Intracellular osteopontin regulates the lineage commitment of lymphoid subsets

Abstract

Methods for diagnosing and prognosing autoimmune diseases and T cell lymphomas are provided, for example by measuring levels of intracellular osteopontin (OPN-i). Also provided are screening methods for identifying activators and inhibitors of the transcription factor Bcl6, which is involved in T cell activation/differentiation. Other aspects of the disclosure provide methods for enhancing adoptive T cell transfer.

We claim: 
     
         1 . A method comprising:
 selecting a subject suspected of having an autoimmune disease or T cell lymphomas; measuring expression level of intracellular osteopontin (OPN-i) in a follicular helper T (TFH) cells sample obtained from the subject.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), psoriasis, multiple sclerosis, Crohn's disease, inflammatory bowel disease (IBD), asthma, rheumatoid arthritis, and psoriatic arthritis. 
     
     
         4 . The method of  claim 3 , wherein the autoimmune disease is SLE. 
     
     
         5 - 7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the follicular helper T (TFH) cells are isolated from peripheral blood mononuclear cells (PBMC). 
     
     
         9 . The method of  claim 8 , wherein the follicular helper T (TFH) cells are isolated using immunofluorescence or fluorescence activated cell sorting (FACS). 
     
     
         10 . The method of  claim 1 , wherein OPN-i mRNA or protein expression level is measured. 
     
     
         11 . The method of  claim 10 , wherein OPN-i mRNA expression level is measured using quantitative RT-PCR. 
     
     
         12 . The method of  claim 10 , wherein OPN-i protein expression level is measured using Western blot or enzyme-linked immunosorbent assay (ELISA). 
     
     
         13 . The method  claim 1 , further comprising: measuring expression level of inducible costimulator (ICOS) receptor in the follicular helper T (TFH) cells sample. 
     
     
         14 . A method for identifying Bcl6 inhibitors or Bcl6 activators comprising:
 (a) combining regulatory p85α subunit of phosphatidylinositol-3-OH kinase or a fragment thereof with OPN-i or fragment thereof in presence or absence of a test compound;   (b) labelling p85α or fragment thereof with a fluorescence donor and labelling OPN-i or fragment thereof with a fluorescent acceptor, wherein binding of OPN-i to p-85α is detected by proximity-based luminescence detection; and   (c) identifying the test compound as a Bcl6 inhibitor when the proximity-based luminescence detection signal is decreased in the presence of the test compound relative to the signal in the absence of the test compound; or   identifying the test compound as a Bcl6 activator when the proximity-based luminescence detection signal is increased in the presence of the test compound relative to the signal in the absence of the test compound.   
     
     
         15 - 39 . (canceled) 
     
     
         40 - 50 . (canceled) 
     
     
         51 . A method for identifying Bcl6 inhibitors or Bcl6 activators comprising:
 (a) combining OPN-i or a fragment thereof with Bcl6 RD2 domain in presence or absence of a test compound;   (b) labelling OPN-i or fragment thereof with a fluorescence donor and labelling Bcl6 RD2 domain with a fluorescent acceptor, wherein binding of OPN-i to Bcl6 RD2 domain is detected by proximity-based luminescence detection; and   (c) identifying the test compound as a Bcl6 inhibitor when the proximity-based luminescence detection signal is decreased in the presence of the test compound relative to the signal in the absence of the test compound; or
 identifying the test compound as a Bcl6 activator when the proximity-based luminescence detection signal is increased in the presence of the test compound relative to the signal in the absence of the test compound. 
   
     
     
         52 - 61 . (canceled) 
     
     
         62 . A method for identifying Bcl6 inhibitors comprising:
 (a) combining cells expressing fluorescently labelled Bcl6 fusion protein and p85α subunit with OPN-i or fragment thereof in the presence or absence of a test compound; and   (b) identifying the test compound as a Bcl6 inhibitor when fluorescence signal is decreased in the presence of the test compound relative to the signal in the absence of the test compound.   
     
     
         63 . A method for identifying Bcl6 modulators comprising:
 (a) combining OPN-i or a fragment thereof with Bcl6 RD2 domain in presence or absence of a test compound, wherein binding of OPN-i to Bcl6 RD2 domain is detected by ELISA-based assay; and   (b) identifying the test compound as a Bcl6 modulator when the ELISA signal is decreased or increased in the presence of the test compound relative to the signal in the absence of the test compound.   
     
     
         64 . A method of enhancing adoptive T cell transfer in a subject, said method comprising
 isolating CD4+ T cells from peripheral blood from a subject in need thereof; transducing the isolated CD4+ T cells by contacting the CD4+ T cells with retroviral vectors expressing OPN-i;   expanding the transduced CD4+ T cells by growing them in a culture medium until the number of transduced CD4+ T cells increases by at least 5%; and   administering the expanded transduced CD4+ T cells to the subject.   
     
     
         65 - 67 . (canceled) 
     
     
         68 . A method of enhancing adoptive T cell transfer in a subject, said method comprising
 isolating CD4+ T cells from peripheral blood from a subject in need thereof;   treating the isolated CD4+ T cells with cell-permeable OPN-i or fragments thereof;   expanding the treated CD4+ T cells by growing them in a culture medium until the number of treated CD4+ T cells increases by at least 5%; and   administering the expanded treated CD4+ T cells to the subject.   
     
     
         69 - 72 . (canceled)