SYNTHETIC CONSTRUCTS AND METHODS FOR PROCESSING ANTIGEN-SPECIFIC CELLS
The present disclosure provides methods for detecting antigen-specific cells, such as T cells or B cells, using a proximity ligation assay (PLA) in which at least one of the PLA probes is a peptide-major histocompatibility (MHC) multimer or a B cell specific antigen bound to an oligonucleotide. Also provided are kits for use in such methods.
1 . A method for detecting a target cell in a sample, comprising:
contacting the target cell with a cell selector component for which the target cell is specific, the cell selector component being bound to a first oligonucleotide;
contacting the target cell with a targeting component specific for a receptor on the target cell, the targeting component being bound to a second oligonucleotide; and
forming a nucleic acid template by ligating the first oligonucleotide and the second oligonucleotide.
2 . The method of claim 1 , wherein the first oligonucleotide is directly bound to the cell selector component.
3 . The method of claim 1 or 2 , wherein the second oligonucleotide is directly bound to the targeting component.
4 . The method of claim 1 or 2 , wherein the first oligonucleotide is bound to a first secondary antibody that is bound to the cell selector component.
5 . The method of claim 4 , wherein the first secondary antibody is bound to a myc-tag, a his-tag, or a fluorescent tag on the cell selector component.
6 . The method of claim 3 , wherein the second oligonucleotide is bound to a second secondary antibody that is bound to the targeting component.
7 . The method of any one of claims 1-6 , wherein the cell selector component comprises a peptide-MHC multimer.
8 . The method of claim 7 , wherein the peptide-MHC multimer comprises a peptide-MHC tetramer
9 . The method of any one of claims 1-7 , wherein the peptide-MHC multimer comprises streptavidin or dextran.
10 . The method of claim 9 , wherein the first oligonucleotide is bound to streptavidin or dextran.
11 . The method of any one of claims 1-6 , wherein the cell selector component comprises a B cell specific antigen.
12 . The method of any one of claims 1-11 , wherein the targeting component comprises an antibody or antigen binding fragment thereof, a fusion protein, an aptamer, or a combination thereof.
13 . The method of any one of claims 1-10 or 12 , wherein the receptor is CD3 or the T cell receptor (TCR).
14 . The method of any one of claims 1-10, 12, or 13 wherein the target cell is a T cell.
15 . The method of any one of claims 1-6 or 11 , wherein the receptor is CD79 of the B cell receptor (BCR).
16 . The method of any one of claims 1-6, 11, or 15 wherein the target cell is a B cell.
17 . The method of any one of claims 1-16 , wherein the cell selector component is no more than 50 nanometers (nm) from the targeting component.
18 . The method of any one of claims 1-17 , wherein the cell selector component is no more than 40 nm from the targeting component.
19 . The method of any one of claims 1-18 , wherein the cell selector component is no more than 35 nm from the targeting component.
20 . The method of any one of claims 1-19 , wherein the cell selector component is no more than 30 nm from the targeting component.
21 . The method of any one of claims 1-20 , wherein the cell selector component is no more than 25 nm from the targeting component.
22 . The method of any one of claims 1-21 , further comprising contacting the first and second oligonucleotides with a connector oligonucleotide.
23 . The method of any one of claims 1-22 , further comprising amplifying the nucleic acid template.
24 . The method of claim 23 , wherein the amplifying the nucleic acid template comprises rolling circle amplification.
25 . The method of claim 23 or 24 , wherein the amplifying the nucleic acid template comprises producing an amplification product.
26 . The method of claim 25 , further comprising detecting the amplification product.
27 . The method of claim 26 , wherein the detecting the amplification product comprises forming a labeled amplification product by contacting the amplification product with a probe comprising a fourth oligonucleotide and a label, the fourth oligonucleotide having a sequence that is substantially complementary to at least a portion of the amplification product.
28 . The method of claim 27 , wherein the label is a fluorescent label.
29 . The method of claim 27 , wherein the label is a heavy metal tag.
30 . The method of claim 27 , wherein the label is an oligonucleotide tag or a DNA barcode.
31 . The method of claim 27 or 28 , wherein the detecting the amplification product comprises imaging the labeled amplification product using flow cytometry.
32 . The method of claim 27 or 29 , wherein the detecting the amplification product comprises using time-of-flight mass spectrometry.
33 . The method of claim 27 or 30 , wherein the detecting the amplification product comprises using sequencing or PCR.
34 . The method of any one of claims 1-33 , wherein the ligating the first oligonucleotide and the second oligonucleotide comprises using a protein ligase.
35 . The method of any one of claims 1-33 , wherein the ligating the first oligonucleotide and the second oligonucleotide comprises using a chemical ligase.
36 . The method of any one of claims 1-33 , wherein the ligating the first oligonucleotide and the second oligonucleotide comprises using a nucleic acid ligase.
37 . The method of any one of claims 1-36 , wherein the sample is a tissue sample or a cell sample.
38 . The method of any one of claims 1-37 , further comprising sequencing the target cell receptor.
39 . A kit for detecting a target cell in a sample comprising:
a cell selector component for which the target cell is specific, the cell selector component being bound to a first oligonucleotide;
a targeting component specific for a second antigen on the target cell, the targeting component being bound to a second oligonucleotide;
a ligation reagent; and
written instructions for using the cell selector component and the targeting component to detect the target cell.
40 . The kit of claim 39 , further comprising a connector oligonucleotide.
41 . The kit of claim 39 or 40 , further comprising a probe comprising a fourth oligonucleotide and a label.
42 . The kit of claim 41 , wherein the label is a fluorescent label.
43 . The kit of claim 41 , wherein the label is a heavy metal tag.
44 . The kit of claim 41 , wherein the label is an oligonucleotide tag or a DNA barcode.
45 . The kit of any one of claims 39-44 , wherein the ligation reagent comprises a protein ligase.
46 . The kit of any one of claims 39-44 , wherein the ligation reagent comprises a chemical ligase.
47 . The kit of any one of claims 39-44 , wherein the ligation reagent comprises a nucleic acid ligase.
48 . A synthetic construct, comprising:
a cell selector component;
a first oligonucleotide bound to the cell selector component;
a second oligonucleotide bound to the first oligonucleotide; and
a targeting component bound to the second oligonucleotide.
49 . The synthetic construct of claim 48 , wherein the first oligonucleotide is bound directly to the cell selector component.
50 . The synthetic construct of claim 48 or 49 , wherein the second oligonucleotide is directly bound to the targeting component.
51 . The synthetic construct of claim 48 or 49 , wherein the first oligonucleotide is bound to a first secondary antibody that is bound to the cell selector component.
52 . The synthetic construct of claim 51 , wherein the first secondary antibody is bound to a myc-tag, a his-tag, or a fluorescent tag on the cell selector component.
53 . The synthetic construct of claim 48 or 50 , wherein the second oligonucleotide is bound to a second secondary antibody that is bound to the targeting component.
54 . The synthetic construct of any one of claims 48-53 , wherein the cell selector component comprises a peptide-MHC multimer.
55 . The synthetic construct of claim 54 , wherein the peptide-MHC multimer comprises a peptide-MHC tetramer
56 . The synthetic construct of claim 54 , wherein the peptide-MHC multimer comprises streptavidin or dextran.
57 . The synthetic construct of claim 56 , wherein the first oligonucleotide is bound to streptavidin or dextran.
58 . The synthetic construct of any one of claims 48-53 , wherein the cell selector component comprises a B cell specific antigen.
59 . The synthetic construct of any one of claims 48-58 , wherein the targeting component comprises an antibody or antigen binding fragment thereof, a fusion protein, an aptamer, or a combination thereof.
60 . The synthetic construct of any one of claims 48-57 or 59 , wherein the receptor is CD3 or the T cell receptor (TCR).
61 . The synthetic construct of any one of claims 48-57, 59 or 60 , wherein the target cell is a T cell.
62 . The synthetic construct of any one of claims 48-53 or 58 , wherein the receptor is CD79 or the B cell receptor (BCR).
63 . The synthetic construct of any one of claims 48-53, 58, or 62 , wherein the target cell is a B cell.
64 . The synthetic construct of any one of claims 48-63 , further comprising a connector oligonucleotide bound to the first and second oligonucleotides.