Analysis of the Breadth of the HCV-specific CD8+ Lymphocyte Response in a Subject with Resolving Acute HCV Infection.
A 42-yr-old intravenous drug user presented to the hospital with acute HCV infection, characterized by elevated ALT and HCV RNA positivity. ALT subsequently normalized, and HCV RNA became negative. Blood samples were available from this subject during and after acute illness such that the development of HCV-specific T cell responses during a successful elimination of the virus could be studied in detail. In an initial set of experiments, the CTL epitopes recognized were identified and characterized by in vitro expansion and cloning of PBMCs from a week 4 blood sample after stimulation with autologous B-LCLs infected with recombinant HCV-vaccinia viruses (see Materials and Methods). 120 clones were raised and screened for CTL recognition of autologous B-LCLs infected with recombinant HCV-vaccinia viruses. All HCV-specific CTL clones were further characterized using a panel of overlapping peptides representing the expressed gene products of HCV-1. A total of 80 HCV-specific CTL clones were generated and found to recognize one of 8 epitopes: 2 restricted by HLA-A2 (NS3 1073–1081 and NS5B 2594–2602), 1 by HLA-B37 (NS4 1966–1976), and 5 by HLA-A25 (NS2 832–841, NS4 1744–1754, NS4 1758–1766, NS4 2225–2233, and NS5A 2225–2233). This method was repeated for PBMCs from weeks 0, 10, and 24, yielding clones of the same specificities, but no additional epitopes were identified.
Except for the HLA-A2–restricted epitope NS3 1073, all defined epitopes recognized by subject 1 had not been described previously. demonstrates the cytolytic activity of individual clones using target cells pulsed with the optimal epitopes. The newly identified HLA-A2–restricted NS5B 2594 response targets a peptide conforming to the defined HLA-A2 motif, with a leucine at the COOH terminus and at position 2 35
. The A25-restricted responses all have a motif similar to that described in a conserved peptide from HIV p24 gag 36
, which includes a tryptophan at the COOH terminus. Together, these data indicate that the CTL response in this subsequently controlled infection was simultaneously targeted at eight different epitopes.
Figure 1 Cytotoxic activity of HCV-specific clones derived in acute infection. CTL clones from subject 1 (week 4) were expanded on recombinant HCV-vaccinia vector–infected B-LCLs and tested on truncated peptides to determine their fine-specificity (A–H). (more ...)
Longitudinal Quantification of the CTL Response in Resolving Acute HCV Infection.
The availability of longitudinal samples in subject 1 allowed for the detailed quantification of CTL responses over the course of resolving acute hepatitis. Comparison was made using two separate techniques. Initially, CTL responses to naturally processed viral proteins expressed by recombinant HCV-vaccinia viruses were determined using an IFN-γ ELISPOT assay. This assay confirmed the multispecificity of the response, and demonstrated that responses peaked between weeks 4 and 10, at a time when ALT levels had already normalized and no viral RNA was detectable in blood ( A). These results were confirmed using a second IFN-γ ELISPOT assay, in which PBMCs were stimulated with the individual optimal peptides as identified using cloned CTLs ( B). Both assays showed that the magnitude of the overall response was maximal at week 10 after onset of jaundice. Relative responses to individual proteins were similar with both ELISPOT assays. In the peptide ELISPOT, the HLA-A25–restricted response to NS4 1744 was maximal at the earliest time point tested ( B), although the overall response to NS4 (containing another two identified epitopes) peaked between weeks 5 and 10, similar to the other proteins ( A). Assuming that 10% of PBMCs are CD8+ lymphocytes, the total number of IFN-γ–producing cells was estimated to represent ~6% of CD8+ lymphocytes at the peak of the response. These results indicate that the magnitude of the HCV-specific IFN-γ−producing CTLs in resolving acute hepatitis continued to increase at a time when the ALT was decreasing and the HCV RNA had become negative.
Figure 2 Antigen-specific IFN-γ production of HCV-specific T lymphocytes during resolution of acute HCV infection. PBMCs from subject 1 were tested for HCV-specific IFN-γ release in an ELISPOT assay at the time points shown after presentation with (more ...)
Phenotypic Characterization of the Antiviral CD8+ Lymphocyte Response.
The above studies provide a functional analysis of the CTL response in terms of IFN-γ production, but do not assess for the presence of CTLs that may be unable to mediate this specific effector function. The use of peptide–HLA tetramers to directly visualize antigen-specific CD8 cells by flow cytometry allows a more precise quantification of the full complement of antigen-specific cells. The HLA-A2–restricted CTL response was studied in subject 1 using such tetrameric complexes to accurately define the dynamics and phenotype of these populations. Fluorescently labeled tetramers specific for four HLA-A2–restricted epitopes (NS3 1073–1081, NS3 1406–1415, NS4B 1807–1816, and NS5B 2594–2602) were used to stain antigen-specific CD8+
lymphocytes in PBMCs ( A). PBMCs were also stained for the presence of the activation markers CD38, MHC class II, and CD69, as well as the chemokine receptor CCR5. A very strong CD8+
lymphocyte response was observed directed against the epitope NS5B 2594, comprising 7.40% of circulating CD8+
lymphocytes at the first time point when the patient was jaundiced ( and ). These high levels of tetramer-positive cells, which expressed the activation markers CD38 and HLA class II, occurred at the time of maximal ALT elevation, reflecting destruction of hepatocytes (). The initial NS5B 2594–specific CD8+
response was also associated with the expression of high levels of the chemokine receptor CCR5, which is mainly found on Tc1 cells (37
; E). The frequency and activation status of NS5B 2594–specific CD8+
lymphocytes decreased parallel to the rapid drop of ALT. Although the NS5B 2594–specific CD8 response persisted at a level of ~2% of CD8 cells, these cells no longer expressed elevated levels of CD38 and HLA class II, as had been observed during the peak of the response. The CD8+
response against the A2-restricted epitope NS3 1073 was subdominant at the time of initial analysis, with 1.1% of CD8 cells reacting with this tetrameric complex. It also peaked later than that against NS5B 2594 (maximum at week 10 = 2.35% of CD8+
lymphocytes; A) and remained roughly at this level throughout follow-up. These cells demonstrated lower levels of CD38 expression and only slightly elevated HLA class II expression compared with total CD8+
lymphocytes, and peaked at a time when ALT levels had already normalized (). No lymphocytes stained with tetramers for the two HLA-A2–restricted HCV epitopes NS3 1406 and NS4B 1807, consistent with the lack of detection of these responses in the cloning assays. It should be noted that acute hepatitis was also associated to a lesser extent with increased expression of CD38, HLA class II, and CCR5 on total CD8+
lymphocytes (). These tetramer-negative CD38+
HLA class II+
cells detected during clinical illness are likely to include HCV-specific T cells recognizing epitopes other than the HLA-A2–restricted one, e.g., those restricted by HLA-A25, for which no tetramers were available. In addition, these cells may also represent activation of bystander (non–HCV-specific) CD8+
lymphocytes. This possibility is suggested by the detection of EBV-specific cells expressing elevated levels of CD38 and HLA class II at early time points ( and ). Expression of CD69 was not elevated on tetramer-positive cells compared with total CD8+
lymphocytes at any time point examined (data not shown).
Figure 3 Tetramer analysis of PBMCs during and after acute disease. FACS® analysis data of PBMCs from subject 1 over time are shown: (A) percentage of CD8+ lymphocytes that were tetramer-positive at each time point; (B) serum ALT levels and RT-PCR (more ...)
IFN-γ ELISPOT responses to both naturally processed NS5B protein and peptide NS5B 2594 identified the peak to be at week 10, whereas tetramer analysis showed that the response to NS5B 2594 was highest at week 0 ( B and 3 A). This result suggested that the early dominant CTL response may have had impaired effector function at the level of IFN-γ production, as has been described in murine chronic viral infections and in melanoma patients 2228
. To test whether the NS5B 2594–specific CD8+
lymphocytes obtained at the time of acute infection showed impaired IFN-γ production in vitro, intracellular IFN-γ was measured in tetramer-positive cells after stimulation with PMA and ionomycin. Whereas NS5B 2594–specific cells obtained at weeks 10 and 32 readily produced IFN-γ after stimulation, IFN-γ production was severely impaired at week 0 compared with tetramer-negative cells ( A). Consistent with this, in vitro peptide-induced upregulation of CD69 was detectable at weeks 4 and 24 but not at week 0 ( B). These findings indicate that a high percentage of the NS5B 2594–specific CTLs detected early in infection was unresponsive both to nonspecific and antigen-specific stimuli and would have been undetected or greatly underestimated using IFN-γ ELISPOT assays on their own.
Figure 4 Functional analysis of HCV-specific PBMCs. (A) Intracellular IFN-γ staining. PBMCs from subject 1 were stimulated with PMA and ionomycin for 6 h or left unstimulated and subsequently stained with PE-labeled NS5B 2594 tetramers, PerCP-conjugated (more ...)
Since unresponsiveness is a feature of cells exposed to high levels of antigen 222638
, we looked for corroborative evidence that the NS5B 2954–specific cells had recently been exposed to antigen. One measure of this is the expression of tetramer-binding TCR and CD8 available at the cell surface, both of which are downregulated after antigen exposure 39404142
. Interestingly, at the first time point evaluated, a percentage of NS5B 2594–specific cells showed a reduced intensity of anti-CD8 antibody and tetramer staining compared with later time points, a feature that was not seen on NS3 2073– or EBV-specific lymphocytes (). These latter cells also showed lower levels of CD38 and HLA class II expression consistent with a lower level of activation. To investigate the proliferative and cytolytic potential of these cells, PBMCs from week 0 were placed in culture for 9 d with peptide and IL-2. After this period, no expansion of the tetramer-positive population was seen (total 5% of CD8+
lymphocytes, data not shown), nor was CD69 upregulated. However, these cells showed a very marked lytic capacity ( C). These results indicate that at the time of acute illness, the NS5B 2594–specific cells were not able to produce IFN-γ or respond to peptide in culture, possibly due to recent encounter of antigen in vivo; however, after cultivation they showed strong lytic capacity. Due to a limited number of cells, we could not test if the NS5B 2594–specific cells also exhibited lytic activity when tested directly after isolation.
Figure 5 Relative expression of CD8 and tetramer-binding TCR over time. To follow the course of TCR downregulation over time, the tetramer-positive CD8+ lymphocyte population is illustrated, and the percentages of gated cells that expressed a tetramer-low CD8 (more ...)
Antiviral T Helper Response in Acute Resolving HCV Infection.
In subject 1, we were also able to study the antiviral T helper response during the acute phase of the disease (). IFN-γ ELISPOT assays were performed on PBMCs, using recombinant proteins derived from HCV as target antigens. Unlike IFN-γ–secreting CTL responses, antigen-specific T helper responses were maximal at the first time point and remained high up to week 10, after which the responses decreased ~5–10-fold but still remained readily detectable. The response was multispecific at all times, a feature previously associated with clearance after acute infection 232443
, and was calculated to comprise ~3% of CD4+
lymphocytes at the time of maximal response. These results indicate that the peak magnitude of the detectable IFN-γ T helper cell response preceded the peak magnitude of the IFN-γ CTL response.
Figure 6 Analysis of T helper responses during and after acute infection. T helper responses of subject 1 to designated recombinant HCV proteins were tested in an IFN-γ ELISPOT assay. Time points are as for and . The y-axis shows numbers of (more ...)
Analysis of HCV-specific T Lymphocyte Responses in Patients with Recent Acute HCV.
Two additional subjects were identified with acute HCV infection, in whom blood for immunologic analyses was first available a few weeks or months after documented symptomatic illness. These two HLA-A2+ subjects had, as did subject 1, an acute self-limiting illness, with jaundice and elevation of ALT after exposure to HCV. Both subjects became HCV PCR–negative before blood was taken for T cell analysis. In subject 2, the first sample was obtained at week 4, and in subject 3, the first sample was available at week 36. Using tetramer staining of PBMCs, antiviral CD8+ responses were detected against two (subject 2) or three (subject 3) of the three tested HLA-A2–restricted epitopes, respectively. Responses to the HLA-A2–restricted epitope NS5B 2594 could not be determined because at the time of analysis this epitope had not yet been characterized and therefore no tetramers were available. In both subjects, switches in the dominance of HCV-specific T cell responses occurred over time, even weeks after the original illness and after clearance of HCV from the blood by PCR and normalization of ALT (). As in subject 1, the tetramer-positive populations in subjects 2 and 3 persisted well beyond the clearance of viremia. Interestingly, expansions of HCV-specific lymphocytes after resolution of acute illness, despite their magnitude (in subject 2 especially), did occur without elevation of ALT, and the tetramer-positive cells showed low expression of CD38 and HLA class II (<20% for CD38 and <7% for HLA class II in both cases; not shown), similar to what was seen at later stages in subject 1. Potential responses to other peptides could not be tested because of the lack of available cells for additional tetramer or ELISPOT analysis. However, sufficient PBMCs from subject 3 were available at week 93. We were able to demonstrate IFN-γ secretion in PMA-stimulated tetramer-positive CD8+ lymphocytes as well as in peptide IFN-γ ELISPOT assays at this time point (peptides NS3 1073 and NS3 1406; data not shown). In addition, a multispecific T helper response was detected using recombinant proteins in an IFN-γ ELISPOT assay, with responses to NS3, NS4, and NS5 (not shown). This indicates that the secretory capacity of the antiviral CTLs as well as T helper cells was preserved in this subject, similar to the later time points of subject 1. These results suggest that patients with resolving acute HCV infection generate strong and multispecific T helper cell as well as CTL responses that are maintained after resolution of viremia and normalization of ALT.
Figure 7 Tetramer analysis of PBMCs from two subjects after resolution of acute hepatitis infection. Results of tetramer stainings of PBMCs are illustrated as in . Subjects 2 and 3 had developed acute disease, but the first blood samples available were from (more ...)
Analysis of HCV-specific T Lymphocytes in Long-Term HCV-seropositive Persons.
persons who had been HCV-seropositive for several years (see Materials and Methods) were also screened using HLA-A2 tetramers. These subjects were divided into two groups, depending on the continued presence or not of virus in blood, as detected by PCR. In those who remained PCR-positive, irrespective of ALT or treatment history, the percentage of HCV-specific CD8+
lymphocytes in peripheral blood was at or below the limit of detection by tetramers (). This is consistent with previous studies showing that CTL precursor frequencies in chronic hepatitis C infection are relatively low 45
. In those patients where HCV-specific cells could be detected (n
= 9/19), the average percentage of tetramer-positive CD8+
lymphocytes was 0.07 (). In persons who were persistently antibody-positive but PCR-negative, either spontaneously or after treatment, similarly low levels of tetramer-positive CD8+
lymphocytes were seen (average 0.09%) compared with those observed during or after acute infection, although in two cases, levels >0.2% were seen. In one subject tested on two occasions >1 yr apart, this population was maintained (not shown).
Figure 8 Comparison of HCV-specific CD8+ lymphocyte responses in subjects with acute HCV infection and in long-term HCV-seropositive subjects. Tetramer staining was performed on PBMCs from subjects 1, 2, and 3 with documented primary acute HCV infection. Peak (more ...)
Compared with the patients who remained PCR-positive, there was overall a greater proportion of positive tetramer responses observed in persons who cleared their RNA (18/59 vs. 11/65 tests; P = 0.07 by Fisher's exact test). There was also an excess of responses seen to the NS5B 2594 epitope (4/8 vs. 0/10 persons; P = 0.02 by Fisher's exact test). In the PCR-positive group, only one subject (1/19) showed a response against more than one tested epitope, whereas in the PCR-negative group 50% of the responding persons had CTLs specific for more than one tested epitope (P = 0.04 by Fisher's exact test). By comparison, the percentage for EBV-specific cells in those patients who had detectable EBV-specific CD8+ T lymphocytes (n = 22/36) was 0.32, thus higher in most patients than the responses seen against HCV (P = 0.047 by Wilcoxon signed rank test).
In both PCR-positive and -negative persons, CD38 expression on HCV and EBV tetramer-positive CD8+ lymphocytes was always <15%, and HLA class II expression was <7% (data not shown). These results indicate that CTL responses were more common in PCR-negative subjects than in PCR-positive subjects, but these responses were weak compared with patients who had recently resolved acute HCV infection and did not express activation markers.