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Volume 9 Supplement 2

AIDS Vaccine 2012

Evaluation of the dual IFNγ/IL-2 fluorospot-assay with flow cytometry for detection of HLA-restricted HIV-specific T-cell responses in HIV controllers


The IFNγ ELISpot assay is used widely for high through-put screening of HIV-specific responses in studies of HIV infection and vaccine studies. However, dual production of IFNγ/IL-2 and increased proliferative capacity may be associated with better natural control of HIV infection. We evaluated a novel fluorospot assay enabling the identification of dual IFNγ/IL-2 producing antigen-specific cells and compared it with intracellular cytokine staining by standard flow cytometry in individuals with natural control of HIV-infection.


PBMC from five untreated HIV-infected individuals were stimulated overnight with HIV peptides or controls in dual IFNγ/IL-2 pre-coated plates. Peptide arrays were specifically selected based on the individual HLA type. Secreted IFNγ and IL-2 were detected using fluorescent-conjugated antibodies. Fluorescent spots were enumerated on the iSpot AID reader. Responses were considered positive if >50 spots/million cells SFU after background subtraction.Positive responses were then evaluated by flow cytometry using the Gallios flow cytometer.


Dual IL-2/IFNγ producing cells were detected to anti-CD3-stimulated PBMC from all patients. IFNγ responses alone were detected to 35 of 136 HLA-restricted peptides tested (median =73, 52-4190 SFU) across the 5 patients (1/19, 5/19, 7/28, 9/15 and 13/55 for each patient), while IL-2 responses were either low grade or undetectable for the majority of HIV peptides tested. Dual IFNγ/IL-2 producing HIV-specific T cells were not detected using the fluorospot assay. 24/35 peptides induced CD8 T cell-IFNγ production by flow cytometry.


HIV-specific mono-IFNγ responses were detected using the novel fluorospot assay. However limited HIV-specific dual IFNγ/IL-2 responses were detected in this patient group. A greater number of epitope-specific positive responses were detected in the fluorospot compared with flow cytometry suggesting the fluorospot may be more sensitive in detecting a greater breadth of epitope-specific T cell responses, and therefore better for screening purposes than flow cytometric methods.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Keane, N., Almeida, C., Roberts, S. et al. Evaluation of the dual IFNγ/IL-2 fluorospot-assay with flow cytometry for detection of HLA-restricted HIV-specific T-cell responses in HIV controllers. Retrovirology 9 (Suppl 2), P243 (2012).

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  • Natural Control
  • Intracellular Cytokine Staining
  • Peptide Array
  • Flow Cytometric Method
  • Standard Flow Cytometry