DETERMINING ANTIGEN-SPECIFIC T-CELLS

• US 20150275296A1
• Filed: 04/01/2014
• Published: 10/01/2015
• Est. Priority Date: 04/01/2014
• Status: Active Grant

• Current Assignee: Adaptive Biotechnologies Corp.
• Original Assignee: Sequenta LLC
• Inventors: Klinger, Mark, Faham, Malek
• Granted Patent: US 10,066,265 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The invention is directed to methods for determining antigen-specific T cells. In some embodiments, methods of the invention may be implemented by the steps of reacting under interaction conditions one or more antigens with T cells in a plurality of subsets of a tissue sample, such as peripheral blood; sorting antigen-interacting T cells from other T cells; separately sequencing for each subset recombined nucleic acid encoding a segment of a TCR chain from a sample of T cells prior to exposure to antigen and from a sample of T cells isolated based on their interaction with antigen, thereby forming a clonotype profile for the former sample and the latter sample for each subset; and identifying as antigen-specific T cells those T cells associated with a clonotype whose frequency increases in the latter sample relative to its frequency in the former sample.

Claims

1. A method of determining antigen-specific T cells in a tissue sample, comprising the steps of:

exposing a tissue sample to an antigen in a reaction mixture;

enriching T cells from the reaction mixture that interact with the antigen into a subset;

sequencing recombined nucleic acids encoding a T-cell receptor chain or a portion thereof from a sample of enriched T cells to provide sequence reads from which clonotypes are determined;

sequencing recombined nucleic acids encoding a T-cell receptor chain or a portion thereof from the tissue sample of from T cells that do not interact with the antigen to provide sequence reads from which clonotypes are determined; and

determining antigen-specific T cells in the tissue sample as T cells whose clonotype frequencies increase in the subset of enriched T cells relative to the frequencies of the same clonotypes in the tissue sample or in the T cells that do not interact with the antigen.

2. The method of claim 1 wherein said step of exposing includes incubating said antigen with said tissue sample for at least a predetermined interval.

3. The method of claim 2 wherein said antigen is a protein or a plurality of peptides derived from a protein.

4. The method of claim 1 wherein said step of sequencing includes isolating individual molecules of said recombined nucleic acids on a solid surface and sequencing the isolated individual molecules or clones thereof to provide said sequence reads.

5. The method of claim 4 wherein said method further includes a step of coalescing a plurality of said sequence reads to determine each of said clonotypes.

6. The method of claim 1 wherein said step of enriching includes sorting said T cells from said reaction mixture that interact with said antigen.

7. The method of claim 6 wherein said step of sorting is implemented with an antigen reagent capable of forming a complex with a T cell specific for said antigen or wherein said step of sorting is implemented with a binding compound specific for an activation marker.

8. The method of claim 7 wherein said antigen reagent is a peptide-MHC multimer complex wherein the peptide of the complex is derived from said antigen.

9. The method of claim 1 wherein said tissue sample comprises tissue samples from a plurality of individuals.

10. The method of claim 1 wherein said tissue sample comprises peripheral blood or is derived from peripheral blood.

11. A method of determining receptors of antigen-specific T cells in a tissue sample, the method comprising the steps of:

forming a plurality of subsets from a tissue sample containing T cells;

exposing under interaction conditions the T cells of each subset to an antigen;

isolating the antigen-interacting T cells of each subset;

sequencing recombined nucleic acids encoding T-cell receptor α

chains in each subset to provide sequence reads from which α

chain clonotypes are determined;

sequencing recombined nucleic acids encoding T-cell receptor β

chains in each subset to provide sequence reads from which β

chain clonotypes are determined;

identifying as antigen-specific T cell receptors with those pairs of α

chain clonotypes and β

chain clonotypes that for every subset either (i) both α

chain and β

chain clonotypes are present in a subset or neither α

chain nor β

chain clonotypes are present in a subset, and (iii) both the α

chain and β

chain clonotypes are present in at least one subset and the α

chain and β

chain clonotypes are not present in at least one subset.

12. The method of claim 11 wherein said tissue sample contains a population of said T cells, the population having a size and each different T cell of the population having a frequency within the population, and wherein said plurality of said subsets depends on said size of the population and the frequency of said T cells whose T-cell receptor α

chains and T-cell receptor β

chains are to be determined.

13. The method of claim 11 wherein a size of said tissue sample and said plurality of subsets are selected so that said T cells of said sample are distributed among said subsets in accordance with a binomial model.

14. The method of claim 11 wherein said step of reacting includes incubating said antigen with said tissue sample for at least a predetermined interval.

15. The method of claim 11 wherein said antigen is a protein or a plurality of peptides derived from a protein.

16. The method of claim 11 wherein said step of isolating is implemented with an antigen reagent or with a binding compound specific for an activation marker.

17. The method of claim 11 wherein said tissue sample comprises tissue samples from a plurality of individuals.

18. The method of claim 11 wherein said tissue sample comprises peripheral blood or is derived from peripheral blood.

19. A method of determining clonotypes of antigen-specific T cells in a tissue sample, the method comprising the steps of:

(a) forming a plurality of subsets from a tissue sample containing T cells;

(b) exposing under interaction conditions T cells in a subplurality of subsets to one or more antigens so that T cells specific for any of the one or more antigens are capable of interacting therewith, wherein each different antigen is exposed to T cells in a different subplurality;

(c) enriching the antigen-interacting T cells of each subset of a subplurality;

(d) sequencing recombined nucleic acids encoding a T-cell receptor chain or a portion thereof from said enriched T cells in each subset of the subplurality to provide sequence reads from which clonotypes are determined;

(e) sequencing recombined nucleic acids encoding T-cell receptor chain or a portion thereof from said T cells in each subset of the subplurality prior to said step of enriching or from non-enriched T cells in each subset of the subplurality to provide sequence reads from which clonotypes are determined; and

(f) identifying a clonotype of a T cell specific for an antigen of the one or more antigens as a clonotype whose frequency increases in substantially every subset of a subplurality corresponding to the antigen and does not increase in substantially every other subset.

20. The method of claim 19 wherein said tissue sample contains a population of said T cells, the population having a size and each different T cell of the population having a frequency within the population, and wherein said plurality of said subsets depends on said size of the population and the frequency of said T cells whose clonotypes are to be determined.

21. The method of claim 19 wherein said step of exposing includes reacting under activation conditions said antigens with said T cells.

22. The method of claim 21 wherein said step of reacting further includes incubating said antigens with said T cells for at least a predetermined interval.

23. The method of claim 19 wherein said antigen is a protein or a plurality of peptides derived from a protein and wherein said tissue sample comprises peripheral blood or is derived from peripheral blood.

24. The method of claim 19 wherein said step of isolating is implemented with an antigen reagent with a binding compound specific for an activation marker.