T-cell reactivity platform

Abstract

Methods for assaying antigen-specific T-cell activation in vitro, comprising the steps of (a) providing a phagocytable particle, having a candidate antigen polypeptide tightly associated thereto, wherein the particle with the associated polypeptide has been subjected to a denaturing wash resulting in an endotoxin level low enough to not interfere with the subsequent steps; (b) providing a viable antigen-presenting cell; (c) contacting the washed particle with the antigen-presenting cell under conditions allowing phagocytosis of the particle by the antigen-presenting cell; (d) providing a T-cell sample to be assayed comprising viable T-cells; (e) contacting the T-cell sample with the antigen-presenting cell contacted with the particle under conditions allowing specific activation of T-cells in response to an antigen presented by an antigen-presenting cell; and (f) determining the degree of T-cell activation in the T-cell sample.

1 - 34 . (canceled). 
     
     
         35 . A method for assaying antigen-specific T-cell activation, comprising the steps of:
 a. providing a phagocytable particle, having a candidate antigen polypeptide tightly associated thereto, wherein the particle with the associated polypeptide has been subjected to a denaturing wash resulting in an endotoxin level low enough to not interfere with steps b-f;   b. providing a viable antigen-presenting cell;   c. contacting the washed particle with the antigen-presenting cell in vitro under conditions allowing phagocytosis of the particle by the antigen-presenting cell;   d. providing a T-cell sample to be assayed, the T-cell sample comprising viable T-cells;   e. contacting the T-cell sample with the antigen-presenting cell contacted with the particle in vitro under conditions allowing specific activation of T-cells in response to an antigen presented by an antigen-presenting cell; and   f. determining the degree of T-cell activation in the T-cell sample.   
     
     
         36 . A method for assaying antigen-specific T-cell activation, comprising the steps of:
 a. providing a phagocytable particle;   b. tightly associating a candidate antigen polypeptide to the particle;   c. subjecting the particle with the associated polypeptide to a denaturing wash resulting in an endotoxin level low enough to not interfere with steps d-h;   d. providing a viable antigen-presenting cell;   e. contacting the washed particle with the antigen-presenting cell in vitro under conditions allowing phagocytosis of the particle by the antigen-presenting cell;   f. providing a T-cell sample to be assayed, the T-cell sample comprising viable T-cells;   g. contacting the T-cell sample with the antigen-presenting cell contacted with the particle in vitro under conditions allowing specific activation of T-cells in response to an antigen presented by an antigen-presenting cell; and   h. determining the degree of T-cell activation in the T-cell sample.   
     
     
         37 . The method according to  claim 35 , further comprising the step of comparing the degree of T-cell activation to a relevant reference, whereby a higher degree of T-cell activation in the sample compared to the reference indicates that the candidate antigen results in antigen-specific T-cell activation in the sample. 
     
     
         38 . The method according to  claim 35 , wherein determining the degree of T-cell activation in the T-cell sample comprises determining the fraction of activated T-cells in the sample. 
     
     
         39 . The method according to  claim 35 , wherein several candidate antigens are assayed against the same T-cell sample. 
     
     
         40 . The method according to  claim 39 , wherein at least 10 candidate antigens are assayed against the same T-cell sample. 
     
     
         41 . The method according to  claim 35 , wherein the antigen-presenting cell and the T-cell sample are derived from a single individual. 
     
     
         42 . The method according to  claim 41 , wherein the antigen-presenting cell and the T-cell sample are derived from the same blood sample from the individual. 
     
     
         43 . The method according to  claim 41 , wherein the antigen-presenting cell and the T-cell sample are derived from a PBMC-sample from the individual. 
     
     
         44 . The method according to  claim 35 , wherein the T-cell sample is derived from a tumour. 
     
     
         45 . The method according to  claim 35 , wherein the T-cell sample is derived from ascites. 
     
     
         46 . The method according to  claim 35 , wherein the T-cell sample comprises CD4+ T-cells, CD8+ T-cells, or both. 
     
     
         47 . The method according to  claim 35 , wherein the washed particle, the antigen presenting cell and the T-cell sample are contacted concurrently. 
     
     
         48 . The method according to  claim 35 , wherein determining the degree of T-cell activation in the T-cell sample comprises determining the fraction of T-cells contacted with the antigen-presenting cell responding by secretion of IFN-γ, IL-17, IL-22, or combinations thereof. 
     
     
         49 . The method according to  claim 35 , wherein determining the degree of T-cell activation in the T-cell sample comprises determining the fraction of T-cells contacted with the antigen-presenting cell responding by secretion of IL-17, IL-22, or both. 
     
     
         50 . The method according to  claim 35 , wherein the candidate antigen polypeptide comprises at least 50 amino acids. 
     
     
         51 . The method according to  claim 35 , wherein the T-cell sample is from a human and the candidate antigen polypeptide sequence is derived from a human. 
     
     
         52 . The method according to  claim 35 , wherein the candidate antigen polypeptide is derived from a polypeptide:
 a. known to be highly expressed in a tissue or cell affected in an autoimmune disease;   b. known to be associated with a neoplastic disease;   c. known to be associated with an autoimmune disease;   d. known to be associated with an infectious disease; or   e. known to be associated with allergy or similar hypersensitivity.   
     
     
         53 . The method according to  claim 35 , comprising the steps of:
 a. providing a phagocytable particle, having a candidate peptide auto-antigen tightly associated thereto, wherein the particle with the associated polypeptide has been subjected to a denaturing wash resulting in an endotoxin level low enough to not interfere with steps b-e;   b. providing a peripheral blood mononuclear cell (PBMC) sample from a test subject, the PBMC sample comprising viable T-cells and viable antigen-presenting cells;   c. contacting the PBMC sample with the particle in vitro under conditions allowing phagocytosis of the particle by an antigen-presenting cell and allowing specific activation of T-cells in response to an antigen presented by an antigen-presenting cell;   d. quantitating the fraction of activated T-cells in the contacted PBMC sample; and   e. comparing the quantitated fraction to a comparable quantitated fraction from a healthy subject, wherein a higher fraction of activated T-cells in the test subject as compared to the healthy subject is indicative of autoreactivity against the candidate auto-antigen in the test subject.