The results of immunohistochemical studies have confirmed the presence of activated CD8 T cells in the liver, and the results of flow cytometric studies have shown a higher frequency of HCV-specific CD8 T cells in the liver than in blood of chronically infected subjects (8
). Most studies evaluating intrahepatic HCV-specific T cells rely on ex vivo expansion with either recombinant HCV proteins or HCV peptides or on nonspecific stimulation using phytohemagglutinin or anti-T-cell antibodies prior to analysis (14
). An alternate way to directly analyze unexpanded cells is through the use of MHC class I tetramers (8
). Although our group has used these reagents with some success (18
), this method requires the use of the majority of freshly isolated cells available from a liver biopsy specimen, and the low frequencies of these cells after virus is cleared are typically below the limit of detection by flow cytometry. Compounding these limitations are the lack of availability of all MHC class I alleles, the requirement that the epitopes to be evaluated need to be precisely known in order to synthesize tetramers, and the requirement that the flow analysis take place in real time on small numbers of freshly isolated cells. In this study, we used real-time PCR to perform a longitudinal analysis of T-cell clonotype expansion on frozen needle biopsy liver samples.
In both animals, the rapid resolution of viremia after rechallenge temporally coincided with massive expansion of the dominant memory T-cell clonotype, highlighting the importance of memory CD8+
T cells to the outcome of infection. While immunological memory conferred by the spontaneous resolution of acute hepatitis C does not protect against reinfection, it does significantly reduce the time of viremia upon reexposure. Here we show that despite the effective depletion of CD8+
T cells, each animal was able to clear virus, albeit at a lower rate. In a previous study (18
), we documented that viral clearance was associated with the return of a detectable number of CD8+
T cells in the periphery and with the ability to expand HCV-specific T cells from the liver and peripheral blood. Here we show that at the clonotype level, the number of HCV-specific T cells remaining after depletion was several logs lower in peripheral blood than in the liver. These cells persisted in the liver after rechallenge, and in the case of animal CB0556, the slower clearance of virus was associated with a smaller peak frequency of these clonotypes, followed by a gradual decay in frequency.
The inability of these clonotypes to expand robustly after HCV challenge in the setting of CD8+ depletion may be due to the persisting effect of the depleting antibody in these animals. In animal CB0556, the predepletion CD8+ T-cell number was 1,328 cells/mm3. Even though this animal cleared virus by day 42, the absolute number of CD8+ T cells was only 5 cells/mm3 by day 42, and 12 months after the depletion, CD8+ T cells in the periphery had only recovered to 634 cells/mm3. For animal CB0572, the predepletion CD8+ T-cell number was 1,073 cells/mm3. This animal had a CD8+ T-cell nadir of 1 CD8+ T cell/mm3 after antibody-mediated CD8+ T-cell depletion and also cleared virus completely by day 42, at which time the absolute CD8+ T-cell number was 146 cells/mm3. Twelve months after the depletion, CD8+ T cells had only recovered to 256 cells/mm3. We did not track the frequencies of total CD8+ T cells in the liver of these animals, but in other animals given the cM-T807 antibody with the same infusion protocol, there was a virtual absence of CD8 alpha and beta chain transcripts in the liver through day 56 after the first infusion (data not shown). Our continued ability to detect these epitope-specific T-cell clonotype transcripts in the liver suggests that they made up a significant fraction of the CD8+ T cells during peak viremia.
Despite the potential advantages of real-time PCR for tracking T-cell clonotypes, there are a few caveats to the interpretation of these data. Since we are measuring RNA transcripts, it is possible that activated T cells may generate more TCR transcripts per cell than resting memory cells, in which case this method would overestimate the actual T-cell frequency. However, our results tracked closely with the actual tetramer frequency in peripheral blood (1%, 0.58%, and 0.2% at week 4, 8, and 24, respectively) and the liver (4.4%, 3.4%, and 1.8% at week 4, 8, and 24, respectively) of chimpanzee CB0572 over the course of infection (18
). It is also possible that primer efficiencies can differ when primer selection is based on the limited number of nucleotides present within the CDR3 region of the TCR beta chain. For the four primers described here, this did not appear to be a significant problem, and the efficiency of amplification was equal to or greater than that of our TCR beta chain constant region primers. Furthermore, the hierarchy of TCR sequence frequency obtained from direct sorting of tet+
T cells was identical to that measured by real-time PCR (Fig. , , and ).
Tracking of individual T-cell clonotypes has been used in studies of immune-based neurological disorders (15
) and can be adapted to the study of T-cell clonotype expansion within peripheral blood or tissue from stored samples in any system where T cells are characterized at a time after the samples are obtained. Recent studies have characterized TCR clonotypes implicated in the pathogenesis of aplastic anemia (16
) and have assessed the repertoire and frequency of melanoma-specific T-cell clonotypes in peripheral blood and tumors after therapeutic vaccination (4
). TCR transcript quantitation in these studies was performed via limiting dilution PCR; however, real-time PCR allows a rapid simultaneous quantitative assessment of clonotypes in peripheral blood and tissues and does not rely on prior knowledge of epitope specificity.
In this study, we confirmed that the rapid expansion of virus-specific T cells occurs as quickly as 7 days postinfection, the kinetics of clonotype expansion and contraction coincide with the clearance of viremia, and the frequencies of HCV-specific clonotypes are always higher in the liver than in PBMC. Even in the setting of robust CD8+ T-cell depletion, these clonotypes persist at low levels in the liver throughout challenge. The ability to perform such experiments on stored samples will greatly enhance our understanding of T-cell kinetics and homing during acute and memory immune responses.