Determining antigen-specific t-cells

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.

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.