This study has developed a quick and efficient means of measuring antigen-specific functional CD8+
T cells from patients irrespective of their HLA types. Qualitatively and quantitatively measuring antigen-specific CD8+
T cells is crucial in monitoring the immune status during disease and in assessing treatment efficacy. Conventional assays have analyzed the bulk population of T cells for cytotoxicity by 51
Cr release assays. This method requires the expansion of the antigen-specific T-cell population in vitro. Researchers have used primary B-lymphoblastoid cells transformed with an Epstein-Barr virus as the autologous stimulators and/or targets, which are either pulsed with specific peptides (37
) or infected with a recombinant vaccinia virus encoding the antigens (23
). The procedures are complicated, and the results usually cannot be compared quantitatively.
Some studies used HLA-matched allogenic cell lines to express a specific antigen as the stimulator and/or target of CTLs (6
). Although this method is more convenient, its use is limited to patients with specific HLA type, so screening of patients before the CTL assays is necessary. The strong alloreaction also often influences the levels of CTL activities, and the results are difficult to analyze.
A novel method was recently developed, using synthetic HLA-peptide tetrameric complexes to directly quantitate antigen-specific CTLs (1
). This method can provide quantitative readouts, since it enumerates antigen-specific T cells without a lengthy in vitro restimulation. The tetramer technique, however, can only identify T cells with single peptide-MHC specificity (18
). The patients suitable for study are limited, and numerous peptides corresponding to various predicted epitope motifs have to be synthesized. Also, the tetramers measure antigen specificity without regard to function. Since some T cells in vivo may represent anergic populations, the use of a functional assay may still be necessary. The enzyme-linked immunospot assay (ELISPOT) and intracellular cytokine assays can also be used to enumerate antigen-specific T cells without lengthy in vitro expansion. However, in contrast to tetramers, both methods measure a functional readout (cytokine [e.g., IFN-γ] production). A major advantage of intracellular cytokine assays over ELISPOT is the ability to concurrently analyze multiple parameters, e.g., CD4, CD8, and activation markers, from every single cell.
This study took advantage of intracellular IFN-γ assays and successfully measured core-specific CD8+
T-cell responses from the peripheral blood of chronically HCV-infected patients undergoing treatment with IFN-α plus ribavirin, by simply using the gene-transduced, autologous PBMCs as stimulators. However, attempts to use the same stimulators to expand core-specific T cells from the PBMCs for standard CTL assays have been less successful (data not shown). The results indicate that inducing peripheral T-cell precursors to proliferate and enriching them to certain levels are technically harder, and often take longer, than directly detecting IFN-γ expression from antigen-activated T cells. Since IFN-γ staining assays analyzed all of the CD8+
T cells that recognized the endogenous peptides presented by various types of MHC class I molecules, the percentages of antigen-specific T cells were expected to be higher than those measured from one specific MHC-peptide. Moreover, the antiviral treatments may also contribute to the higher rate of T cell responses in these patients (13
). Using the autologous PBMCs as targets of CTL assays, our data demonstrated that the HCV core-specific CTLs could be detected by non-antigen-specific stimulation from the liver (Fig. ) but not from the peripheral blood (data not shown). Although the percentages of specific lysis are not particularly high, the low background cytolysis when using autologous targets renders the killing activities on specific targets significantly different from those on nonspecific targets (P
< 0.001). The results are consistent with previous findings (22
) indicating a higher frequency of antigen-specific CTLs in the HCV-infected liver than in the peripheral blood.
Recently, Wong et al. (41
) reported the detection of CTLs from the peripheral blood of chronically HCV-infected patients using Epstein-Barr virus-transformed B-lymphoblastoid cells as autologous stimulators, which were infected with a recombinant vaccinia virus encoding the entire translated proteins of HCV. The method they used also eliminates the need for HLA typing. The advantage of this method is its ability to detect a wider scope of, and probably stronger, T-cell responses against various HCV antigens, which thus enabled those authors to easily identify CTLs in the PBMCs from seven of nine patients. However, since standard 51
Cr release assays were used in that study, a lengthy in vitro stimulation was unavoidable. The CTL responses measured might reflect only the antigen-specific cells that survived during the incubation period rather than the true magnitude of the immune responses present in the PBMCs. In this regard, intracellular IFN-γ staining assays provide faster, more sensitive, and less biased measurements of the CD8+
T-cell responses in the PBMCs of HCV-infected patients. A potential problem with the system described here is that to assess the immune responses against the entire proteins of HCV, several VSV-G-pseudotyped retroviral vectors need to be constructed.
The immune responses against the HCV core antigens of different individuals receiving treatment with IFN-α plus ribavirin treatment varied to a great extent (Fig. ). This study initially evaluated the feasibility of measuring functional immune responses with the use of autologous PBMCs, so the CD8+ responses were measured at random time points during the treatment of patients. Whether the heterogeneity in CD8+ T-cell responses represents a prognosis indicator is therefore unclear. To draw a conclusive answer regarding the CTL responses and treatment efficacy, it would be better to longitudinally monitor individual patients receiving the treatments. A new study has been initiated which will recruit more patients and systematically monitor their CTL responses before, during, and after the treatments. The possibility that the less reactive T-cell responses might come from patients infected with different genotypes of HCV cannot be excluded. Since the sequences of HCV core antigen are quite conserved among different genotypes of HCV, however, this is unlikely.
The CTL responses to HCV infection are multispecific. This study investigated only the core-specific CD8+ T-cell responses. It could certainly be extended to examine other HCV antigens or other virus infections, for measuring the CTL responses in individuals of any HLA types. The method also provides a valuable reference for the study of cancer immunology if the tumor-associated antigens are known. The direct quantitation of HCV-specific CD8+ T cells in different patients in different clinical settings, in conjunction with their virological, biochemical, and histological analysis, should provide further insight into viral pathogenesis and clarify the mechanisms of clearance or persistence of HCV.