Lack of Disease Specificity Limits the Usefulness of In Vitro Costimulation in HIV- and HCV-Infected Patients

Abstract

Measurements of antigen-specific T cell responses in chronic diseases are limited by low frequencies of antigen-specific cells in the peripheral blood. Therefore, attempts have been made to add costimulatory molecules such as anti-CD28 or IL-7/IL-15 to ELISPOT assays to increase sensitivity. While this approach has been successful under certain circumstances, results are often inconsistent. To date, there are no comprehensive studies directly comparing the in vitro effects of multiple costimulatory molecules in different disease settings. Therefore, in the present study we tested the effects of IL-7/IL-15, IFN-α, anti-ICOS, and anti-CD28 on antigen-specific T cell responses in patients infected with HCV or HIV versus healthy individuals. Our data show that none of the aforementioned molecules could significantly increase ELISPOT sensitivity, neither in HCV nor in HIV. Moreover, all of them caused false-positive responses to HCV and HIV antigens in healthy individuals. Our results question the broad use of in vitro costimulation.

Normal Distribution of CD8+ T-Cell-Derived ELISPOT Counts within Replicates Justifies the Reliance on Parametric Statistics for Identifying Positive Responses

“…The PBMC were tested at 1, 2, 4 and 8 × 10 5 cells per well, with 12 replicates for each cell number. Panels A and B in Figure 5 show the responses of Donors 1 and 2, respectively, with and without added human recombinant IL-7 as an assay enhancer in the CTL Test Plus™ medium [ 20 ]. When 12 replicates were used, both donors had a detectable IFN-γ response to BMLF1 (259 – 267), over the medium control background.…”

“…The antigens were dissolved in CTL-Test™ medium (CTLT-005). In a previous work, we observed that adding IL-7 can improve the signal-to-noise performance of ELISPOT assays [ 20 ]. Therefore, where specified, the IL-7-supplemented CTL-Test Plus™ medium was used (CTLTP-005).…”