivers of age-related CD8 + T-cell decline. Primary responses to the D b NP 366-374 and D b PA 224-233 epitopes (derived from nucleoprotein and acid polymerase proteins, respectively) are immunodominant in young adult mice, whereas those to a polymerase B subunit 1 epitope (K b PB1 [703][704][705][706][707][708][709][710][711] ) and an epitope derived from a shifted reading frame of PB1 (D b PB1-F2 62-70 ) are subdominant (21).…”

Receptor revision in CD4 T cells is influenced by follicular helper T cell formation and germinal-center interactions

“…Rag-mediated rearrangement can induce offtarget mutations, creating a potential risk of oncogenesis, a danger that is mitigated by precise regulation of Rag expression during T-cell development (1). It is currently unclear what regulatory processes are in place to dampen the risks incurred by postthymic TCR rearrangement, or TCR revision, a process known to occur in both mice (3)(4)(5)(6)(7)(8)(9)(10)(11) and humans (12)(13)(14)(15).…”