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US 12,449,419 B1Granted
Methods for detecting binding of peptide-MHC monomers to T cells
Assignee10x Genomics, Inc.
InventorsMichael John Terry Stubbington, Luz Montesclaros, Katherine Pfeiffer, Sarah E. B. Taylor, Alvaro Martinez Barrio
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Abstract
Featured are devices, systems, and methods of use for detecting binding of polynucleotide-peptide conjugate-major histocompatibility complex (pMHC) monomers to a T cell receptors (TCR) on a T cell, and the use of a peptide library to detect binding of antigenic peptides in a pMHC monomer to a T cell receptor (TCR) on a T cell.
Claims20
A method for detection of binding of a peptide to a T cell comprising:
- (a) providing a peptide Major Histocompatibility Complex conjugate (pMHC) complex comprising a MHC monomer and a peptide, and wherein the MHC monomer comprises a reporter polynucleotide covalently attached thereto, wherein the reporter polynucleotide comprises a reporter sequence that identifies the peptide of the pMHC;
- (b) presenting the pMHC monomers to T cells to generate a pMHC bound to a T cell, wherein a T cell receptor (TCR) of the T cell binds a pMHC monomer to form a pMHC-TCR complex, wherein the pMHC-TCR complex is stabilized by:
- (i) using a chemical fixative;
- (ii) reducing diffusion of the pMHC monomer from the TCR;
- and/or
- (iii) retaining and/or increasing the rate of internalization of the pMHC-TCR complex into T cells;
- (c) partitioning the T cell and a plurality of nucleic acid barcode molecules comprising a cell barcode sequence into a partition;
- and
- (d) generating a barcoded nucleic acid molecule comprising (i) the reporter sequence or a reverse complement thereof and (ii) the cell barcode sequence or a reverse complement thereof.
6 more elements
A system for detection of binding of a peptide to a T cell comprising:
- (a) a peptide-MHC conjugate (pMHC) complex comprising an MHC monomer and a peptide, and wherein the MHC monomer comprises a reporter polynucleotide covalently attached thereto, wherein the reporter polynucleotide comprises a reporter sequence that identifies the peptide of the pMHC;
- and
- (b) a pMHC-TCR stabilization agent comprising:
- (i) a chemical fixative;
- (ii) an agent configured to reduce diffusion of the pMHC monomer from a TCR;
- and/or
- (iii) an agent capable of retaining and/or increasing the rate of internalization of the pMHC-TCR complex into a T cell.
4 more elements
Dependencies are inferred from the claim text.