WO2021208035A1

WO2021208035A1 - Methods and reagents for high-throughput detection of nucleic acid sequence of single t cell surface receptor

Info

Publication number: WO2021208035A1
Application number: PCT/CN2020/085185
Authority: WO
Inventors: Wenqi ZHU
Original assignee: Singleron Nanjing Biotechnologies Ltd
Current assignee: Singleron Nanjing Biotechnologies Ltd
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2021-10-21
Anticipated expiration: 2022-10-16

Abstract

Provided is a reagent and method for high-throughput detection of the TCR sequence at single cell level.

Description

METHODS AND REAGENTS FOR HIGH-THROUGHPUT DETECTION OF NUCLEIC ACID SEQUENCE OF SINGLE T CELL SURFACE RECEPTOR

Technical field

The present disclosure involves methods and reagents for high-throughput detection of nucleic acid sequences of single cell T cell receptor.

Background

B lymphocytes and T lymphocytes participate in the acquired immune responses ^[1] . Human T cells develop in the thymus from progenitors originating in hematopoietic tissues. During their development, T cells acquire the ability to recognize foreign antigens and provide protection against many different types of pathogens. This functional flexibility is facilitated by the expression of highly polymorphic surface receptors called T cell receptors (TCRs) ^[2] . The diversity of T cell receptors (TCRs) , B cell receptors (BCRs) and secreted antibodies constitutes the core of a complex immune system and serves as a key defense component that protects the body from invasion by viren, bacteria and other foreign substances ^[3] . TCR is a heterodimer--αβ chain (～ 95%, TRA, TRB) or γδ chain (～ 5%) . Each chain can be divided into variable and constant domains ^[4] . Each peptide chain can be divided into variable region (V region) , constant region (C region) , transmembrane region and cytoplasmic region. The variable region of α chain is encoded by V and J gene fragments. The variable region of the β chain is encoded by three gene segments: V, D, and J. The V regions (Vα, Vβ) of the two peptide chains, α and β, have three hypervariable regions: CDR1, CDR2, and CDR3, of which the CDR3 region (also called hypervariable region) has the largest variation, which directly determines the antigen binding specificity of TCR ^[5, 6] .

Due to the rearrangement of the V (D) J gene and the random deletion of germline nucleotides, the TCR profiles are extremely diverse. In humans, it is theoretically estimated that the diversity of TCR-αβ receptors exceeds 10 ¹² in the thymus, and the diversity directly determines the antigen binding specificity of TCR ^[7] .

In recent years, due to the, advances of gene sequencing technologies, high-throughput sequencing technology (such as RNA-Seq) has been used to detect the diversity of immune receptors, and Immune repertoire sequencing can be applied to the fields of vaccine and pharmaceutical research and development, discovery of biomarkers ^[8] , detection of Minimal Residual Disease (MRD) ^[9, 10] , research of autoimmune diseases ^[11] and post-transplant monitoring ^[12] . For example in the study of disease-specific biomarkers, disease-specific CDR3 can be found in people with the same disease through high-throughput sequencing. After verification, these CDR3 sequences can be used as Biomarker representing the disease and can be found in peripheral blood; Research on autoimmune diseases such as rheumatoid arthritis, can identify potential autologous clones by high-throughput sequencing to quantify the T cell repertoire of peripheral blood of early or diagnosed rheumatoid arthritis, as a basis for the early diagnosis of medication. It can promote the development of vaccines for different populations by analyzing the effects of people of different ages after injection of vaccines. For tumor research, disease guidance can be monitored by comparing changes in the immune repertoire of patients before and after medication to prevent tumor recurrence.

However, the traditional RNA-seq measures the average expression level of tissue samples or cell populations, which makes the difference between cells likely masked by the average value, and cannot specifically describe the diversity of lymphocytes or clonotypes that constitute the immune response. On the other hand, bulk RNA-seq cannot determine which TCRA and TCRB chains combine to form a specific TCR, which is essential for many functional and therapeutic applications ^[7] . Therefore, the establishment of a method for detecting the diversity of TCR at single cell level is particularly important for promoting the application of immune receptors sequencing in early clinical diagnosis, efficacy evaluation, and prognosis judgment.

At present, there are several methods and reagents for single cell immune receptors detection, such as SMARTer Human scTCR a /b Profiling Kit kit from Takara/Clontech, through sorting single cells by manual or flow cytometry into 96-well PCR Plate, each well is an independent reaction, through the processes of cell lysis, reverse transcription, and PCR amplification, the enrichment of immune receptor sequences was achieved. However, the disadvantages of Clontech are as follows: Clontech generally relies on plate-or well-based microfluidics and is therefore limited in the number of cells that can be processed, typically 10–100. Additionally, a large number of sequencing reads are generally required to computationally reconstruct paired antigen receptors ^[13] . As such, the cost per cell is relatively high, estimated at $50–$100 USD ^[14] .

Chromium Single Cell V (D) J Reagent Kits launched by 10X Genomics has greatly improved the detection throughput compared to Clontech's products. By encapsulating single cells and hydrogel beads containing cell barcode in individual droplets, TCR from thousands of single cells can be processed and then detected in parallel. However, the disadvantages of Clontech are as follows: The mapping rate of TCR sequencing is relatively low, the Median UMI detection value of TCR a chain is relatively low resulting the Low detection rate of TCR a chain.

Summary

The purpose of the present disclosure is to provide a reagent and method for high-throughput detection of the TCR sequence at single cell level. First, we use probe binding to TCR sequence combined with oligo-dT to capture mRNA, improving the capture efficiency of TCR sequences. The probe and oligo-dT contain the same PCR handle sequence, so that TCR can be amplified by multiplex PCR. Optionally, the probe and oligo-dT can be combined with a oligonucleotide sequence that can act as cell barcode to distinguish each single cell from other cells, so that thousands or more of single cells can be analyzed in parallel. This method can also be used in combination with a microfluidic system where each cell in a sample can be partitioned to individual micro-chambers. Single cells can be lyzed in the micro-chambers; mRNA and TCR sequences can be captured at the same time.

Brief description of drawings

Figure 1 Schematic diagram of the present disclosure.

Figure 2 Schematic diagram of the embodiment of the present disclosure where cell barcoding capture magnetic bead is used to capture mRNA and TCR sequence.

Figure 3 shows the amplified cDNA map.

Figure 4 shows the TCR target enrichment 1 map.

Figure 5 shows the TCR target enrichment 2 map.

Figure 6 shows the TCR libray map.

Detailed description

To overcome the drawbacks of the current single cell TCR analysis methods, we use probe binding to TCR sequence combine with oligo-dT to capture mRNA while improving the capture efficiency of TCR sequences. The probe and polyT oligo contain the same PCR handle sequence, which can act as priming site for RT reactions and TCR Target enrichment reactions.

One embodiment of the present disclosure is to add probe binding to TCR sequence to the 3’ end of oligo-dT. This way one can capture and reverse transcribe both mRNA and TCR sequence captured by probe. The resulting cDNA can be used as template to enrich TCR sequence by multiplex PCR. With unique cell barcodes in conjunction with the oligo-dT sequence, cDNA molecules from the same single cell can be labeled and a group of single cells can be processed in parallel. There is great potential synergy in pairing TCR sequences (which can reveal information about T-cell ancestry and antigen specificity) with information about expression of genes characteristic of particular T-cell functions. Integrating these two types of information can allow one to comprehensively profile a given T cell.

GEXSCOPE Single Cell RNAseq Library Construction kit (Singleron Biotechnologies) was used to demonstrate the technical feasibility and the utility of the present disclosure in massively parallel single cell ncRNA sequencing. The experiment was conducted according to manufactuer’s instructions with modifications described below.

Cell barcoding Magnetic bead synthesis:

The primers on all beads comprise a common sequence used for PCR amplification, a bead-specific cell barcode, a unique 8 molecular identifier (UMI) , a oligo-dT sequence for capturing polyadenylated mRNAs and probe sequence annealing to TCR constant Region for capturing TCR mRNA.

The sequence of the TCR constant region is as follows:

Human T Cell R1-1 TGAAGGCGTTTGCACATGCA

Human T Cell R1-2 TCAGGCAGTATCTGGAGTCATTGAG

Human T Cell R2-1 AGTCTCTCAGCTGGTACACG

Human T Cell R2-2 TCTGATGGCTCAAACACAGC

The complementary sequence is as follows:

PolyA R1-1 CAAACGCCTTCAAAAAAAAAAAAA

PolyA R1-2 GATACTGCCTGAAAAAAAAAAAAA

PolyA R2-1 AGCTGAGAGACTAAAAAAAAAAAA

PolyA R2-2 TGAGCCATCAGAAAAAAAAAAAAA

Briefly, single cell suspension of PBMC was loaded onto the microchip to partition single cells into individual wells on the chip. Cell barcoding magnetic beads were then loaded to the microchip and washed. Only one bead can fall into each well on the microchip based on the diameters of the beads and well (about 25um and 40um, respectively) .

Loading 100ul cell lysis buffer into the chip and let incubate at room temperature for 20 minutes to lyse cells and capture RNAs. After 20 minutes, the magnetic beads, together with captured RNAs, were taken out of the microchip and subject to RT, template switching, cDNA amplification, and a part of cDNA was used to construct Gene expression library using reagents from the GEXSCOPE kit and following manufacturer’s instructions.

The rest of cDNA were used to enrich TCR sequence.

1. Frist-round of enrichment: Take 10ng cDNA as the template for the first round of TCR enrichment by multiplex nested PCR using QIAGEN Multiplex PCR kit.

1) Primer design: TCR V region primer (TRV Reaction1) combined with the universal sequence (Target 1F) . TCR V region primer (TRV Reaction1) including 38 TRA V regions and 36 TRB V region primers, total 74 primers.

2) Configure the PCR mix according to the following system and mix thoroughly:

Component	volume (ul)
2x QIA master mix	25ul
TRV Reaction1 primer (50um)	4.44ul
Target 1F primer (10um)	1.5ul
Dnase/Rnase-Free Water	19.06-x
Template cDNA	x
Total	50ul

The final concentration of each TCR V-region primer is 0.06 μM, Target 1F primer is 0.3 μM.

3) Perform the reaction on the PCR instrument

	Temperature	Time
1	95℃	15min
2	94℃	30s
3	62℃	90s
4	72℃	90s
5	GOTO Step 2, 9X
6	72℃	10min
7	4℃	hold

4) Product purification: 0.8x purification of the obtained PCR product (purification method is the same as above)

2. Second-round of enrichment: a 10-μl aliquot of the first reaction was used as a template for second 50-μl PCR using QIAGEN Multiplex PCR kit;

1) Primer design: TCR V region primer (TRV Reaction2) combined with the universal sequence (Target 2F) , TCR V region primer (TRV Reaction2) includes 36 TRA V region primers, 36 TRB V region primers, total 72 primers.

1) Configure the PCR mix according to the following system and mix thoroughly:

Component	volume (ul)
2x QIA master mix	25ul
TRV Reaction1 primer (50uM)	0.6ul
Target 2F primer (10uM)	1.5ul
Dnase/Rnase-Free Water	12.9ul
aliquots of the first-round PCR products	10ul
Total	50ul

Note: V primers 0.6 μM, Target 2F primer 0.3 μM.

2) Perform the reaction on the PCR instrument

3) Product purification: 0.8x purification of the obtained PCR product (purification method is the same as above)

3. Amplification and library construction: Take 20ng of the second-round enrichment products and use KAPA HiFi PCR kit for amplification and library construction by multiplex PCR

1) Configure the PCR mix according to the following system and mix thoroughly:

Component	volume (ul)
5X HiFi with Mgcl2 buffer	10ul
10mM dNTP Mix	1.5ul
HiFi HotStart	1ul
Target 1F (10uM)	1.5ul
PCR-N7XX primer	1.5ul
Dnase/Rnase-Free Water	34.5-x ul
aliquots of the second-round PCR	x
Total	50

2) Perform the reaction on the PCR instrument

	Temperature	Time
1	95℃	3min
2	98℃	20s
3	64℃	30s
4	72℃	1min
5	GOTO Step 2, 5X
6	72℃	5min
7	4℃	hold

1) Product purification: The obtained PCR product was purified 0.8x (the purification method is the same as above) , add 20 μl of Nuclease-Free water to elute DNA.

The resulting single cell RNAseq library was sequenced on Illumina NovaSeq with PE150 mode and analyzed with scopeTools bioinformatics workflow (Singleron Biotechnologies) .

(1) Amplified cDNA map

Figure 3 shows the amplified cDNA map.

(2) TCR Target Enrichment 1 map

Figure 4 shows the TCR target enrichment 1 map.

(3) TCR Target Enrichment 2 map

Figure 5 shows the TCR target enrichment 2 map.

(4) TCR libray map

Figure 6 shows the TCR libray map.

(5) TCR Mapping

(6) Top 10 Clonotype Frequencies

Our data shows that the mapping rate of TCR can reach more than 90%, and the detection rate of TRA and TRB paired cells also reaches 62%.

The number of T cells annotated in the transcriptome data is consistent with the number of T cells detected in the TCR enrichment library.

The basic principles, main features and advantages of the present disclosure are shown and described above. All technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present disclosure.

Reference

[1] Tonegawa S. Somatic generation of antibody diversity. Nature 1983; 302: 575-81.

[2] Marco D S , Grazisa R, Massimiliano P. Single Cell T Cell Receptor Sequencing: Techniques and Future Challenges [J] . Frontiers in Immunology, 2018, 9: 1638.

[3] Lefranc, M. -P., and G. Lefranc, 2001a The Immunoglobulin Factsbook. Academic Press, San Diego.

[4] Benichou, J., R. Ben-Hamo, Y. Louzoun, and S. Efroni, Rep-Seq: uncovering the immunological repertoire through nextgeneration sequencing. Immunology, 2012.135: 183–191.

[5] Robins HS, Campregher PV, Srivastava SK., et al. Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells [J] . Blood, 2009, 114 (19) : 4099.

[6] Zvyagin IV, Pogorelyy MV, Ivanova ME., et al. Distinctive properties of identical twins’ TCR repertoires revealed by high-throughput sequencing. Proc Natl Acad Sci USA. (2014) 16: 5980–5.

[7] Bryan Howie., et al. High-throughput pairing of T cell receptor a and b sequences. Sci Transl Med (2015) ; 7, 301ra131

[8] Looney T, Topacio-Hall D, Lowman G., et al. TCR convergence in individuals treated with immune checkpoint inhibition for cancer. Front Immunol, 2020 (09) .

[9] Velden V H J V D , Dongen J J M V . MRD Detection in Acute Lymphoblastic Leukemia Patients Using Ig/TCR Gene Rearrangements as Targets for Real-Time Quantitative PCR [J] . Methods in Molecular Biology.

[10] Logan A C , Vashi N , Faham M., et al. Immunoglobulin and T Cell Receptor Gene High-Throughput Sequencing Quantifies Minimal Residual Disease in Acute Lymphoblastic Leukemia and Predicts Post-Transplantation Relapse and Survival [J] . Biology of Blood &Marrow Transplantation, 2014, 20 (9) : 1307-1313.

[11] Oftedal, Bergithe E, Ardesj Lundgren., et al. T cell receptor assessment in autoimmune disease requires access to the most adjacent immunologically active organ [J] . Journal of Autoimmunity: S0896841116303444.

[12] Jang-Hee Cho, Young-Deuk Yoon, Hye Min Jang., et al. Immunologic Monitoring of T-Lymphocyte Subsets and HLA-DR-Positive Monocytes in Kidney Transplant Recipients [J] . Medicine, 2015, 94 (44) : e1902.

[13] Rizzetto S , Eltahla A A , Lin P , et al. Impact of sequencing depth and read length on single cell RNA sequencing data of T cells [J] . Scientific Reports, 2017, 7 (1) : 12781.

[14] Ziegenhain C , Vieth B , Parekh S , et al. Comparative Analysis of Single-Cell RNA Sequencing Methods [J] . Molecular Cell, 2017, 65 (4) : 631-643.

Claims

A method for analyzing TCR sequence, at single cell level, wherein said method comprising:

a) capture the RNA from a single cell with an oligo-dT primer combined with probe sequence that binding to TCR RNA sequence;

b) reverse transcribe the RNA to cDNA with the oligo-dT primer and TCR-recognizing sequence;

c) amplify cDNA;

d) amplify TCR sequence;

e) analyze amplified cDNA.
The method of Claim 1, wherein the primer sequence additionally comprises a sequence that acts as cell barcode that identifies each single cells; a sequence that can be used as PCR primer-binding sequence for amplification of the cDNA.
The method of Claim 1, wherein the primer sequence comprise a unique molecular index (UMI) sequence that can be used to quantify cDNA.
The method of Claim 1, wherein the probe sequence is added by using an enzyme.
The method of Claim 1, wherein the probe sequence is added chemically.
The method of Claim 4, wherein the enzyme is a ligase, to add specific sequence to the 3’ of oligo-dT.
The method of Claim 4, wherein the enzyme is a DNA polymerase, to add specific sequence to the 3’ of PolyT.
The method of Claim 1, wherein the target enrichment method is PCR.
The method of Claim 8, wherein the PCR used in Target Enrichment is annealing to TCR variable Region.
The method of Claim 1, wherein the analysis method is sequencing.
A product that includes reagents needed to enable the process as described in Claim 1.