Influence of three successive antiviral treatments on viral heterogeneity in nonresponder chronic hepatitis C patients.

The purpose of the present study was to assess the viral diversity of hepatitis C virus (HCV) in six nonresponder patients during three unsuccessful treatments. These patients were treated successively with IFN-alpha2a (IFN-alpha) at a posology of 3.10(6) units (MIU) three times a week, 10 MIU three times a week, and a combination of IFN-alpha (3 MIU) plus ribavirin (1,000 mg/day). However, only two chronically infected patients could be included in the study due to the persistence of HCV RNA during the three successive treatments. The viral diversity was analysed by cloning and sequencing the HVR-1 region. The treatment of the two nonresponder patients was associated with the persistence of a wide diversity in the viral population and with the emergence of new or minor variants. Under the influence of standard doses of IFN-alpha, a rearrangement of the quasispecies present was observed at this time point. No significant change in viral load or in the complexity of the quasispecies was observed. A second treatment with a high dose of IFN-alpha induced a significant decrease in the associated viral load and, in one case, resulted in a radical change of the viral diversity. Administration of a combination of IFN-alpha and ribavirin did not affect the evolution of the variants but was followed by the emergence of various multiple variants. These results reinforce the hypothesis of the presence of preexisting quasispecies best adapted to the host environment, and therefore resistant to any current therapy.

Individuality of Ag-selected and preimmune TCR repertoires.

We have combined flow cytometry and single-cell PCR to characterize the TCRBV repertoires selected by individual mice in a model CD8 response against a defined peptide/MHC complex (CW3 170-1 79/Kd). Ourresults established thatdifferent mice select individually distinct yet structurally similar CW3-specific repertoires. Repertoire selection appears to be flexible depending on the immunizing cell dose. Using a single-donor, matched-pair-recipient adoptive transfer strategy, we demonstrated that the CW3-specific TCR repertoires of normal mice are already distinct at the preimmune level. We combine our data with computer simulations to test models for the composition of an Ag-specific preimmune repertoire and its selection during an immune response.

Comparative T cell receptor repertoire selection by antigen after adoptive transfer: a glimpse at an antigen-specific preimmune repertoire.

The low frequency of precursor cells specific for any particular antigen (Ag) makes it difficult to characterize preimmune T cell receptor (TCR) repertoires and to understand repertoire selection during an immune response. We have undertaken a combined adoptive transfer single-cell PCR approach to probe the Ag-specific preimmune repertoires of individual mice. Our strategy was to inject paired irradiated recipient mice with normal spleen cells prepared from individual donors and to compare the TCR repertoires subsequently selected during a CD8 response to a defined model Ag. We found that although some TCRs were shared, the TCR repertoires selected by mice receiving splenocytes from the same donor were not identical in terms of the TCRs selected and their relative frequencies. Our results together with computer simulations imply that individual mice express distinct Ag-specific preimmune TCR repertoires composed of expanded clones and that selection by Ag is a random process.

A quantitative, single-cell PCR analysis of an antigen-specific TCR repertoire selected during an in vivo CD8 response: direct evidence for a wide range of clone sizes with uniform tissue distribution.

The development of T cell effector and memory responses against foreign antigens (Ags) involves the activation, differentiation and proliferation of naive T cells expressing distinct Ag-specific TCRs. Understanding the complexity of Ag-selected TCR repertoires in individual responders in terms of the sequences selected and their relative frequencies may provide indications about how a repertoire is established and suggest ways to influence the outcome of an immune response. Most methods of repertoire analysis are unsuitable for calculating the relative in vivo frequencies of Ag-specific clones (expressing distinct TCRs) selected during an immune response, whereas sequence data obtained by single-cell PCR analysis directly reflect cell frequencies if a sufficiently large number of cells is sampled. Using a CD8 T cell response in normal mice in which Ag-selected cells are identified by cell surface phenotype and rearranged TCRBV sequences are determined by PCR amplification of genomic DNA directly from single cells, we have analyzed a large number (>200 per animal) of structurally-related Ag-specific TCRs to calculate the frequencies of distinct TCRs selected by individual mice. We found that each responder selects a unique Ag-specific TCR repertoire in which the various TCRBV sequences are present in a wide range of frequencies. However, the overall distribution of sequences is quite similar for different responder animals. Moreover, an individual's selected TCR repertoire is uniformly represented among Ag-specific CD8 cells circulating in the blood or localized in the spleen or liver. Relatively few sequences make up the bulk of the repertoire and account for the oligoclonality observed in earlier studies. We discuss various models that could account for this skewed distribution of an Ag-selected TCR repertoire.