The αβ T cell receptor (TR) is a membrane-anchored heterodimer expressed on the surface of T cells that mediates specific recognition of antigen in the form of major histocompatibility complex (MHC) molecules presenting endogenously-derived peptides bound in the α1α2 groove. Surface expression of such peptide-MHC (pMHC) antigens provides a display library that enables T cells to detect abnormal intracellular processes through TR-mediated surveillance, which in turn is the pivotal event that dictates the initiation of a T cell response. Each α and β chain comprises three hypervariable complementarity-determining regions (CDRs), which determine the antigen specificity of the heterodimeric TR. Three mechanisms govern the generation of diversity within the TR repertoire that is necessary to recognize the myriad potential antigenic peptides that a host may encounter (Nikolich-Zugich et al., 2004
). First, each TR chain is formed by the genetic rearrangement of variable (V), diversity (D; β chain only) and joining (J) germline gene segments, which vary in number between species, with a TRC gene. Second, nucleotide insertions and deletions at the V(D)J junctions (N-diversity) provide an additional level of variability within the CDR3 of each chain; the CDR1 and CDR2 regions are germline-encoded by the V genes. Third, pairing of individual α and β chains further amplifies the potential number of TRs that can be generated. After thymic selection, which operates to ensure a degree of MHC bias and delete autoreactive TRs, it has been estimated that approximately 2.5 × 107
unique human (Arstila et al., 1999
) and 2×106
murine (Casrouge et al., 2000
) TRs populate the periphery and are available to respond to antigenic challenges. However, these numbers are dwarfed by the potential number of antigenic pMHC combinations. By necessity, then, an intrinsic degree of cross-reactivity is incorporated within the TR recognition system to enable sufficient coverage and also to increase the likelihood that a given antigen will be recognized by a cognate TR within a time frame that facilitates an effective response (Mason, 1998
). Thus, an individual clonotype, i.e. a T cell defined by the singular expressed TR, can recognize multiple ligands; similarly, individual pMHC ligands can productively engage multiple TRs. As a consequence, any antigen-specific T cell response can comprise multiple clonotypes, which in turn dictate the functional qualities of the T cell population. The role of this protocol is to enable the accurate and quantitative analysis of constituent clonotypes within T cell populations specific for defined antigens. A detailed understanding of clonal selection within the memory and effector T cell pools is essential to further our understanding of the factors that influence effective T cell immunity and has direct implications for the rational design of vaccines and immunotherapies (Appay et al., 2008
; Price et al., 2009
Critical Parameters and Troubleshooting
Meticulous attention to detail is essential for the successful and reliable application of this protocol. Reagents should be DNase/RNase-free and PCR-grade plastics should be used throughout. Dedicated hoods in a "clean room" free from high copy number plasmids should be used for mRNA extraction, cDNA synthesis and PCR set-up. Benches and pipettes should be cleaned with 10% bleach solution and an RNase-inactivator (e.g. RNase AWAY; Sigma-Aldrich) both before and after each procedure to minimize contamination. Disposable sleeves and gloves should be used throughout. Refer to UNIT 10.20 for detailed recommendations on optimal work practices in a molecular biology laboratory.
Template purity and quality
As discussed in section (1) above, this protocol is entirely dependent on the isolation of good quality RNA from viable, healthy and pure T cell populations. The sequence output, due to its sensitivity and linearity, will reflect the nature of the starting template. Thus, erroneously captured T cells will lead to the overestimatation of clonality and diversity. Similarly, exclusion of specific T cells, for example due to antigen-induced cell death, can lead to the loss of true clonotypes from the starting template and subsequent underestimation of clonality and diversity.
Related to template quality, adequate sampling of the repertoire under investigation is necessary to ensure reproducibility. Thus, as many cells as possible should be sorted from the initial pool of T cells and the number of sequences generated should reflect the heterogeneity of constituent TRs within the pool. In practice, based on empirical replicate analyses, adequate sampling is achieved for largely oligoclonal pMHC tetramer-sorted T cell populations with >500 cells and >50 sequences. These minimal guidelines should be adjusted, however, according to the nature of the population under investigation. Thus, lower frequency clonotypes will be detected as more sequences are generated; however, the intrinsic sequence error rate will limit the reliable detection of very low frequency clonotypes. Sequence comparisons should account for such issues (Venturi et al., 2008a
; Venturi et al., 2007
Unsuccessful PCR amplification
If the 5'-RACE PCR does not generate bands of the expected size, repeat the procedure using more cDNA (e.g. 13 µl). Further cDNA can be made from the stored RNA for additional attempts. However, unsuccessful amplification generally reflects a poor starting template, in which case RNA concentration can be useful (see below). Do not use additional PCR cycles or nested approaches to amplification, as these procedures increase the error rate.
- Microcon Centrifugation Filters (Millipore)
- DNase/RNase-free water, molecular biology grade (Sigma)
- Bring the remaining mRNA up to 20 µl with water.
- Place the Microcon filter into a collection tube and add the sample to the red side of the column. This contains a glycerol-coated filter that allows solutes and fluid through, but retains the RNA.
- Close the lid and spin at 14,000 rpm for 3 min.
- Transfer the Microcon filter to another clean collection tube.
- Add 6 µl of water to the red side.
- Flip over the filter into the collection tube so that the red side is now facing the bottom of the tube.
- Spin the filter and collection tube for 3 min at 3,300 rpm. The concentrated mRNA is now in the collection tube.
- Use all 6 µl of mRNA for cDNA synthesis, doubling reagent volumes accordingly.
Poor transformation efficiency
If the 5'-RACE PCR gave a distinct band of the expected size but few bacterial colonies contained an insert, product may have been lost during gel extraction. Most notably, ensure that buffer NT3 contains the appropriate quantity of ethanol; this can be reduced due to differential evaporation after reconstitution, especially if the bottle is opened frequently. Ligation efficiency can also be compromised due to loss of adenine overhangs if the PCR product is not processed promptly; thus, always proceed to ligation as quickly as possible once the 5'-RACE PCR is completed. Finally, the quality of the competent bacteria should be verified.
This protocol allows the rapid and comprehensive analysis of all expressed TRs within any given T cell population and offers significant advantages over previous approaches (Rufer, 2005
). Multiple facets of TR usage can be assessed depending on the particular issues under investigation. Indeed, fundamental aspects of T cell immunobiology have already been illuminated with this approach (Davenport et al., 2007
; Venturi et al., 2008b
). It should be noted that allelic exclusion does not operate at the α locus; thus, clonotypic assessment is defined by molecular analysis of TRB gene products.
With experience and good organization, samples can be processed from mRNA extraction to overnight ligation in one day. An approximate time-guide is: mRNA extraction, 90 min; cDNA synthesis and clean-up, 3 hr; RACE-PCR amplification of rearranged TR products, 2 hr 45 min; gel extraction and ligation, 2 hr plus overnight incubation; transformation, 3 hr; colony PCR, 4 hr; preparation of samples for sequencing, 1.5 hr. Up to 4 samples can comfortably be handled in parallel. However, caution should be exercised as scaling up increases the risk of cross-contamination.