Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

The T cell receptor (TCR) dictates T cell fate decision during development, steady state/homeostasis, and antigenic stimulation in periphery^1,2,3. 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 vivo^4,5,6,7. 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 sequence^4,5,6,7. 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 TCRs^8,9,10,11. 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 chains^9,11. 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.

Protokół

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.

1. Mix PBMCs 1:1 with MHC-matched feeder cells, and label with 5 µM CFSE cell division dye.
2. Plate 2-2.5 x 10⁵ cells per well in a 96-well round-bottom culture plate in 200 µL 10% FCS complete RPMI media.
   NOTE: Complete RPMI contains 2 mM l-glutamine, 1 mM sodium pyruvate, 100 µM MEM nonessential amino acids, 5 mM HEPES, 5.5 × 10^{− 5} units of 2-mercaptoethanol, 100 U mL^{− 1} penicillin and 100 µg mL^{− 1} streptomycin.
3. Stimulate cells with 25 µM peptide antigen.
4. On the fourth day of culture, carefully remove and replace 100 µL of media.
   NOTE: A more optimal approach for antigen-specific T cell identification is the use of peptide-HLA tetramers¹³. Tetramer staining allows for simultaneous assessment of antigen specificity, as well as HLA-restriction.

2. Set up the single cell sort.

1. Perform all procedures in a hood or a designated area free of amplified template. Aliquot the reagents to avoid contamination, clean all work surfaces and, if feasible, equipment with 10% bleach prior to PCR setup. Use filter tips, RNAse and DNAse free reagents and plastics in all the PCR and vector cloning steps. Key reagents can be found in the Table of Materials.
   NOTE: The multiplex PCR reaction is highly sensitive, and low levels of contamination can result in amplified product.
2. Generate the reverse transcription master mix by combining 10x RT reaction buffer, 25X DNTPs, 9 U RT enzyme, 0.01% Triton-X, 0.7 U RNAse inhibitor, and 383 nM of each RT-TCR primer (listed in Table 1) in a sterile pipetting reservoir. For each 96-well plate, make up enough master mix for 10 additional reactions (total 106), to compensate for loss of liquid during pipetting. See Table 2 for reaction components and amounts per well.
3. Prepare a 96-well PCR collection plate for cell sorting by pipetting 6 µL of the reverse transcription master mix per well with a multichannel pipette. Keep the plate on ice or in a cold block.
4. Label cells with antibodies to cell surface markers (such as CD4 and CD3, or CD8 and CD3, each at 2 µg/mL, in combination with cell division dye or tetramer staining).
5. Sort T cells at one cell per well, skipping the twelfth row, which will serve as a negative control.
   NOTE: The efficiency of the sort will depend on precise collection plate alignment within the plate holder, as well as maintaining the cells and plates at a low temperature prior to cDNA reaction. Collection plates should be kept on ice at all times, except during sorting, when they are fixed within the cooling plate. Not all sorters are equipped with a temperature regulated plate holder; however, the use of one is strongly recommended. Since the sort generally takes about 10 - 15 min per plate, there is an increased potential for RNA degradation if the plate is not maintained at a low temperature.
6. As a positive control, add a larger number of cells (100 cells) manually with a pipette to one of the wells at this stage. Alternatively, previously isolated RNA from pooled cells can be used as a positive control at 10 ng per well.
   NOTE: Pipette purified control RNA with precision and care in order to avoid cross-contamination of other wells and false positive results.
7. Seal the plate with adhesive film, and spin down at 1000 x g for 5 min at 4 °C.
8. Immediately transcribe RNA to cDNA by incubating the plate at 25 °C for 10 min, 37 °C for 45 min, and 85 °C for 10 min.
   NOTE: To ensure efficient PCR reaction, it is recommended that the first PCR reaction be performed the same day. Alternatively, the transcribed plates can be kept in -80°C for up to 2 months.

3. Perform multiplex-nested PCR

NOTE: Prepare all master mixes in a clean template-free area to avoid contamination.

1. Reconstitute each variable region primer to 100 µM with DNAse and RNAse free H₂O. Next, combine 20 µL of each of the 44 primers to generate the V-alpha primer pool. To generate the V-beta primer pool combine 20 µL of each of the 40 V-beta primers plus 80 µL of DNAse and RNAse free H₂O. Primer sequences are listed in Table 1. Once pooled, the concentration of each primer in the mix is 2.3 µM.
2. Reconstitute the constant region primers to 100 µM stock solution with DNAse and RNAse free H₂O. Dilute the constant region primers to 10 µM working solution and aliquot for use.
3. Prepare two separate master mixes for TCR-alpha and TCR-beta amplification. Generate the master mixes by combining 5X buffer, 80 µM dNTPs, 3% DMSO, 2 µM primer pool (final concentration of 46 nM for each primer), 200 nM TCR-constant region reverse primer, 1 U DNA polymerase, and DNAse and RNAse free H₂O (Table 2). For each 96-well plate, make up enough master mix for 10 additional reactions (total 106), in order to compensate for loss of liquid during pipetting.
   NOTE: The calculations are based on the final PCR volume of 25 µL per well, where 22.5 µL master PCR mix is combined with 2.5 µL cDNA template.
4. Keep the plate with cDNA on ice or in a cold block. Use a multichannel to pipette 22.5 µL of TCR-beta reaction into a new 96-well PCR plate. Then use a multichannel to transfer 2.5 µL of cDNA into the PCR plate.
5. Use a multichannel to pipette 22.5 µL of TCR-alpha reaction directly into the plate containing the remainder of the cDNA.
6. Seal the plates with adhesive, gently vortex, and spin down at 1000 x g for 5 min at 4 °C.
7. Run the multiplex PCR reaction with the following cycles: 5 min at 95 °C followed by 34 cycles of 20 s at 95 °C, 30 s at 54 °C, and 1 min at 72 °C, followed by 7 min at 72 °C.
8. Carry out the nested PCR reaction in a final volume of 25 µL, using 2.5 µL of the multiplex PCR mixture as template.
   1. Reconstitute the internal and adaptor primers to 100 µM with DNAse and RNAse free H₂O. Make 10 µM aliquots for the working stock.
   2. Prepare two separate master mixes for TCR-alpha and TCR-beta amplification. Mix 5X buffer, 25X DNTP, 3% DMSO, 200 nM forward adaptor primer, 200 nM reverse nested primer, 1 U DNA polymerase, and DNA polymerase and nuclease-free H₂O for a final volume of 22.5 µL (Table 2). For each 96-well plate, make up enough master mix for 10 additional reactions (total 106), to compensate for loss of liquid during pipetting.
      NOTE: In the second PCR reaction use the 5X PCR buffer containing loading dye to facilitate agarose gel loading.
   3. Using a multichannel, pipette the master mixes for the TCR-alpha and TCR-beta reactions at 22.5 µL per well into two new 96-well PCR plates.
   4. Add the multiplex PCR reaction template at 2.5 µL per well.
      NOTE: The remaining first PCR reaction should be sealed, and stored at -20 °C to serve as a source of additional template, if it becomes necessary (see step 4.3 Note).
   5. Seal the plates, gently vortex, and spin down at 1000 x g for 5 min at 4 °C.
   6. Run the nested PCR reaction with the following cycles: 5 min at 95 °C followed by 34 cycles of 20 s at 95 °C, 30 s at 56 °C, and 1 min at 72 °C, followed by 7 min at 72 °C.
   7. Run 5 µL of the final reaction out on a 1% agarose gel containing ethidium bromide (including the negative and positive control wells).
      NOTE: The expected band should run at around 500 bp size. An example reaction is shown in Figure 2.
   8. Purify the remainder of the second reaction (20 µL) using a 96-well format PCR purification kit, elute with 15 µL of 70 °C H₂O per well, and perform Sanger sequencing. Use the appropriate TCR-alpha or TCR-beta adaptor forward primer for sequencing (Table 1).
      NOTE: Sequences can be analyzed with online available immunogenetics software.

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).

1. Digest the purified PCR products from the beta nested reaction with BstbI and MfeI (Table 3).
   NOTE: Do not exceed 5 µg insert.
2. In parallel, digest the template vector with BstbI and MfeI (Table 3). Use 1-2 µg of the template vector per TCR. Run the digested vector on an agarose gel, and isolate the larger band (~7500 bp). Purify the vector with a gel DNA purification kit.
   NOTE: During the restriction enzyme digest, the template murine TCR-beta variable region is removed, allowing for the addition of the human TCR-beta variable region.
3. Purify the digested PCR products using a PCR purification kit.
   NOTE: Verify the DNA purification capacity of the columns included in the kit; use several columns if necessary. The minimum amount of a purified insert necessary for a successful ligation reaction is 50 ng at a minimum concentration of 10 ng/µL. If the purified insert amount is not sufficient for a successful ligation, the second PCR reaction can be repeated.
4. Treat the template vector with 10 U CIP enzyme for 1 h at 37 °C to prevent self-ligation, followed by heat inactivation for 10 min at 75 °C. Purify the DNA with a PCR purification kit (Table 3).
5. Ligate TCR-beta insert and vector using DNA ligase in a 20 µL total volume at 6 molar excess of the insert for 30 min at room temperature.
   NOTE: In total, the ligation reaction should contain about 150 ng of DNA (Table 3).
6. For transformations, mix 8 µL of the ligation reaction with 80 µL of chemically competent E.coli DH5α cells (Table of Materials) and incubate on ice for 30 min. Heat shock at 42 °C for 45 s. Add 500 µL Super Optimal broth with Catabolite suppression (S.O.C.) medium and shake for 1.5 h at 37 °C.
7. Plate out 250 µL of bacterial culture on an ampicillin containing Lysogeny Broth (LB) agar plate. Incubate the plate overnight (~16 h) at 37 °C.
8. The next day pick bacterial colonies, and grow them up in 3 mL LB medium containing ampicillin overnight. Isolate the plasmid DNA using DNA plasmid purification kit.
9. Confirm the successful insertion of the beta chain by a small-scale test-digest with the restriction enzymes BstbI and MfeI. In a 12 µL total reaction volume, combine 200-500 µg plasmid DNA, 0.25 µL of each enzyme, and 10X enzyme buffer (Table 3). Run the reaction out on a 1% agarose gel containing ethidium bromide to confirm the size of the insert band (~500 bp).
10. After confirming the presence of the insert, sequence the TCR-beta section of the vector using the IRES reverse primer (Table 1).
11. Following sequence confirmation of the TCR-beta insert, perform a similar process for insertion of the alpha variable region. Digest the purified PCR products from the alpha nested reaction, and the newly generated TCR-beta insert containing vector, with SnaBI and SacII.
    NOTE: During the restriction enzyme digest, the template murine TCR-alpha variable region is removed, allowing for the addition of the human TCR-alpha variable region.
12. Ligate alpha inserts into TCR vectors encoding the corresponding beta variable region using DNA ligase in a 20 µL total volume at 6 molar excess of the insert for 30 min at room temperature.
    NOTE: In total, the ligation reaction should contain about 150 ng of DNA (Table 3).
13. For transformations, mix 8 µL of the ligation reaction with 80 µL of chemically competent E.coli DH5α cells (Table of Materials) and incubate on ice for 30 min. Heat shock at 42 °C for 45 s. Add 500 µL S.O.C. medium and shake for 1.5 h at 37 °C.
14. Plate out 250 µL of bacterial culture on an ampicillin containing Lysogeny Broth (LB) agar plate. Incubate the plate overnight (~16 h) at 37 °C.
15. The next day pick bacterial colonies, and grow them up in 3 mL LB medium containing ampicillin overnight. Isolate the plasmid DNA using DNA plasmid purification kit.
16. Confirm the successful insertion of the alpha chain by a small-scale test-digest with the restriction enzymes SnaBI and SacII. In a 12 µL total reaction volume combine 200-500 µg plasmid DNA, 0.25 µL of each enzyme, and 10x enzyme buffer (Table 3). Run the reaction out on a 1% agarose gel containing ethidium bromide to confirm the size of the insert band (~500 bp).
17. After confirming the presence of the insert, sequence the TCR-alpha section of the vector using the MSCV2-forward primer (Table 1).
18. Once the alpha chain sequence is validated, verify the complete TCR insert by sequencing with TCR-beta-reverse and IRES-reverse primer (Table 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).

1. Plate out HEK293T cells in 12-well tissue culture plates at 1 x 10⁵ cells per well in 1 mL complete DMEM media containing 5% FCS, and culture overnight at 37 °C. Plate out enough cells to have designated separate wells for each new TCR, control template TCR vector, CD3 vector only, control TCR vector only, and a non-transfected well.
   NOTE: Transfection with a fully mouse template TCR vector (mTCR-pMIA) in combination with mCD3εγδζ-pMIG vector, mTCR-pMIA vector alone, and mCD3εγδζ-pMIG vector alone are used as positive and negative controls.
   NOTE: Complete DMEM contains 2 mM L-glutamine, 1 mM Sodium Pyruvate, 100 µM Non-essential Amino Acids, 5 mM HEPES buffer, 5.5 x 10^-5 units of 2-mercaptoethanol, 100 U/ml Penicillin/Streptomycin.
2. The following day, transfect the cells with 1 µg chimeric h/mTCR-pMIA vector, 1 µg mouse CD3εγδζ-pMSCVII-GFP (pMIG) vector, and a liposome-based transfection reagent following manufacturer instructions.
3. For each TCR, add 6 µL transfection reagent to 100 µL room temperature, serum-free DMEM. Briefly vortex, and incubate at room temperature for 15 min.
4. In separate 1.5 mL tubes combine 1 µg of TCR-pMIA vector with 1 µg mouse CD3εγδζ-pMSCVII-GFP (pMIG) vector, or 1 µg of each vector separate for controls.
5. Dropwise, add the transfection reagent/DMEM solution to the tubes containing the vector DNA. Incubate at room temperature for 15 min.
6. Dropwise, add the transfection reagent/DMEM/DNA solution to the HEK293T cells. Gently tap the plate to mix. Incubate at 37 °C for 24 h.
7. After 24 h replace the media, and keep cells in culture for additional 24 h.
8. Remove cells from the plate by vigorous pipetting, and stain the cells with anti-mouse CD3 antibody (2.5 µg/mL) to verify surface expression of the chimeric-TCR by flow cytometry.
9. In order to compare CD3 expression between cells that were transfected to a similar level, gate the analysis on cells expressing a similar high level of fluorescent reporter molecules (GFP for CD3 complex, and Ametrine for TCR expression vector). Optimally, the analysis should be gated on GFP+Ametrine+ cells within 10⁴ to 10⁵ fluorescent intensity (Figure 3A).
   NOTE: The level of CD3 expression in cells transfected with the chimeric construct should be comparable to the control mouse (original template) vector (Figure 3A). The generated TCR retroviral vectors are compatible with in vivo expression systems as previously described⁴.

Wyniki

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 ...

Dyskusje

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 produc...

Materiały

Name	Company	Catalog Number	Comments
CFSE	eBioscience	65-0850-84	5 mM
anti-human CD4 (OKT4) BV421	Biolegend	317433	2 mg/mL
anti-human CD3 (OKT3) PerCP/Cy5.5	Biolegend	317336	2 mg/mL
anti-mouse CD3 AF647	Biolegend	100322	2.5 mg/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