Method of measuring adaptive immunity
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.
1. A method for generating a biomarker set of rearranged T cell receptor (TCR) and/or immunoglobulin (Ig) sequences for a disease, the method comprising:
(a) generating a plurality of clonotype profiles of rearranged T cell receptor (TCR) and/or immunoglobulins (Ig), wherein each clonotype profile is generated from a biological sample of a patient having the disease, and wherein generating each clonotype profile comprises:
(i) combining a plurality of at least 14 V segment primers each complementary to a single functional V segment or small family thereof and a plurality of at least 3 J segment primers each complementary to a single functional J segment with DNA from a biological sample comprising T cells and/or B cells, wherein the V segment primers for TCR amplification are selected from the group consisting of the primers of SEQ ID NOs: 1-45, the J segment primers for TCR amplification are selected from the group consisting of 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 are selected from the group consisting of the primers of SEQ ID NOs: 452-467, wherein the terminal “n” position in SEQ ID NOs: 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;
(ii) 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;
(iii) 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
(iv) 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 of TCRs and/or Igs in the biological sample; and
(b) identifying rearranged TCR and/or Ig sequences shared between clonotype profiles generated from biological samples of patients having the disease, wherein the identifying comprises comparing the clonotype profiles generated from biological samples of the patients having the disease, thereby generating a biomarker set of rearranged TCR and/or Ig sequences for the disease.
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 45 primers selected from the group consisting of SEQ ID NOs: 58-102 and the J segment primers for TCR amplification comprise 11 primers selected from the group consisting of SEQ ID NOs: 103-113.
4. The method of claim 1 , wherein the total diversity of rearranged TCRs is greater than 1×10 6 .
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.
10. The method of claim 1 , wherein the disease is a cancer, an autoimmune disease or an infectious disease.
11. The method of claim 10 , wherein the cancer is an early stage tumor.
12. The method of claim 10 , wherein the autoimmune disease is multiple sclerosis, Type I diabetes or rheumatoid arthritis.
13. The method of claim 1 , further comprising treating the disease in a subject in need thereof by targeting with a drug T cells or B cells expressing one or more of the biomarker set of TCR and/or Ig sequences for the disease.
14. The method of claim 1 , wherein the selected V segment primers and the selected J segment primers do not cross an intron/exon boundary.
15. The method of claim 1 , wherein the selected V segment primers and the selected J segment primers anneal to areas of sequence conservation in the V or J segment.
16. The method of claim 1 , further comprising step (i)′ of combining a universal C segment primer with the V and J segment primers.
17. A method for generating a clonotype profile of rearranged T cell receptor (TCR) and/or immunoglobulin (Ig) sequences for a disease, the method comprising:
(a) generating a plurality of clonotype profiles of rearranged T cell receptor (TCR) and/or immunoglobulins (Ig), wherein each clonotype profile is generated from a biological sample of a patient having the disease, and wherein generating each clonotype profile comprises:
(i) combining a plurality of at least 14 V segment primers and a plurality of at least 3 J segment primers with genomic DNA or cDNA from a biological sample comprising T cells and/or B cells, wherein the V segment primers for TCR amplification are selected from the group consisting of the primers of SEQ ID NOs: 1-45, the J segment primers for TCR amplification are selected from the group consisting of 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 are selected from the group consisting of the primers of SEQ ID NOs: 452-467, wherein the terminal “n” position in SEQ ID NOs: 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;
(ii) amplifying rearranged TCR and/or Ig using the plurality of V segment primers and J segment primers by multiplex polymerase chain reaction (PCR) to produce amplified rearranged TCR and/or Ig DNA molecules;
(iii) 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
(iv) sequencing the TCR and/or Ig DNA molecules in the template clusters to produce sequence reads that encompass a CDR3 encoding region of the TCR and/or Ig thereby generating a clonotype profile of TCRs and/or Igs in the biological sample; and
(b) identifying rearranged TCR and/or Ig sequences shared between clonotype profiles generated from biological samples of patients having the disease, wherein the identifying comprises comparing the clonotype profiles generated from biological samples of the patients having the disease, thereby generating a clonotype profile of rearranged TCR and/or Ig sequences for the disease.
18. The method of claim 17 , wherein the selected V segment primers and the selected J segment primers do not cross an intron/exon boundary.
19. The method of claim 17 , wherein the selected V segment and the selected J segment primers anneal to areas of sequence conservation in the V or J segment.
20. The method of claim 17 , further comprising the step (i)′ of combining a universal C segment primer with the V and J segment primers.