Method for detection and quantification of T-cell receptor Vbeta repertoire

• US 20070117134A1
• Filed: 11/20/2006
• Published: 05/24/2007
• Est. Priority Date: 11/18/2005
• Status: Active Grant

• Current Assignee: Shanghai Abelzeta Limited
• Original Assignee: Zhong Kou
• Inventors: Kou, Zhong
• Granted Patent: US 7,375,211 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

The invention is a method for detecting and measuring T-cell receptor (TCR) repertoires from mammalian lymphocytes. The method is based on the use of the multiple sets of unique primers to amplify 22 regions of the TCR Vβ region and thereby detect clonal expansions related to antigen stimulation of the immune system. Kits containing sets of primers and specialized analytical statistical software for use in determining clonal expansion in humans and mice are disclosed. The reliability, efficiency and short assay time in using the method is well suited to monitoring immune response to vaccination and therapeutic treatments for immune disorders.

Claims

1. A method for detecting T-cell clonality, comprising:

synthesizing first strand cDNA from a sample containing T-lymphocytes;

amplifying the cDNA to produce nested PCR products;

further amplifying the nested PCR products with at least 59 primers each specific for one of 22 regions of T-cell Vβ

gene CDR3 regions;

separating amplified nested PCR products from among 22 Vβ

gene families; and

detecting T-cell clonality as at least one of a monoclonal repertoire, an oligoclonal repertoire, or a polyclonal repertoire of Vβ

clonal expansion.

2. The method of claim 1 wherein the sample is a human sample.

3. The method of claim 1 wherein the sample is a mouse sample.

4. The method of claim 2 wherein the at least 59 primers are identified as having the sequence of SEQ ID NOs;

60-118.

5. The method of claim 3 wherein the at least 59 primers are identified as having the sequence of SEQ ID NOs:

1-59.

6. The method of claim 1 wherein the primers bind to non-overlapping regions of CDR3 T-cell Vβ

human or mouse gene.

14. A method for monitoring changes in clonality associated with immune system disorders, comprising

determining clonality by the method of claim 1 from a tissue sample in a subject suspected of having an immune system disorder;

obtaining sequence lengths of amplified DNA products of 22 TCR Vβ

repertoires from T-lymphocytes in the sample;

spectratyping the DNA products;

analyzing the spectratyped DNA products to provide a quantitative profile of said products;

repeating the clonality and profile determinations on a tissue sample from the subject after treatment for the immune system disorder; and

comparing clonality changes before and after treatment

wherein clonal expansion provides monitoring of changes associated with treatment of immune system disorders.

15. The method of claim 14 wherein the subject is human.

16. The method of claim 14 wherein the subject is murine.

17. The method of claim 14 wherein the immune system disorder is selected from the group consisting of myelodysplasia, graft versus host disease, aplastic anemia, HIV, diabetes and infectious diseases.

18. The method of claim 14 wherein the immune system disorder is cause by a parasite, virus or bacterium.

7. A method for measuring T-cell receptor (TCR) repertoire profiles, comprising,

(a) amplifying cDNA encoding TCR third complementarity determining regions of a single Vβ

family with a T-cell family-specific Vβ

primer and a Vβ

primer;

(b) further amplifying nested DNA templates from step (a) with at least 59 primers comprising Vβ

primers and Jβ

primers between about 15 and about 28 bases each hybridizing with a non-overlapping region of Vβ

or Jβ

;

(c) separating amplified DNA segments from step (b);

(d) measuring length of separated amplified DNA fragments from step (c);

(e) spectratyping the DNA from step (d); and

(f) determining a quantitative TCR Vβ

profile by analytical statistical analysis of spectratyping data from step (e).

8. The method of claim 7 wherein the eDNA is from human T-lymphocytes.

9. The method of claim 7 wherein the CDNA is from mouse T-lymphocytes.

10. The method of claim 8 wherein the at least 59 primers are identified as having the sequence of SEQ ID NOs;

60-118.

11. The method of claim 9 wherein the at least 59 primers are identified as having the sequence of SEQ ID NOs:

1-59.

12. A kit for detecting T-cell receptor (TCR) Vβ

repertoires, comprising;

DNA primers comprising primers having the sequence of SEQ ID NOs;

1-118 PCR amplification buffers, and

Instructions for use.

13. The kit of claim 12 further comprising analytical statistical software for quantitation of the detected TCR Vβ

repertoires and additional instructions therefor.