Materials

1. Lymphoprep (Axis-Shield, Oslo, Norway).

2. RPMI 1640 (Euroclone Life Sciences Division, Pero, Italy).

3. Fetal bovine serum (FBS) (Cambrex, East Rutherford, NJ).

4. Penicillin/streptomycin (Bristol-Myers Squibb, Sermoneta, Italy).

5. l-glutamine (Life Technologies, Milan, Italy).

6. FACS antibodies: anti-CD4 FITC conjugated, anti-CD25 PE conjugated, anti-CD69 FITC conjugated, anti-CD40L FITC conjugated, anti-CD28 PE conjugated (BD Bioscience, San Diego, CA), anti-GITR monoclonal antibody (MAb) (R&D Systems, Minneapolis, MN), anti-CTLA-4 biotin conjugated (BD Bioscience), antihuman Foxp3 PE conjugated (clone PCH 101) (e-bioscience, San Diego, CA).

9. CD4 T cell isolation kit II (Miltenyi Biotech, Bergisch Gladbach, Germany).

10. CD25 microbeads (Miltenyi Biotech).

11. CD45RO microbeads (Miltenyi Biotech).

12. MACS buffer: PBS pH 7.2, 2 mM EDTA, 0.5% human serum (Cambrex).

13. PHA (Roche Diagnostic, Roche Applied Science, Mannheim, Germany).

14. Proleukin (IL-2) (Chiron Corporation, Emeryville, CA).

15. [3H]-thymidine deoxyribose ([3H]-TdR) (Amersham Biosciences, Uppsala, Sweden).

16. Anti-CD3 MAb (clone OKT3) (Orthoclone Janssen-Cilag, Cologno Monzese, Italy).

17. Anti-CD28 MAb (clone V5T CD28.05) (BD Bioscience).

18. Th1/Th2 Cytometric bead array (CBA) (BD Bioscience).

19. Trypsin-EDTA (Life Technologies).

20. Recombinant human IL-15 (rhIL-15) (R&D Systems).

21. Recombinant human IL-10 (rhIL-10) (BD Bioscience).

22. Recombinant human IFN (rhIFN)-a (PeproTech, Rocky Hill, NJ).

23. Recombinant human IL-4 (rhIL-4) (R&D Systems).

24. Recombinant human IL-12 (rhIL-12) (R&D Systems).

25. Anti-human IL-4 MAb (clone MP4-25D2) (BD Bioscience).

26. Anti-human IL-12 MAb (clone C8.6) (BD Bioscience).

27. CD3, CD8, CD14 and CD19 beads for depletion (Dynal, Oxoid, Milan, Italy).

28. 2-Mercaptoethanol (Bio-Rad Laboratories, CA).

29. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) (Immunotools, Friesoythe, Germany).

30. Tetanus Toxoid (Calbiochem, Bioscience, Inc., La Jolla CA; Alexis Biochemicals, Switzerland).

31. Paraformaldehyde (Fluka Chemicals AG, Switzerland).

32. Saponin (Sigma Aldrich CO, St. Louis MO).

33. Brefeldin A (Sigma).

34. PE-labeled anti-hIL-4, anti-hIL-2, or anti-hIL-10, and FITC-coupled anti-hIFN-y (BD Bioscience).

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