The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.
Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.
The T cell receptor (TCR) dictates T cell fate decision during development, steady state/homeostasis, and antigenic stimulation in periphery1,2,3. Recent expansion of deep sequencing technologies has uncovered a previously underappreciated TCR diversity within antigen specific T cell responses. TCR diversity suggests a potential for functionally broad T cell responses. In order to integrate the TCR sequence repertoire analysis with TCR functional assays, the sequencing approaches should be designed to be compatible with experimental systems and in vivo models utilized for subsequent functional analysis of select TCRs. We have developed an efficient approach for human TCR sequence isolation and streamlined sub-cloning into a chimeric human/mouse TCR template vector compatible with TCR expression in HLA-humanized mice4. Isolation of corresponding alpha and beta chains of heterodimeric TCRs requires PCR amplification of both chains from a single cell. Although, several single-cell TCR cloning protocols have been developed and utilized, none so far have been easily compatible with high-throughput streamlined direct cloning of unknown Vα/Vβ TCRs into retroviral vectors necessary for re-expression in vivo4,5,6,7. Previous studies have utilized two major approaches, either to selectively amplify a limited portion of the TCR sufficient to extrapolate the sequence, or to amplify the entire TCR sequence4,5,6,7. Downstream functional analysis of TCR sequences obtained via the first approach requires costly in silico assembly and de novo construction of the TCR. While the second approach provides the complete TCR sequence, the human constant region is not compatible with in vivo expression of the cloned TCRs in mouse models. Our approach is specifically designed to be compatible with in vivo functional analysis of TCRs in mouse models. We developed an efficient and streamlined single cell PCR protocol that allows for direct sub-cloning of PCR fragments into the template expression vector.
Our approach utilizes a highly sensitive multiplex-nested PCR reaction that is performed in two steps. In the first multiplex reaction step a pool of 40 primers specific for all the V-beta chains and a pool of 44 primers specific for all the V-alpha chains are used to amplify the TCR-alpha or TCR-beta without the prior knowledge of the sequence (Table 1). The forward primer has an adaptor sequence, which is incorporated into the 5' of the PCR product. The reverse primer is based on the constant region of the TCR. We have identified unique restriction sites that are absent from human variable or junction TCR regions, and incorporated these into newly redesigned TCRα and TCRβ primers (Figure 1). In the second nested reaction, a primer specific for the adaptor sequence and a nested reverse primer within the constant region are used to further amplify the TCR chains with increased specificity (Table 1, Figure 1). After single cell isolation, two rounds of PCR (first reaction with a pool of both Vα and Vβ primers, and second with adaptor primers) result in a PCR product that can be directly sub-cloned into the template retroviral vector. The final TCR construct will encode, in a single open reading frame (ORF), human variable regions combined with mouse constant regions connected by the 'self-cleaving' protein sequence, P2A (hVα-mCα-P2A-hVβ-mCβ)8. The P2A sequence has been used in multiple systems, and has been extensively tested specifically for the expression of TCRs8,9,10,11. Although, after translation most of the P2A sequence remains attached to the C-terminus of the alpha chain connected by a flexible linker, while the beta chain signal sequence has an additional proline, this modification has no detrimental effect on TCR function. The mouse constant region is used in the construct instead of human to avoid potential altered interactions with downstream signaling components when re-expressed in mouse cells. The single ORF will result in stoichiometric separate expression of alpha and beta chains9,11. The current protocol is based on reagents and approaches that are widely available, and is designed to be performed in a streamlined and efficient fashion. Although we have specifically used this technique to assay TCRs from self-reactive T cells implicated in autoimmune diabetes, we anticipate that this protocol can be widely applicable for identification and functional assessment of human TCRs specific for autoimmune epitopes, cancer epitopes, or responses to pathogens and vaccines.
1. Identify the T cell population of interest.
NOTE: Antigen specific proliferation in combination with cell division dye (like Carboxyfluorescein succinimidyl ester, CFSE) can be used to isolate T cells based on their proliferation in response to antigenic stimulation. If starting from PBMCs, a 7-day in vitro expansion should be sufficient to identify an antigen specific CFSE low population12.
2. Set up the single cell sort.
3. Perform multiplex-nested PCR
NOTE: Prepare all master mixes in a clean template-free area to avoid contamination.
4. Sub-cloning alpha and beta PCR chains.
NOTE: The pool of the forward primers utilized in the first reaction, and the reverse primers in the second PCR reaction are used to incorporate restriction sites. Therefore, the obtained alpha and beta chain PCR products are ready to be sequentially sub-cloned into the template retroviral vector (Figure 1).
5. Verify TCR cell surface expression and specificity.
Note: HEK293T cells are used to test successful TCR chain paring and cell surface expression (Figure 3A)8. Peptide-MHC tetramer staining can also be performed on the transfected HEK293T cells to test for retention of antigenic specificity post TCR gene isolation (Figure 3B).
The efficiency of the multiplex-nested PCR reaction is checked in step 3.2.7 (Figure 2) by running out 5µL of the second reaction on an agarose gel. On average the efficiency of TCR-beta amplification is expected to be around 80%, while the efficiency of the TCR-alpha reaction is usually lower, at around 50%4. Only paired TCR-alpha and TCR-beta chains can be used for TCR expression; however, all PCR products could be sequenced to obtain TCR sequence and repertoire information.
Gene silencing will only allow a single TCR-beta chain to be expressed in a given T cell. However, a T cell is capable of rearranging a second TCR-alpha chain, and about 25% of T cells will expressing a second in-frame TCR-alpha chain 14. It is expected that some proportion of the isolated TCR-alpha chains will be the secondary alpha instead of the "correct" alpha chain. The secondary alpha chain is often not an optimal binding partner with the TCR-beta chain; consequently, it is expected that not all cloned TCRs will form a stable complex and express on cell surface. Therefore, the proper TCR chain pairing and cell surface expression has to be confirmed by transfecting HEK293T cells (Figure 3A).
Figure 1. Streamlined multiplex-nested PCR and retroviral construct development from single T cells. Two rounds of PCR result in amplification of corresponding TCR alpha and beta chains. Restriction sites embedded into the primers allow streamlined sub-cloning and generation of human/mouse chimeric vector for expression in mouse cells. The template mouse vector can be used to easily switch out variable mouse regions for human, generating a chimeric human/mouse TCR retroviral vector compatible with expression in mouse cells. Please click here to view a larger version of this figure.
Figure 2. Example of single cell PCR. Multiplex nested PCR amplification of T cell receptor beta and alpha chains from single human T cells sorted into one 96-well plate. Shown are corresponding (top and bottom) PCR products from single cell amplified TCR beta and TCR alpha chains from human PBMCs. NC – no template control, PC – positive control. By running a small portion of the reaction on an agarose gel (5 µL), the efficiency of the single cell alpha and beta chain PCR reactions is confirmed prior to PCR product sequencing. Please click here to view a larger version of this figure.
Figure 3. Verification of cell surface chimeric TCR expression and specificity. (A) HEK293T cell were transfected with mouse TCR or a human/mouse chimeric TCR in combination with mouse CD3εγδζ. Cells surface TCR expression was measured with anti-mCD3ε antibody. Gating strategy: First, the analysis is gated on Ametrine and GFP double positive cells (G2). In the case of single vector controls the gating has to be based on the single fluorescence (G1). Second, the cells from G1 and G2 are analyzed for the level of CD3ε expression. (B) TCR specificity is confirmed by tetramer staining of 293T cells with DRB1*0401:GAD115-127 tetramer. Analysis is gated on GFP+Ametrine+CD3ε + cells, as shown in (A). Please click here to view a larger version of this figure.
TARGET GENE | PRIMER ORIENTATION | SEQUENCE | |||
Reverse Transcription | |||||
TRAC cDNA | AGCTGGACCACAGCCG | ||||
TRBC1 cDNA | GAAATCCTTTCTCTTGACCATG | ||||
TRBC2 cDNA | GCCTCTGGAATCCTTTCTCT | ||||
TCR alpha PCR reaction 1 | |||||
TRAV1-1 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGTGGGGAGCTTTCCTTCTCTATGTTT | |||
TRAV1-2 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGTGGGGAGTTTTCCTTCTTTATGTTTC | |||
TRAV2 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGCTTTGCAGAGCACTCTGG | |||
TRAV3 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGCCTCTGCACCCATCTCG | |||
TRAV4 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAGGCAAGTGGCGAGAGTGATC | |||
TRAV5 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAAGACATTTGCTGGATTTTCGTTC | |||
TRAV6 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGAGTCATTCCTGGGAGGTGTTT | |||
TRAV7 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGAGAAGATGCGGAGACCTGTC | |||
TRAV8-1 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGCTCCTGTTGCTCATACCAGTGC | |||
TRAV8-2 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGCTCCTGCTGCTCGTCCC | |||
TRAV8-3 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGCTCCTGGAGCTTATCCCACTG | |||
TRAV8-7 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGCTCTTAGTGGTCATTCTGCTGCTT | |||
TRAV9-1 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAATTCTTCTCCAGGACCAGCG | |||
TRAV9-2 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAACTATTCTCCAGGCTTAGTATCTCTGATACTC | |||
TRAV10 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAAAAAGCATCTGACGACCTTCTTG | |||
TRAV12-1 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGATATCCTTGAGAGTTTTACTGGTGATCC | |||
TRAV12-2 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGATGAAATCCTTGAGAGTTTTACTAGTGATCC | |||
TRAV12-3 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGATGAAATCCTTGAGAGTTTTACTGGTG | |||
TRAV13-1 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGACATCCATTCGAGCTGTATTTATATTCC | |||
TRAV13-2 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGCAGGCATTCGAGCTTTATTT | |||
TRAV14 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGTCACTTTCTAGCCTGCTGAAGGTG | |||
TRAV16 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAAGCCCACCCTCATCTCAGTG | |||
TRAV17 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGAAACTCTCCTGGGAGTGTCTTTG | |||
TRAV18 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGCTGTCTGCTTCCTGCTCAGG | |||
TRAV19 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGCTGACTGCCAGCCTGTTGAG | |||
TRAV20 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGAGAAAATGTTGGAGTGTGCATTC | |||
TRAV21 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGAGACCCTCTTGGGCCTG | |||
TRAV22 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAAGAGGATATTGGGAGCTCTGCT | |||
TRAV23 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGACAAGATCTTAGGAGCATCATTTTTAG | |||
TRAV24 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGAGAAGAATCCTTTGGCAGCC | |||
TRAV25 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGCTACTCATCACATCAATGTTGGTCTTAT | |||
TRAV26-1 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAGGCTGGTGGCAAGAGTAACTG | |||
TRAV26-2 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAAGTTGGTGACAAGCATTACTGTACTCC | |||
TRAV27 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGTCCTGAAATTCTCCGTGTCC | |||
TRAV29 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGCCATGCTCCTGGGGG | |||
TRAV30 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGAGACTCTCCTGAAAGTGCTTTCAG | |||
TRAV34 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGAGACTGTTCTGCAAGTACTCCTAGG | |||
TRAV35 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGCTCCTTGAACATTTATTAATAATCTTGTGG | |||
TRAV36 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGATGAAGTGTCCACAGGCTTTACTAGC | |||
TRAV38-1 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGACACGAGTTAGCTTGCTGTGGG | |||
TRAV38-2 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGCATGCCCTGGCTTCCT | |||
TRAV39 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAAGAAGCTACTAGCAATGATTCTGTGG | |||
TRAV40 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGAACTCCTCTCTGGACTTTCTAATTCTGA | |||
TRAV41 | Forward | CGGTTCAGCAGGAATGCCtacgtaATGGTGAAGATCCGGCAATTTTTG | |||
TRAC External | Reverse | CAGACAGACTTGTCACTGGATTTAGAGTCTC | |||
TCRbeta PCR reaction 1 | |||||
TRBV2 | Forward | CAGAAGACGGCATACGAGATcaattgATGGATACCTGGCTCGTATGCTGG | |||
TRBV3-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCTGCAGGCTCCTCTG | |||
TRBV4-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCTGCAGGCTGCTCTG | |||
TRBV5-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCTCCAGGCTGCTCTGTT | |||
TRBV5-3 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCCCCGGGCTCC | |||
TRBV5-4 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCCCTGGGCTCCTCT | |||
TRBV5-8 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGACCCAGGCTCCTCTTCT | |||
TRBV6-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGAGCATCGGGCTCCTGTGC | |||
TRBV6-2 | Forward | CAGAAGACGGCATACGAGATcaattgATGAGCCTCGGGCTCCTGTG | |||
TRBV6-4 | Forward | CAGAAGACGGCATACGAGATcaattgATGAGAATCAGGCTCCTGTGCTGTG | |||
TRBV6-6 | Forward | CAGAAGACGGCATACGAGATcaattgATGAGCATCAGCCTCCTGTGCTG | |||
TRBV7-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCACAAGGCTCCTCTGC | |||
TRBV7-2 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCACCAGGCTCCTCTTCT | |||
TRBV7-3 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCACCAGGCTCCTCTG | |||
TRBV7-6 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCACCAGTCTCCTATGCTG | |||
TRBV7-7 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGTACCAGTCTCCTATGCTGGG | |||
TRBV7-9 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCACCAGCCTCCTCTG | |||
TRBV9 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCTTCAGGCTCCTCTGCT | |||
TRBV10-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCACGAGGCTCTTCTTCTATG | |||
TRBV10-2 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCACCAGGCTCTTCTTCTATG | |||
TRBV10-3 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCACAAGGTTGTTCTTCTATGTG | |||
TRBV11-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGAGCACCAGGCTTCTCTGCTG | |||
TRBV11-3 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGTACCAGGCTCCTCTGCTG | |||
TRBV12-3 | Forward | CAGAAGACGGCATACGAGATcaattgATGGACTCCTGGACCTTCTGCTGT | |||
TRBV12-4 | Forward | CAGAAGACGGCATACGAGATcaattgATGGACTCCTGGACCCTCTGCTG | |||
TRBV12-5 | Forward | CAGAAGACGGCATACGAGATcaattgATGGCCACCAGGCTCCTCTG | |||
TRBV13 | Forward | CAGAAGACGGCATACGAGATcaattgATGCTTAGTCCTGACCTGCCTGACTC | |||
TRBV14 | Forward | CAGAAGACGGCATACGAGATcaattgATGGTTTCCAGGCTTCTCAGTTTAGTGT | |||
TRBV15 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGTCCTGGGCTTCTCCACT | |||
TRBV16 | Forward | CAGAAGACGGCATACGAGATcaattgATGAGCCCAATATTCACCTGCATCA | |||
TRBV17 | Forward | CAGAAGACGGCATACGAGATcaattgATGGATATCTGGCTCCTCTGCTGG | |||
TRBV18 | Forward | CAGAAGACGGCATACGAGATcaattgATGGACACCAGAGTACTCTGCTGTGC | |||
TRBV19 | Forward | CAGAAGACGGCATACGAGATcaattgATGAGCAACCAGGTGCTCTGCTG | |||
TRBV20 | Forward | CAGAAGACGGCATACGAGATcaattgATGCTGCTGCTTCTGCTGCTTCT | |||
TRBV24-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGGCCTCCCTGCTCTTCTTCTG | |||
TRBV25-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGACTATCAGGCTCCTCTGCTACATGG | |||
TRBV27 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGCCCCCAGCTCCTTG | |||
TRBV28 | Forward | CAGAAGACGGCATACGAGATcaattgATGGGAATCAGGCTCCTCTGTCG | |||
TRBV29-1 | Forward | CAGAAGACGGCATACGAGATcaattgATGCTGAGTCTTCTGCTCCTTCTCCT | |||
TRBV30 | Forward | CAGAAGACGGCATACGAGATcaattgATGCTCTGCTCTCTCCTTGCCCT | |||
TRBC External | Reverse | GTGGCCAGGCACACCAGTGTG | |||
TCR alpha PCR reaction 2 | |||||
TRAC Internal | Reverse | CAGCTGGTACAccgcggGGTCAGGGTTCTG | |||
TRAV Adaptor | Forward | CGGTTCAGCAGGAATGCCtacgtaATG | |||
TCR beta PCR reaction 2 | |||||
TRBC Internal | Reverse | CTCTGCTTCTGATGGttcgaaCACAGCGACCTCGG | |||
TRBV Adaptor | Forward | CAGAAGACGGCATACGAGATcaattgATG | |||
Sequencing primers | |||||
pMSCV-II | Forward | CCTCCTCTTCCTCCATCCGCC | |||
TCRbeta | Reverse | GCCAAGCACACGAGGGTAGCC | |||
IRES | Reverse | AACGCACACCGGCCTTATTCC | |||
*Lower case letters within the primer sequences indicate the incorporated restriction enzyme cut sites |
Table 1: Reverse transcription, PCR, and sequencing primers.
Step 2.2 | amounts per well | ||
RT-PCR reaction (6μL) | (μL) | ||
10x buffer | 0.6 | ||
2x dNTP | 0.24 | ||
10% Triton X | 0.06 | ||
3 TCR-specific primers (10mM ea.) | 0.23 ea. (x 3 = 0.69) | ||
Enzyme | 0.18 | ||
Rnase inhibitor | 0.28 | ||
NF(nuclease-free)-H2O | 3.95 | ||
Step 3.1 | |||
a PCR reaction 1 (25μL) | (μL) | b PCR reaction 1 (25μL) | (μL) |
5x buffer | 5 | 5x buffer | 5 |
dNTP (10mM) | 1 | dNTP (10mM) | 1 |
DMSO | 0.75 | DMSO (3%) | 0.75 |
a pool (F primer) (2.3μM) | 0.3 | b pool (F primer) (2.3μM) | 0.5 |
a external R primer (10uM) | 0.6 | b external R primer (10μM) | 1 |
DNA polymerase | 0.2 | Go Taq polymerase | 0.2 |
RT-PCR cDNA | 2.5 | RT-PCR cDNA | 2.5 |
NF-H2O | 14.65 | NF-H2O | 14.05 |
Step 3.8 | |||
PCR reaction 2 – same for a and b (25μL) | (μL) | ||
5x buffer | 5 | ||
dNTP (10mM) | 1 | ||
DMSO | 0.75 | ||
Adaptor F primer (10μM) | 1 | ||
Internal R primer (10μM) | 1 | ||
DNA polymerase | 0.2 | ||
PCR rxn 1 product | 2.5 | ||
NF-H2O | 13.55 |
Table 2. Reverse transcription and PCR reactions.
Steps 4.2 and 4.3 | |||
TRBV digest reaction (50μL) | (μL) | mTCR-pMIA vector digest (500μL) | (μL) |
DNA (~20μg) | DNA (~20μg) | ||
MfeI | 2 | MfeI | 20 |
BstbI | 2 | BstbI | 20 |
10x Buffer | 5 | 10x Buffer | 50 |
H2O | H2O | ||
Step 4.4 | |||
CIP reaction (100μL) | (μL) | DNA(~1.5mg) | |
Digested & purified vector DNA | 84 | ||
10x Buffer | 10 | ||
CIP enzyme | 6 | ||
Step 4.5 | |||
Ligation reaction (20μL) | (μL) | ||
Digested/CIPed vector DNA | (total insert and vector amount is ~150ng) | ||
Insert DNA | (6 molar access to vector) | ||
2x Ligase buffer | 10 | ||
Ligase enzyme | 1 | ||
H2O | |||
Step 4.9 | |||
Test Digest reaction (12μL) | (μL) | ||
DNA (100-300ng) | 1 | ||
MfeI | 0.25 | ||
BstbI | 0.25 | ||
10x Buffer | 1.2 | ||
H2O | 9.3 | ||
Step 4.11 | |||
TRAV digest reaction (60μL) | (μL) | Beta containing – mTCR-pMIA vector digest (120μL) | |
DNA(~1.5μg) | DNA (~3μg) | ||
SnaBI | 3 | MfeI | 6 |
SacII | 2 | BstbI | 4 |
10x Buffer | 6 | 10x Buffer | 10 |
H2O | H2O |
Table 3. Vector cloning reactions.
In the current protocol, we describe an efficient method for single cell TCR amplification and subsequent sub-cloning of paired TCR alpha and beta chains into a template retroviral expression vector. Although, several single cell PCR protocols have been developed, none so far have been compatible with immediate sub-cloning into an expression vector. In most cases, a partial sequence encompassing the highly variable CDR3 regions is amplified, with enough sequence to extrapolate the variable region. This smaller PCR product size supports higher PCR efficiency, but necessitates de novo TCR gene synthesis for functional studies. The current protocol maintains the efficiency of previously published CDR3-focused single cell TCR sequencing protocols, while at the same time yielding a longer amplicon containing the whole variable region suitable for cloning. Therefore, the system described has an increased flexibility in giving a quantitative sequence, as well as functional output.
Depending on the scientific question addressed, the source and specificity of T cells analyzed can vary. However, if the end goal of the project is the analysis of the in vivo TCR function, it is necessary to know the HLA restriction of isolated T cells. The current protocol has been specifically designed to be compatible with in vivo TCR expression in HLA-humanized mice. Many of these mice have been generated and are now commercially available, including HLA-A2.1, HLA-DQ8 (HLA-DQA1*301,HLA-DQB1*302), HLA-DQ6 (DQA1*0102,HLADQB1*0602), HLA-DR4 (DRB1*0401), HLA-DR1 (DRA*0101, DRB1*0101), HLA-A11 (A*11:01/K), HLA-B2705, HLA-A24, and HLA-B*0702 transgenic strains. This protocol can be successfully combined with the previously published protocol for retroviral mediated bone marrow stem cell expression of human TCRs4,15,16. We expect that the humanized TCR retrogenic system will be applicable and highly useful for functional analysis of human TCRs in various autoimmune, cancer, and infectious models.
The current single cell PCR protocol describes the critical steps and gives suggestions necessary for successful amplification of paired TCR-alpha and TCR-beta sequences. The first critical step is a timely performance of cDNA transcription, and subsequent first round of multiplex PCR. All the steps involved should be performed in a timely manner, and all the reagents and cells kept on ice to prevent RNA and cDNA degradation. Secondly, because the protocol is designed to be highly sensitive to low levels of the template, it is highly susceptible to contamination. Therefore, it is imperative to work in a template-free area, away from amplified PCR products or TCR vector cloning. A separate room or hood should be used to set up cDNA and PCR reactions. Template from the first PCR reaction should be added in a different area from where the PCR is set up. It is advisable that a separate set of aliquoted reagents be used for the single cell PCR experiments.
This protocol has been specifically optimized using the reagents listed in Table of Materials, and any substitutions in reagents will require additional optimization. The initial identification of antigen-specific cells can be modified to use peptide-MHC tetramers. Tetramer staining allows for simultaneous assessment of antigen specificity, as well as HLA-restriction. Alternative methods for the detection of antigen reactivity, such as upregulation of early activation markers or secretion of inflammatory cytokines can be considered in the absence of tetramer reagents. For example, dual specificity fusion antibodies specific for a T cell antigen and IFNγ can be combined with magnetic bead isolation to enrich for antigen reactive IFNγ producing cells.
While the chimeric human/mouse construct described in our protocol ensures compatibility with mouse cells, the level of its compatibility with human CD3 complex is unknown. Although not formally tested by us, others have shown that chimeric TCR constructs with murine constant regions can be expressed on the surface of human lymphocytes17. This indicates that murine TCR constant regions can support interaction with the human CD3 signaling complex. However, if the goal is to express the identified TCRs in human primary cells or human cell lines, such as Jurkat cells, a more optimal approach may be to use a fully human construct. This can be accomplished by swapping the murine constant regions within the vector with human constant regions.
If an identified TCR variable region contains one of the restriction sites used for cloning, the TCR should be inserted into a shuttle vector and subsequently mutated using a site directed mutagenesis kit to induce a silent nucleotide change within the restriction cut site. An alternative approach is de novo synthesis of the TCR variable region of interest modified to exclude the unwanted restriction site.
The main limitation of this technique is the inability to eliminate amplification of secondary ‘cytoplasmic’ alpha chains. TCR-alpha genes do not go through allelic exclusion like TCR-beta genes, thus a significant proportion of T cells will express a secondary ‘cytoplasmic’ alpha chain14. The described PCR protocol only results in amplification of one TCR alpha chain, but if the amplified alpha chain is the ‘cytoplasmic’ alpha chain it may not pair with the amplified beta chain. Therefore, it is imperative to test TCR cell surface expression in HEK293T cells to ensure successful chain pairing.
The authors have nothing to disclose.
This study was funded by JDRF 1-FAC-2014-243-A-N, ADA 7-14-JF-07, NIH 5 P30 DK079638-05 PILOT PJ, and The Robert and Janice McNair Foundation.
We thank Sandra Pena and Andrene McDonald for patient recruitment, Samuel Blum for technical assistance, Dr. George Makedonas for the gift of antibodies and control DNA used for PCR optimization.
CFSE | eBioscience | 65-0850-84 | 5 μM |
anti-human CD4 (OKT4) BV421 | Biolegend | 317433 | 2 μg/mL |
anti-human CD3 (OKT3) PerCP/Cy5.5 | Biolegend | 317336 | 2 μg/mL |
anti-mouse CD3 AF647 | Biolegend | 100322 | 2.5 μg/mL |
Recombinant RNAse inhibitor biotech grd 2.5KU | VWR | 97068-158 | 0.7 U |
Applied Biosystems High capaity cDNA RT kit | Invirtogen | 4368814 | 9 U (RT enzyme) |
NF-H2O: Nuclease-free water 1000mL | Invirtogen | AM9932 | |
Gotaq DNA polymerase 2500U | VWR | PAM3008 | 1 U |
96 Well Half Skirt PCR Plate | Phenix | MPX-96M2 | |
MicroAmp Clear Adhesive Film | ThermoFisher | 4306311 | |
UltraPure Agarose | Invirtogen | 15510-027 | |
SnaBI | New England Biolabs | R0130 | 15 U (insert) 30 U (vector) |
SacII | New England Biolabs | R0157 | 40 U (insert) 80 U (vector) |
MfeI | New England Biolabs | R3589S | 40 U (insert) 80 U (vector) |
BstBI | New England Biolabs | R0519 | 40 U (insert) 80 U (vector) |
Calf Intestinal Phosphatase (CIP) | New England Biolabs | M0290S | 10 U |
Quick ligase | New England Biolabs | M2200S | |
Wizard Plus minipreps DNA purification systems | Promega | A1460 | |
Wizard Plus midipreps DNA purification systems | Promega | A7640 | |
DNA clean & concentrator-25 kit | Genesee Scientific | 11-304C | |
ZR-96 DNA clean & concentrator-5 kit | Genesee Scientific | 11-306A | |
QIAquick gel extraction kit | Qiagen | 28704 | |
QIAquick PCR purification kit | Qiagen | 28104 | |
Subcloning Efficiency DH5a Competent Cells | ThermoFisher | 18265017 | |
Transit LT-1 Reagent 1mL (transfection reagent) | Mirus | MIR2300 | |
BD FACS Aria II (sorter) | BD | ||
BD Fortessa (flow cytometer) | BD |