Hepatitis C virus immune escape via exploitation of a hole in the T cell repertoire

Abstract

Hepatitis C virus (HCV) infection frequently persists despite eliciting substantial virus-specific immune responses. Thus, HCV infection provides a setting in which to investigate mechanisms of immune escape that allow for viral persistence. Viral amino acid substitutions resulting in decreased MHC binding or impaired Ag processing of T cell epitopes reduce Ag density on the cell surface, permitting evasion of T cell responses in chronic viral infection. Substitutions in viral epitopes that alter TCR contact residues frequently result in escape, but via unclear mechanisms because such substitutions do not reduce surface presentation of peptide-MHC complexes and would be expected to prime T cells with new specificities. We demonstrate that a known in vivo HCV mutation involving a TCR contact residue significantly diminishes T cell recognition and, in contrast to the original sequence, fails to effectively prime naive T cells. This mutant epitope thus escapes de novo immune recognition because there are few highly specific cognate TCR among the primary human T cell repertoire. This example is the first on viral immune escape via exploitation of a "hole" in the T cell repertoire, and may represent an important general mechanism of viral persistence.

Figure 1

Decreased recognition of the methionine variant (M_p5) of the HLA A*0201 restricted T-cell epitope NS3₁₄₀₆ is not due to loss of antigen processing and presentation or masking of low level responses. A. IFN-γ ELISpot was performed using PBMC obtained from Subject 28 one year following HCV infection when M_p5 was the dominant form of virus. Responses for PBMC incubated with the L_P5 peptide (circles), which was present upon initial viremia, and M_p5 peptide (triangles), which was dominant six months following infection demonstrate decreased recognition of the M_p5 variant. B. Constructs containing either the L_p5 or M_p5 sequence were introduced via electroporation into EBV transformed cells from Subject 28. Immunogenicity of the construct-containing EBV cells (Black bars labeled mRNA) was assessed via intracellular cytokine staining (ICS) for IFN-γ using NS3₁₄₀₆ specific-T cell lines generated from Subject 28. As a positive control, the same T cell line was tested for recognition of L_p5 and M_p5 peptides pulsed onto the surface of the autologous EBV transformed cells (Gray bars labeled peptide pulsed). There was no recognition of mock transfected EBV transformed cells (Black Control) or EBV transformed cells not pulsed with peptide (Grey Control). Recognition of the constructs as well as control constructs known not to represent processing mutations were similar to recognition when the peptides were pulsed onto the cell surface at a concentration of 0.001M, indicating that the M_p5 substitution did not prevent processing.

Figure 2

Prolonged CD8 T cell restimulation with M_p5 peptide failed to select a T cell line which recognized M_p5 better than L_p5A. PBMC were obtained from Subject 28 approximately six months after initial viremia when the M_p5 form of the virus was dominant. Those PBMC were stimulated in vitro with either the L_p5 (top panel) or M_p5 -peptide (lower panel) and supplemental interleukin-2 for two 10 day cycles and were subsequently tested for recognition of the M_p5 or L_p5-peptide. Independent of the peptide used for stimulation, both T cell lines recognized targets pulsed with the L_p5 peptide better than the M_p5 peptide in an ELISpot assay for IFN-γ. B. CD8 T cells isolated from Subject 28 after he developed M_p5 as the dominant form of the virus were restimulated with either L_p5 (top panel) or M_p5 pulsed (lower panel) DC followed by two 7 day rounds of stimulation with autologous PBMC pulsed with the same peptides as were pulsed on the DC. The response of both the L_p5 and M_p5 peptide stimulated CD8 T cells was subsequently assessed via IFN-γ ICS using L_P5 (circles) or M_p5 (triangles) as antigens. L_p5 remained better recognized. C. PBMC form Subject 28 taken from the one-year time point were cultured in the presence of either L_p5 or M_p5 peptide. The precursor frequency of T cell specific for L_p5 or M_p5 was extremely low at this time point, however, after two rounds of stimulation with the peptide or the variant peptide, cells were tested for recognition of both peptides as demonstrated by IFN-gamma production and CD107a degranulation, in serial peptide dilutions in an intracellular cytokine assay. D. PBMC from an additional subject were used to generate an L_p5 specific T cell line as described in 2c. The T cell line recognized L_p5 peptide better than the M_p5 peptide, as measured by IFN-gamma production and degranulation.

Figure 3

TCR from Subject 28 have decreased avidity for the M_p5/HLA-A*0201 complex. A. Binding of TCR from Subject 28 to L_p5 and M_p5 on HLA A*0201 was assessed by dissociation rates using the T cell lines described in Figure 2. Tetramer off-rate experiments were performed by staining L_p5- and M_p5- peptide primed T cell lines with L_p5- or M_p5- tetramer, washing, then incubating the cells with excess HLA-A2 antibody that prevented rebinding of any tetramer that had fallen off. An aliquot of each sample was removed following 0, 1, 2, 5, 7, 10, 20, 40, and 60 minutes of incubation and the MFI of L_p5- and M_p5- tetramer bound to the TCR for each line was recorded. The data were used to determine the time it took for half the tetramer to dissociate (t_½) and demonstrated higher avidity of both L_p5 and M_p5 primed T cell lines for the L_p5-tetramer than the M_p5-tetramer. B. Serial dilution of tetramers were performed using half-log dilutions of tetramer and again demonstrated higher avidity of both L_p5 and M_p5 primed T cell lines for the L_p5-tetramer than the M_p5-tetramer.

Figure 4

M_p5 ineffectively primes naïve T cells. CD45RO−/CD8+ (naïve) T cells from HCV-negative individuals were primed under optimal conditions for generation of an antigen specific response with L_p5 or M_p5 and the generation of antigen specific responses assessed by tetramer staining. Priming was performed with five different donors in multiple experiments and in parallel groups for L_p5 and M_p5. An L_p5-specific response was obtained in 36 out of 38 lines and the majority of responses were vigorous. In contrast, a total of 14 out of 38 lines generated using M_p5 had a specific response for that variant as detected by tetramer staining, but nearly all were very low level responses and at the limit of detection in the assay. The L_p5 and M_p5 specific responses were compared via GEE analysis and the L_p5-specific responses were significantly more frequent and of higher magnitude than M_p5-specific responses. (p<.0001)

Figure 5

Responses to Lp5 in HCV naïve individuals derive from the naïve T cell population. A. Immunophenotyping of CD45RA+CCR7+ T-cells sorted cells showed a high purity of CD45RA+CCR7+CD28+CD62L+ naïve T cells (left panel), whereas a CD45RA-CCR7- control population was a CD45RO+ memory subpopulation (right panel). B. CD45RA+CCR7+ and CD45RA−CCR7− sorted T cells were expanded and restimulated for 2 cycles. Only the naïve CD45RA+CCR7+ population yielded a response to the L_p5 HCV epitope, as measured by magnitude (top) or percent positive (bottom). C. CD8+ cells were isolated by negative selection and then positively selected for CD62L or CD45RO expression. No L_p5-specific response was obtained from the CD45RO+ memory population, whereas a statistically significant number of wells were positive in the CD62L+ naïve group. D. Cells isolated by CD45RO depletion followed by CD8 positive selection were compared with CD8 cells obtained from PBMC by negative selection, depleted of CD45RO+ cells, then positively selected for CD62L expression. Following expansion and restimulation for 2 cycles, similar numbers of tetramer+ wells were detected from both populations containing naïve T cells.

Figure 6

M_p5 primes naïve T cells that are primarily cross-reactive. In the right lower quadrant for each T cell line, numbers separated by a slash indicate the percent of T cells specific for NS3₁₄₀₆/MFI. The percent of tetramer bound cells is shown using L_p5 (top row) or M_p5 (bottom row). Representative ICS data are shown for one line to the right of the FACS plots in each panel. A. Priming naïve cells with L_p5 peptide resulted in L_p5-specific responses that were cross-reactive for the M_p5-epitope as shown for three representative lines. The percent of T cells specific for L_p5 was usually higher and the MFI was always higher for L_p5 than M_p5 tetramer. Although cross-reactive, better IFN-γ production was observed in response to L_p5 versus M_p5B. For the T cells lines generated with M_p5 peptide, parallel staining with either M_p5 or L_p5/A*0201-MHC tetramers demonstrated that 8 of these lines were cross-reactive to the L_p5 epitope, displaying a similar or higher MFI, when stained with the L_p5 multimer, as shown for three representative lines. The percent of T cells specific for L_p5 and M_p5 and the MFI were usually equivalent. Responses vigorous enough to assess for cytokine production were very rare, but better or equivalent IFN-γ production was observed in response to M_p5 as compared to L_p5 for the lines that recognized both. C. Six of the 14 lines generated by M_p5 priming demonstrated a higher MFI after staining with the M_p5-multimer than the L_p5-multimer and one a much larger percent of M_p5 specific cells (25 vs. 0.1%), suggesting they were more specific for M_p5 and where assessed, they also produced more IFN-γ in response to M_p5.

Figure 7

M_p5 acts as a partial agonist and not as an antagonist. A. M_p5 fails to boost an L_p5-specific response. Using naïve T cells that had been primed with L_p5 -pulsed DC and expanded with L_p5-pulsed PBMC, a third round of stimulation was performed using either L_p5 (LLL) or M_p5 (LLM) and compared to a third round of culture with media alone (LL-). Both L_p5 and M_p5 bearing tetramers were used to assess the percent of cells specific for each antigen with no significant differences in recognition pattern. The percent of CD8+/L_p5 tetramer positive T cells did not differ significantly between LLM and LL-, demonstrating that M_p5 expanded specific T cells no better than media alone. B. The M_p5 peptide did not boost the L_p5-specific response, but does not have an inhibitory effect relative to a control peptide of comparable HLA-A*0201 binding either. A T-cell line was generated by stimulating naïve T-cells with the L_p5 peptide and the percent of L_p5 variant-specific T cell is shown in the FACS plot labeled (i) as 0.98%. This line was divided and restimulated with equal numbers of autologous PBMC pulsed with one of four peptide combinations; L_p5 only (ii), M_p5 only (iii), an equal mixture of L_p5 and control peptide (iv) or an equal mixture of L_p5 and M_p5 (v). Evaluation by tetramer staining 1 week after stimulation showed that M_p5 peptide did not boost or inhibit the L_p5-specific response relative to control peptide (L_p5 specific T cells 4.94% versus 4.85%).

Figure 8

Priming naïve cells with L_p5 peptide resulted in L_p5-specific responses that were very weakly cross-reactive for the M_p5-epitope. Data are shown for three representative lines in Figure 6a. Although cross-reactive, better IFN-γ (Figure 6a) and TNF-α production, degranulation (CD107a), and upregulation of activation markers (CD137, CD25) was observed in response to the L_p5 as compared to M_p5.