Detection of HDV specific CD8+ T cells in resolved HDV infection

Induction of antiviral state (e.g. production of IFN) upon antigenic exposure is one of the most significant functions of the T cells. This important characteristic of the lymphocytes is

57 used for epitope discovery. In order to identify HDV-specific CD8+ T cell epitopes, therefore, IFN-γ production was measured upon stimulation of PBMCs with HDV specific peptides.

Obviously, PBMCs were isolated from a donner with resolved HDV infection. This approach was also used to confirm the findings of novel HLA-B*27 epitopes (described in previous section, 3.2.1. ). With respect to the HLA-B*27, the first evidence was found in a HLA-B*27 positive patient (Patient A) with resolved HDV infection (HDV RNA negative but anti-HDV antibody positive). The PBMCs of this patient was stimulated with library of overlapping 16-mer peptides spanning L-HD Ag (kindly provided by Dr. Melanie Fiedler). In this preliminary study, HDV peptide library was divided into 8 pools of peptides (A to H) and applied for a 10-day stimulation of the isolated PBMCs from recovered patients. After re-stimulation of the expanded PBMCs on day 10 with the respective pools of HDV peptides, IFN-γ staining was performed and the percentage of IFN-γ-positive CD8+ T cells was measured in each well.

Interestingly, IFN-γ-positive CD8+ T cells were detected in PBMCs from a HLA-B*27 positive patient with resolved HDV infection. In this patient, IFN-γ was induced in PBMCs stimulated with peptide pool D (Figure 3.9). This response was not observed in the same cells stimulated with other pools. In fact, the percentage of IFN-γ-positive CD8+ T cells (0.54%) was approximately 12 times higher than that in the cells stimulated with other peptide pools or with unrelated peptide control (mean 0.045%).

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Figure ‎3.9 FACS staining for IFN-γ production after stimulation of PBMCs (Patient A) by overlapping 16-mer peptides listed in

Table ‎2.1.

PBMCs from a HLA-B*27 positive patient with resolved HDV infection were

stimulated with overlapping peptides spanning the ORF of L-HD Ag with 214 amino acids. The percentage of IFN-γ+/CD8+T cells upon stimulation with peptide pools A to J is given in the upper right corner of each panel.

Next, to investigate which peptide(s) within the pool D could trigger this specific T cell response, the cultured cells were re-stimulated in the presence of single peptides from pool D. As shown in Figure 3.10, peptide D14 was able to induce a proportion of CD8+ T cells to produce IFN-γ (0.25% IFN-γ+ CD8+ T cells vs 0.01-0.02% detected for other peptides or un-re-stimulated control). Interestingly, the 16-mer peptide, D14 (98-113 ERRDHRRRKALENKK K), includes the sequences of both predicted epitopes, aa 99-108 RRDHRRRKAL and aa 103-112 RRRKALENKK, which were able to bind to the HLA-B*27 molecule with high affinities (Figure 3.8).

59 Figure ‎3.10 FACS staining after stimulation with single peptides.

(A) FACS staining for IFN-γ production after stimulation of PBMCs (from patient A) with single peptides from the pool D (B) Amino acid sequences of all single peptides within the pool D (peptide 11a, 12, 13 and 14) are listed and the sequence of HLA-B*27 restricted epitopes are marked with lines in peptide 14.

As demonstrated in Figure 3.10, the two predicted and binding epitopes are only present in one of the overlapping peptides (peptide D14) of this pool which indicates that the IFN-γ induction is probably restricted to, at least, one of these two epitopes. Since the PBMCs of this patient were no longer available, we were not able to pinpoint which one of these two epitopes within this 16-mer (peptide D14) was indeed the main inducer of CD8+ T cells to produce IFN-γ. It is possible that sometimes one epitope may appear in more than one peptide of a library of overlapping peptides. Obviously, this depends on how many amino acid is the library overlapped by. In which case, it would be possible to recognize the minimal epitope after detection of response upon stimulation with two peptides of the library. This was not applicable here in this study.

We, in collaboration with the university hospital of Freiburg, tested PBMCs of another HDV recovered HLA-B*27 positive patient (Patient B) in order to determine the exact minimal CD8+ T cell epitope, against which the T cell response was directed, and check the

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reproducibility of the results obtained from the first patient, whose PBMCs were stimulated with overlapping peptides.

In this assay, PBMCs were isolated and cultured in the presence of peptide D14 (aa 98-113 ERRDHRRRKALENKKK), and after 10 days, the cells were re-stimulated with both predicted epitopes: aa 99-108 RRDHRRRKAL and aa 103-112 RRRKALENKK as well as the 16-mer (peptide D14). Re-stimulation of the PBMCs from patient B with 16-mer (98-113 ERRDHRRRKALENKKK) resulted in IFN-γ induction in 0.50% of the CD8+ T cells (Figure 3.11) which was consistent with the findings in patient A after stimulation with the same peptide (peptide D14). Interestingly, re-stimulation of the cells with the two predicted binding epitopes within this 16-mer revealed that indeed RRDHRRRKAL may be the HDV-specific HLA-B*27-restricted CD8+ T cell epitope targeted in the patient B. In fact, re-stimulation of the cells with the synthetic peptide RRDHRRRKAL (L-HD Ag aa 99-108) was able to induce the same percentage of CD8+ T cells to produce IFN-γ as for the 16-mer, peptide D14; however, aa 103-112 RRRKALENKK did not induce any response in this patient (patient B). This analysis indicates that despite binding capability of aa 103-112 RRRKALENKK to its corresponding HLA molecule (HLA-B*27) in vitro, this peptide cannot be considered as a CTL epitope at least based on the data obtained from patient B from Freiburg. The 10-mer RRDHRRRKAL, in contrast, was able to induce a specific T cell response in this HLA-B*27 positive patient who had resolved HDV infection. Therefore, the results from epitope prediction analysis, MHC binding assay, and, most importantly, functional T cell assay confirm that RRDHRRRKAL is a HDV-specific T cell epitope.

Figure ‎3.11 CD8 T cell assay in patient B to distinguish the exact prototype HDV-specific HLA-B*27-restricted epitope.

Flow cytometry staining of the PBMCs from Patient B (HLA-B*27 positive / HDV resolved patient) stimulated with the 16-mer (first panel from the left) and the 10-mers. The frequency of IFN-γ+/CD8+ T cell determined by ICS is indicated as percentage in right upper corner of each panel.

61 One of the chronically HDV infected subjects who lost HDV RNA during the follow-up was HLA-B*27 positive (Patient H36). Therefore, the PBMC sample from this subject was evaluated by T cell assay. The PBMCs applied for the T cell assay were, obviously, collected from the time-point when the patient was HDV RNA negative. According to the previous data achieved from patient B, we did not use the library of L-HD Ag anymore for this subject (patient C), and performed the assay with focus on overlapping 16-mer peptide (aa 98-113 ERRDHRRRKALENKKK) covering both predicted epitopes as well as single epitopes (aa 99-108 RRDHRRRKAL and aa 103-112 RRRKALENKK) to detect the prototype epitope in this patient. A 10-day culture and stimulation of the cells with the peptides were performed as described for patient B.

Flow cytometry analysis showed that the CD8+ T cells of this patient are induced for IFN-γ production after stimulation with overlapping 16-mer peptide (aa 98-113 ERRDHRRRKALENKKK). This was consistent with the data from patient A and B. However, analysis of single epitopes revealed that the second predicted and HLA-B*27-ligand (aa 103-112 RRRKALENKK) is indeed the minimal epitope targeted by the T cells in this patient and not the previously identified epitope in patient B (aa 99-108 RRDHRRRKAL). As demonstrated in Figure 3.12, 0.55% and 0.75% of the cells were positive for CD8+ and IFN-γ after stimulation with 16-mer peptide (aa 98-113 ERRDHRRRKALENKKK) and (aa 103-112 RRRKALENKK), respectively, whereas, 0.02% of the cells were positive for these two markers after stimulation with the first epitope, (aa 99-108 RRDHRRRKAL).

Figure ‎3.12 CD8 T cell assay in patient C (H36) to distinguish the exact prototype epitope.

Flow cytometry staining of the PBMCs from Patient C (HLA-B*27 positive / HDV resolved patient in the follow-up) stimulated with the 16-mer (first panel from the left) and the 10-mers. The frequency of IFN-γ+/CD8+ T cell determined by ICS is indicated as percentage in each panel.

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