Cell-based microarrays and methods of use
View Patent ↗The invention provides methods and compositions for rapid, sensitive, and highly specific detection of antigen-specific interactions between cytolytic T lymphocytes (CTLs) and antigen presenting cells (APCs). The invention also features compositions, including kits, for use in the methods of the invention.
1. A method for identifying a cytolytic T lymphocyte (CTL) antigen, the method comprising:
contacting a sample comprising a cytolytic T lymphocyte (CTL) with an array comprising a plurality of adherent recombinant antigen-presenting cells (APCs) adhered to a surface of the array at different, discrete locations on the array, wherein the adherent recombinant APCs express different recombinant polynucleotides encoding different polypeptides, and wherein the different, discrete locations on the array correlate with the different recombinant polynucleotides expressed by the adherent recombinant APCs;
washing the array; and
detecting the presence or absence of caspase activity in the plurality of adherent recombinant APCs by detecting the presence of a fluorescent signal generated from a fluorogenic caspase substrate present in said adherent recombinant APCs;
wherein the presence of a fluorescent signal in an adherent recombinant APC is indicative of an antigen-specific interaction between the CTL and the adherent recombinant APC and indicates the recombinant polynucleotide of the adherent recombinant APC encodes a polypeptide that comprises a CTL antigen.
2. The method of claim 1 , wherein the recombinant polynucleotides encode a tumor antigen.
3. The method of claim 1 , wherein the recombinant polynucleotides encode an antigen of an intracellular pathogen.
4. The method of claim 3 , wherein the antigen is a viral antigen, bacterial antigen, antigen of a parasite, or fungal antigen.
5. The method of claim 1 , wherein the recombinant polynucleotides encode an autoantigen.
6. The method of claim 1 , wherein the fluorogenic caspase substrate is a fluorogenic multi-caspase substrate.
7. The method of claim 1 , wherein the CTL is in a biological sample obtained from a subject.
8. The method of claim 1 , wherein the CTL is a CTL clone.
9. The method of claim 1 , wherein said contacting is for about 1 to 4 hours.
10. The method of claim 1 , wherein said contacting is for about 1 hour.
11. The method of claim 1 , wherein the adherent recombinant APCs were produced by plating the APCs onto an array surface comprising the recombinant polynucleotides deposited on an array location, wherein said plating is under appropriate conditions for adherence of the APCs to the array surface and introduction of the recombinant polynucleotide into the adherent APC, and wherein the array location at which the recombinant polynucleotide was deposited corresponds to the location of the adherent APC containing the recombinant polynucleotide.
12. The method of claim 1 , wherein the adherent recombinant APC contains a recombinant polynucleotide encoding a polypeptide fragment.
13. The method of claim 12 , wherein the polypeptide fragment provides a T cell antigen that is presented in Class I MHC for recognition by a CTL specific for that antigen.