Method of measuring adaptive immunity

• US 9,809,813 B2
• Filed: 03/04/2016
• Issued: 11/07/2017
• Est. Priority Date: 06/25/2009
• Status: Active Grant

• Current Assignee: Fred Hutchinson Cancer Center
• Original Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Inventors: Robins, Harlan S., Warren, III, Edus H., Carlson, Christopher Scott
• Primary Examiner(s): Schultz, Doug
• Application Number:
  US15/061,827
• Publication Number:
  US 20160251721A1
• Time in Patent Office: 613 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding

Abstract

A method of measuring immunocompetence is described. This method provides a means for assessing the effects of diseases or conditions that compromise the immune system and of therapies aimed to reconstitute it. This method is based on quantifying T-cell diversity by calculating the number of diverse T-cell receptor (TCR) beta chain variable regions from blood cells.

Claims

1. A method for generating a clonotype profile of rearranged T cell receptor (TCR) and/or immunoglobulins (Ig) in a biological sample comprising:

(a) combining a plurality of V segment primers and a plurality of J segment primers with DNA from a biological sample comprising T cells and/or B cells wherein the V segment primers for TCR amplification comprise the primers of SEQ ID Nos;

1-45, the J segment primers for TCR amplification comprise the primers of SEQ ID Nos;

46-57, the V segment primers for Ig amplification comprise the primers of SEQ ID Nos;

443-451 and the J segment primers for amplification of Ig comprise the primers of SEQ ID Nos;

452-467, wherein the terminal “

n”

position in SEQ ID NO;

1-45 and SEQ ID Nos;

46-57 comprises a universal primer sequence, and wherein SEQ ID Nos;

443-451 and SEQ ID Nos;

452-467 comprise a universal primer sequence;

(b) amplifying rearranged TCR and/or Ig using the plurality of V segment primers and J segment primers in a multiplex polymerase chain reaction (PCR) to produce amplified rearranged TCR and/or Ig DNA molecules;

(c) immobilizing said amplified rearranged TCR and/or Ig DNA molecules on a solid surface and performing solid phase PCR to form template clusters on the solid surface; and

(d) sequencing the TCR and/or Ig DNA molecules in the template clusters to produce sequence reads that encompasses a CDR3 encoding region of the TCR and/or Ig thereby generating a clonotype profile TCRs and/or Igs in the biological sample.

2. The method of claim 1 wherein the universal primer sequence is a GA2 universal primer sequence.

3. The method of claim 2 wherein the V segment primers for TCR amplification comprise SEQ ID NOS:

58-102 and the J segment primers for TCR amplification comprise SEQ ID Nos;

103-113.

4. The method of claim 1 wherein the total diversity of rearranged TCR is greater than 1×

10⁶.

5. The method of claim 1 wherein sequencing in step (d) is done by reversible dye termination chemistry.

6. The method of claim 5 wherein the sequencing reads 30-54 base pair intervals.

7. The method of claim 1 wherein the sample comprises genomic DNA.

8. The method of claim 1 wherein the sample comprises cDNA.

9. The method of claim 1 wherein the biological sample is a blood sample.