Methods for discovering and using lymphocytes with antigen specificity
Described herein is a method for determining a lymphocyte cell receptor chain sequence specific to a unique antigen, comprising: sorting a plurality of antigens into a plurality of reaction mixtures, wherein the sorting comprises adding a unique antigen of the plurality of antigens to a unique subset of the plurality of reaction mixtures such that two different unique antigens are not added to the unique subset; contacting each reaction with a biological sample comprising a plurality of lymphocytes; separating a target lymphocyte from a subset of the plurality of lymphocytes, wherein the target lymphocyte recognizes the unique antigen; after separating the target lymphocyte, sequencing nucleic acids of the target lymphocyte to obtain the lymphocyte receptor chain sequence, wherein the sequencing is performed by single-cell sequencing; and detecting the unique antigen, wherein the detecting comprises: computing a frequency of lymphocyte cells that express the lymphocyte receptor chain sequence.
1 . 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.
2 . The method of claim 1 , wherein the first activated lymphocyte is a T cell or a B cell.
3 . The method of claim 1 , further comprising HLA typing of the biological sample to determine a predicted display of at least one antigen of the plurality of first antigens by an MHC molecule present in the biological sample.
4 . The method of claim 1 , further comprising enriching the plurality of lymphocytes prior to sorting the plurality of first antigens into the plurality of first reaction mixtures.
5 . The method of claim 1 , further comprising enriching the plurality of lymphocytes after providing the condition for the first activated lymphocyte to expand in number and prior to extracting the plurality of second antigen presenting cells.
6 . The method of claim 1 , wherein separating the second activated lymphocyte and the non-activated lymphocyte is performed based on a marker, wherein the marker is selected from the group consisting of CD3, CD4, CD8, CD137, OX40, CD25, PD-L1, CD69, CD154, and a combination thereof.
7 . The method of claim 1 , wherein the second activated lymphocyte recognizes the query antigen by binding an MHC complex comprising the query antigen.
8 . The method of claim 1 , wherein the sorting further comprises applying, using a processor, an error-correcting code configured to determine the unique subset of the plurality of first reaction mixtures that the unique first antigen is added to.
9 . The method of claim 8 , wherein the error-correcting code is a collision free superimposed code configured to allow for detection of at least two unique first antigens specific for the lymphocyte receptor chain sequence.
10 . The method of claim 9 , wherein the collision free superimposed code is determined by a random search method.
11 . The method of claim 9 , wherein the collision free superimposed code consists of:
a plurality of prefix codes, wherein a prefix code of the plurality of prefix codes is assigned to the unique first antigen of the plurality of first antigens, wherein the prefix code identifies an overlap set, wherein the prefix code is identical for more than one first antigen of the plurality of first antigens within the overlap set; and
a plurality of suffix codes, wherein a suffix code of the plurality of suffix codes is assigned to the unique first antigen of the plurality of first antigens, wherein a combination of the prefix code and the suffix code is distinct for the unique first antigen.
12 . The method of claim 1 , wherein the detecting comprises applying, using a processor, a decoding algorithm, wherein the decoding algorithm is configured to detect the unique first antigen specific for the lymphocyte receptor chain sequence when the lymphocyte receptor chain sequence is not substantially present in at least one reaction mixture of the unique subset of the plurality of first reaction mixtures.
13 . The method of claim 12 , wherein the decoding algorithm is a nearest set algorithm.
14 . The method of claim 1 , wherein separating the second activated lymphocyte and the non-activated lymphocyte from the subset of the plurality of final reaction mixtures is performed using multimer sorting.
15 . The method of claim 1 , wherein separating the second activated lymphocyte and the non-activated lymphocyte from the subset of the plurality of final reaction mixtures is performed using fluorescence-based sorting.
16 . The method of claim 1 , wherein separating the second activated lymphocyte and the non-activated lymphocyte from the subset of the plurality of final reaction mixtures is performed using bead-based sorting.
17 . The method of claim 1 , wherein a number of reaction mixtures corresponding to the unique subset of the plurality of first reaction mixtures is a function of a number of expected unique first antigens that are specific to the lymphocyte receptor chain sequence.
18 . The method of claim 1 , wherein the plurality of first reaction mixtures comprises at least one control reaction mixture, wherein the control reaction mixture does not contain any antigens that are added to the biological sample.
19 . The method of claim 1 , wherein the detecting further comprises computing a frequency of lymphocytes that express the lymphocyte receptor chain sequence.