The intracellular detection of MIP-1beta enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell responses in a flow cytometric setting providing a sensitive alternative to the ELISPOT

Abstract

Background: T-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN--based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flowcytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level.

Longitudinal changes in HIV-1-specific T-cell quality associated with viral load dynamic

“…12 Analysis was performed using FlowJo version 8.8.6 (Tree Star, Ashland, OR). According to the differential expression of CD45RA, CD154, IFN-␥, IL-2 and MIP-1␤, 30 responding CD4 and CD8 T-cell subpopulations were identified.…”

“…A threshold level for each subpopulation was calculated following background subtraction. 12 A general threshold of 40 cell counts was applied for all qualitative CD8 and CD4 T-cell analyses to exclude minor responses.…”

Comparison of Immunologic Assays for Detecting Immune Responses in HIV Immunotherapeutic Studies: AIDS Clinical Trials Group Trial A5181

“…In HIV-1 immune-based therapy trials, the standard assays have included the ELISPOT, LP, CP, and ICC assays. Of these, the ELISPOT assay has been considered the gold standard in a number of vaccine trials because of its sensitivity and extensive validation (18,20). However, although the ELISPOT assay is more sensitive than the ICC assay, the latter has the advantage of simultaneously identifying production of many different cytokines, chemokines, and cytotoxic factors (26).…”

La couleur du métal

“…Ces mêmes cellules CD8 + matures corrèlent avec la protection de l'infection chez les primates non humains vaccinés par un cytomégalovi-rus recombinant exprimant les protéines du VIH [7]. Enfin, dans le domaine de la vaccinologie, la cytométrie en flux remplace progressivement les méthodes classiques d'exploration de la réponse immune induite, comme les ELISpot [8]. L'intérêt croissant pour la cytométrie en flux est lié à la capacité d'étudier des paramètres multiples sur des millions de cellules analysées individuellement.…”