Method of identifying CD4+ T cell antigens

1. Method of identifying CD4+ T cell antigens comprising the steps of:

a) providing an antigen encoding nucleic acid from tissue of patients and obtaining antigenic fragments of said nucleic acid, wherein the fragments have a size ranging from about 25 to about 300 bp;

b) expressing said fragments as a fusion protein comprising said fragment and a marker in a suitable host cell, wherein the marker is chloramphenicol acetyltransferase (CAT);

c) contacting said fusion protein expressing cell with antigen presenting cells (APCs) and co-cultivating with antigen-specific CD4+ T cells;

d) determining whether the CD4+ T cells are activated by said fusion protein expressing cells; and

e) identifying the antigen fragments which activate the antigen-specific CD4+ T cells by sequence analysis.

2. The method of claim 1 , wherein the fragments are obtained by cleaving the antigen encoding nucleic acid with one or more frequently cutting restriction enzymes or digested with nucleases.

3. The method of claim 2 , wherein the restriction enzymes are selected from the group consisting of AciI, MnlI, BsaJI and CviJI.

4. The method of claim 2 , wherein the nuclease is DNAseI.

5. The method of claim 1 , wherein the fragments have a size ranging from about 40 to 200 bp.

6. The method of claim 1 , 2 or 5 , wherein said fragments are expressed as a fusion protein by introducing an expression vector, containing the nucleic acid sequence coding for the fragment and a marker, into the host cell.

7. The method of claim 6 , wherein the expression vector is a plasmid.

8. The method of claim 1 , wherein the host cell is selected from prokaryotic and lower eukaryotic cells.

9. The method of claim 8 , wherein the lower eukaryotic cells are yeast cells.

10. The method of claim 8 , wherein the prokaryotic cells are bacterial cells.

11. The method of claim 1 , wherein the nucleic acid coding for the antigen is cDNA obtained by transcribing mRNA isolated from the antigen-expressing cells or tissue into cDNA.

12. The method of claim 1 , wherein the APCs are selected from dendritic cells, macrophages and lymphocytes.

13. The method of claim 12 , wherein the lymphocytes are from a lymphoblastoid cell line (LCL).

14. The method of claim 1 , which further comprises expanding host cells recognized by antigen specific CD4+ T cells.

15. The method of claim 1 , wherein the activation of said CD4+ T cells is measured by determining cytokine secretion or proliferation.

16. The method of claim 1 , wherein the antigen-specific CD4+ T cells are isolated by in vitro stimulation of autologous stimulator cells, wherein the CD4+ T cells are isolated from peripheral blood, secondary lymphoid organs or tissue of patients.

17. The method of claim 16 , wherein the secondary lymphoid organs are lymph nodes.

18. The method of claim 16 , wherein the tissue of patients comprises tumor explants in the case of tumor patients or inflamed tissues in cases of autoimmunity or infection.

19. A method for selecting CD4+ T cell antigen variants comprising the steps of identifying a CD4+ T cell antigen by the method of claim 1 and selecting antigen variants with improved or diminished T cell stimulatory capacity compared to said CD4+ T cell antigen.

20. The method of claim 1 , wherein the tissue of patients consists of tumor explants in the case of tumor patients or inflamed tissues in cases of autoimmunity or infection.

21. The method of claim 1 , wherein the method provides for the identification of alternative open reading frames coding for CD4+ T cell antigens.

22. Method of identifying CD4+ T cell antigens comprising the steps of:

a) providing an antigen encoding nucleic acid from tissue of patients and obtaining antigenic fragments of said nucleic acid, wherein the fragments have a size of about 90 bp;

b) expressing one or more of said fragments as a fusion protein comprising said fragment and a marker in a suitable host cell, wherein the marker is CAT;

c) contacting said fusion protein expressing cell with antigen presenting cells (APCs) and co-cultivating with antigen-specific CD4+ T cells;

d) determining whether the CD4+ T cells are activated by said fusion protein expressing cells; and

e) identifying the antigen fragments which activate the antigen-specific CD4+ T cells by sequence analysis.