US 12,449,419 B1
Methods for detecting binding of peptide-MHC monomers to T cells
Michael John Terry Stubbington, Pleasanton, CA (US); Luz Montesclaros, Pleasanton, CA (US); Katherine Pfeiffer, Oakland, CA (US); Sarah E. B. Taylor, Pleasanton, CA (US); and Alvaro Martinez Barrio, Pleasanton, CA (US)
Assigned to 10x Genomics, Inc., Pleasanton, CA (US)
Filed by 10X Genomics, Inc., Pleasanton, CA (US)
Filed on Feb. 10, 2021, as Appl. No. 17/172,868.
Claims priority of provisional application 62/975,295, filed on Feb. 12, 2020.
Int. Cl. C12N 9/22 (2006.01); B01L 3/00 (2006.01); G01N 1/30 (2006.01); G01N 33/569 (2006.01)
CPC G01N 33/56977 (2013.01) [C12N 9/22 (2013.01); G01N 1/30 (2013.01); B01L 3/502784 (2013.01); B01L 2200/0673 (2013.01); B01L 2300/0829 (2013.01); C12Y 301/22001 (2013.01); G01N 2001/305 (2013.01); G01N 2333/70539 (2013.01)] 20 Claims
OG exemplary drawing
 
1. 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.