Methods for discovering and using lymphocytes with antigen specificity

A method for determining a lymphocyte receptor chain sequence, or a portion thereof, specific for at least two antigens, the method comprising:

• providing a biological sample comprising a plurality of lymphocytes;
• extracting a plurality of first antigen presenting cells from the biological sample;
• dividing the plurality of first antigen presenting cells into a plurality of first reaction mixtures;
• sorting a plurality of first antigens into the plurality of first reaction mixtures, wherein the sorting comprises adding a unique first antigen of the plurality of first antigens to a unique subset of the plurality of first reaction mixtures, and wherein two unique first antigens are not added to any two identical subsets of the plurality of first reaction mixtures;
• contacting each first reaction mixture with the biological sample;
• providing a condition for a first activated lymphocyte in at least one first reaction mixture of the plurality of first reaction mixtures to expand in number such that a plurality of lymphocyte clones is formed;
• extracting a plurality of second antigen presenting cells from the biological sample;
• adding the plurality of second antigen presenting cells into a second reaction mixture;
• adding a plurality of query antigens into the second reaction mixture;
• dividing the second reaction mixture into the plurality of first reaction mixtures to create a plurality of final reaction mixtures;
• separating a second activated lymphocyte and a non-activated lymphocyte from a subset of the plurality of final reaction mixtures, wherein the second activated lymphocyte recognizes a query antigen of the plurality of query antigens, wherein the query antigen is different from any antigen of the plurality of first antigens;
• sequencing nucleic acids of the second activated lymphocyte to obtain the lymphocyte receptor chain sequence;
• and
• detecting the unique first antigen of the plurality of first antigens, wherein the unique first antigen and the query antigen are specific for the lymphocyte receptor chain sequence.