HIV-Specific Ts and CTLA-4 Figure 4. HIV-specific TGF-b and IL-10 positive CD8+ T cells are CTLA-4 negative. PBMC were stimulated with HIV peptides then stained with anti-TGF-b PE (or IL-10 PE), anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, anti-CD8 PE Cy7, anti-CTLA-4 APC, and analyzed by flow cytometry. Gating on the CTLA-4 positive cells was performed using the fluorescence-minus-one (FMO) control for CTLA-4. (A) Representative plots of samples that were first gated on the CD3+/CD4+ and CD3+/ CD8+ lymphocyte population and then the percentages of CTLA-4 positive cells were determined. (B) Representative plots of samples that were first gated on the CD3+/CD8+ lymphocyte population and then the percent of TGF-b and IL-10 positive cells that express CTLA-4 was determined after subtraction of the back ground values. The values marked with an asterisk represent the fraction of TGF-b (or IL-10) positive cells that express CTLA-4 over the total number of TGF-b (or IL-10) positive cells (equivalent to 100%). Plots are from three independent experiments yielding similar results. doi:10.1371/journal.pone.0008194.g004 Further studies should focus on the pathways by which complex immune regulatory mechanisms modulate the adaptive, antiviral immune response. The ability to manipulate and regulate effective References 1. Scotto L, Naiyer AJ, Galluzzo S, Rossi P, Manavalan JS, et al. (2004) Overlap between molecular markers expressed by naturally occurring CD4+CD25+ regulatory T cells and antigen specific CD4+CD25+ and CD8+CD28- T suppressor cells. Hum Immunol 65: 1297–306. 2. Elrefaei M, Ventura FL, Baker CA, Clark R, Bangsberg DR, et al. (2007) HIV- specific IL-10-positive CD8+ T cells suppress cytolysis and IL-2 production by CD8+ T cells. J Immunol 178: 3265–71. 3. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA (2001) CD4+CD25high regulatory cells in human peripheral blood. J Immunol 167: 1245–53. 4. Cosmi L, Liotta F, Lazzeri E, Francalanci M, Angeli R, et al. (2003) Human CD8+CD25+ thymocytes share phenotypic and functional features with CD4+CD25+ regulatory thymocytes. Blood 102: 4107–14. 5. Levings MK, Sangregorio R, Sartirana C, Moschin AL, Battaglia M, et al. (2002) Human CD25+CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells. J Exp Med 196: 1335–46. Figure 5. Removal of CD4+ T cells abrogates the effects of CTLA-4 blockade on HIV-specific CD8+ T Cells. PBMC (or CD4 negative PBMC) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-c FITC, anti- TGF-b PE, IL-10 APC, anti-CD3 Am Cyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-b, IL- 10, and IFN-c positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-b, IL-10, or IFN-c after subtraction of the back ground. Representative plots of (A) Gag and (B) Nef-specific CD8+ T cells expressing TGF-b, IL-10, or IFN-c in the presence or absence of anti-CTLA-4. Data plots shown are representa- tive of three volunteers examined in three independent experiments yielding similar results. doi:10.1371/journal.pone.0008194.g005 antiviral immunity provides an approach toward decreasing the number of infected cells and improving disease outcome. Author Contributions Conceived and designed the experiments: ME HC. Performed the experiments: ME CMB. Analyzed the data: ME. Contributed reagents/ materials/analysis tools: CMB CAB NGJ SB DRB HC. Wrote the paper: ME HC. 6. Weiner HL (2001) Induction and mechanism of action of transforming growth factor-beta-secreting Th3 regulatory cells. Immunol Rev 182: 207–14. 7. Balashov KE, Khoury SJ, Hafler DA, Weiner HL (1995) Inhibition of T cell responses by activated human CD8+ T cells is mediated by interferon-gamma and is defective in chronic progressive multiple sclerosis. J Clin Invest 95: 2711–9. 8. Filaci G, Bacilieri S, Fravega M, Monetti M, Contini P, et al. (2001) Impairment of CD8+ T suppressor cell function in patients with active systemic lupus erythematosus. J Immunol 166: 6452–7. 9. Filaci G, Fravega M, Fenoglio D, Rizzi M, Negrini S, et al. (2004) Non-antigen specific CD8+ T suppressor lymphocytes. Clin Exp Med 4: 86–92. 10. Filaci G, Fravega M, Negrini S, Procopio F, Fenoglio D, et al. (2004) Nonantigen specific CD8+ T suppressor lymphocytes originate from CD8+CD28- T cells and inhibit both T-cell proliferation and CTL function. Hum Immunol 65: 142–56. 11. Kumar V (2004) Homeostatic control of immunity by TCR peptide-specific Tregs. J Clin Invest 114: 1222–6. PLoS ONE | www.plosone.org 6 December 2009 | Volume 4 | Issue 12 | e8194