Methods of high-throughput identification of T cell epitopes by capturing cytokines on the surface of antigen-presenting cells
The present disclosure relates to methods that combine identification of T cell-secreted cytokines by modified antigen-presenting cells (APCs), cell sorting, and next-generation sequencing to identify class I- and class II-restricted epitopes starting from massively-complex peptide-encoding oligonucleotide pools. APCs are modified to express anti-cytokine antibodies, a library of DNA-encoded peptides, and multiple HLA class I or II molecules. Upon co-culture with T cells, these modified APCs form HLA/epitope/TCR complexes, enabling the production of T cell activation-dependent cytokines with the DNA that encodes the presented peptide. After co-culture, the APCs are sorted, and the peptide-encoding DNA is sequenced to determine the identity of the immunogenic peptides. Thus, the disclosure allows pooled screening of thousands of encoded peptides to enable epitope discovery for orphan T cell receptors.
1 . A method of identifying epitopes that activate T cells, comprising:
providing a plurality of antigen presenting cells (APCs), wherein each APC expresses a) a nucleic acid encoding a candidate epitope or a nucleic acid encoding a peptide that may be processed into a candidate epitope, b) a nucleic acid encoding an HLA molecule, and c) a nucleic acid encoding an anti-cytokine antibody;
mixing the plurality of APCs with a plurality of T cells, wherein each T cell expresses a T cell receptor (TCR) on its surface, wherein binding of the TCR on the T cells to the candidate epitope activates the T cells, wherein the activated T cells secrete a cytokine that binds to the anti-cytokine antibody; and
identifying APCs bound to the cytokine; and
sequencing the nucleic acid encoding the candidate epitope contained in the APCs to which the cytokine is bound.
2 . The method of claim 1 , wherein the APCs are professional APCs.
3 . The method of claim 2 , wherein the professional APCs are dendritic cells, macrophages, monocytes or B cells.
4 . The method of claim 1 , wherein the APCs are non-professional APCs.
5 . The method of claim 1 , wherein the APCs are human APCs.
6 . The method of claim 1 , wherein the candidate epitope is an infectious disease-associated candidate epitope, an autoimmune disease-associated candidate epitope, or a tumor-associated candidate epitope, or wherein the APCs are non-professional APCs of an immortalized cell line; or wherein the HLA molecule is a class I HLA molecule.
7 . The method of claim 1 , wherein the HLA molecule is a class II HLA molecule.
8 . The method of claim 1 , wherein the anti-cytokine antibody is an anti-IL-2 antibody, or wherein the anti-cytokine antibody is an anti-INF-γ antibody.
9 . The method of claim 1 , wherein the T cells comprise CD8 + T cells.
10 . The method of claim 1 , wherein the T cells comprise CD4 + T cells.
11 . The method of claim 1 , wherein the identifying comprises contacting the APCs after contact with the plurality of T cells with a detectable label that binds the cytokine; and detecting the label.
12 . The method of claim 11 , wherein the detectable label comprises a fluorescently-labeled, secondary anti-cytokine antibody.
13 . The method of claim 1 , further comprising sorting labeled APCs from non-labeled APCs that do not bear a T cell-activating epitope.
14 . The method of claim 13 , wherein the sorting is conducted by magnetic or flow cytometry; or wherein the sequencing is conducted using next generation sequencing.
15 . The method of claim 1 , wherein the plurality of APCs comprises a library of APCs that expresses a library of the candidate epitopes.
16 . The method of claim 1 , wherein the candidate epitope is 8-24 amino acids in length, or wherein the candidate epitope is 8-15 amino acids in length, or wherein the candidate epitope is 8-12 amino acids in length.
17 . A modified APC, wherein the modified APC expresses a) an exogenous nucleic acid encoding a candidate epitope or an exogenous nucleic acid encoding a peptide that may be processed into a candidate epitope, b) an exogenous nucleic acid encoding an HLA molecule, and c) an exogenous nucleic acid encoding an anti-cytokine antibody.
18 . The modified APC of claim 17 , which is a professional APC.
19 . The modified APC of claim 18 , wherein the professional APC is a dendritic cell, macrophage, monocyte or a B cell.
20 . The modified APC of claim 17 , wherein the APC is a non-professional APC.
21 . The modified APC of claim 20 , wherein the non-professional APC is an immortalized cell line.
22 . The modified APC of claim 17 , wherein the APC is a human APC; or wherein the candidate epitope is an infectious disease-associated epitope, an autoimmune disease-associated epitope, or a tumor-associated epitope; or wherein the HLA molecule is a class I HLA molecule.
23 . The modified APC of claim 17 , wherein the HLA molecule is a class II HLA molecule.
24 . The modified APC of claim 17 , wherein the anti-cytokine antibody is an anti-IL-2 antibody, or wherein the anti-cytokine antibody is an anti-IFN-γ antibody.
25 . A library of APCs of claim 17 , wherein respective APCs comprise different nucleic acids that encode a different candidate epitope, thereby representing a library of candidate epitopes expressed by the library of APCs.
26 . The library of claim 25 , wherein the nucleic acids encoding the different candidate epitopes are obtained from a genomic library of candidate epitopes.