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MHC-I peptides with HLA-A*0201 binding motifs were predicted according to the amino acid sequence of HERV-W env using SYFPEITHI and BIMAS. There were 17 peptides that matched the cut-off score > 20 for SYFPEITHI, and 20 peptides matched the T 1/2 > 10 for BIMAS. Seventeen peptides matched both criteria. Among them, five epitope peptides ( Figure 1) that had a higher SYFPEITHI score were chosen (Table 1).
Figure 1. The positions of the five predicted epitope peptides of HERV-W env. The HERV-W env protein has the characteristics of a typical retroviral envelope protein, including a cleavage site that separates the surface (SU) and transmembrane (TM) proteins which form a heterodimer
Peptide Score Starta Sequence Name SYFEPITHIb BIMASc 263 C L P S G I F F V C 26 5103.03 360 R V A D S L V T L R 25 28.516 367 T L Q D Q L N S L T 28 201.447 371 Q L N S L A A V V Q 24 17.627 447 W I L P F L G P L W 27 56.802 HBc117e125 E Y L V S F G V W E – – Note: aAmino acid start position in the protein; bposition binding scores to HLA-A2.1; cselection criteria: T 1/2 > 10. Table 1. CTL epitopes restricted by the HLA-A2.1 allele and control peptides
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Evidence indicated that peptide affinity to MHC molecules is often correlated with its immunogenicity (Paul et al., 2013). Therefore, the transporter-associated antigen processing (TAP)-deficient and HLA-A*0201+ cell line T2 was used to detect peptide affinity for the HLA-A*0201 molecule. When the peptides were added, the MFI of T2 cells increased significantly (Figure 2A). Of the five candidate peptides, peptides W and Q had the highest affinity to HLA-A*0201, with MFIs of 306 and 314, and FIs of 1.94 and 2.02. For peptide T, the MFI was 299, and the FI was 1.875, and for the peptides C and R, the MFI values were 213 and 188, and their FIs were 1.04 and 0.81, respectively, indicating moderate affinity for the HLA-A*0201 molecule (P < 0.001). The FI of the negative control was only 0.192. From the results, peptides W and Q have the highest affinity, and no weak-affinity peptides were found in the five candidate peptides according to the definition of peptide affinity for the HLA-A*0201 molecule. With increasing time, the MFI of T2 cells in each group decreased, the longer the time, the less stable the complex. Complex stability of peptide W, Q and T were highest. And peptide C and R have a lower stability ( Figure 2B).
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The feasibility of induction and antigen recognition of peptide-specific CTLs was investigated. T cell proliferation rates were measured daily by CCK-8 assay. The T cells stimulated with peptide W started proliferating on the third day. And Q, T group started proliferating on the fourth day. T cells stimulated with the peptides R or without any peptide did not result in proliferation, and had no statistical significance compared with the negative control (Figure 3). On the eighth day, the proliferation of T cells stimulated with peptide W had the highest increase rate, by 2.2 times (P ≤ 0.001), and peptides Q and T had increased by nearly 1.8 times (P ≤ 0.001), compared to the negative control. In conclusion, T cells would proliferate when stimulated with peptides W, Q and T, but peptides C and R would not induce T cell proliferation.
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To determine the immunogenicity of the peptides, PBMCs were stimulated as indicated in the Materials and Methods section. The potential of CTLs for IFN-γ production was detected by IFN-γ ELISpot and ELISA. The SFC stimulation with peptides of W, Q and T was about 82, 66 and 41 peptide-specific spots, whereas the spots for negative peptide were about 20, showing statistically significant increases of 4, 3 and 2 times compared with negative peptide, P ≤ 0.0001 (Figure 4A); while stimulation with peptides C and R showed no significant differences compared with the negative control.
The T cell specifically secreted IFN-γ from the supernatant was determined by ELISA (Figure 4B). In this, for stimulation with peptide W the IFN-γ rose nearly one time, P ≤ 0.05, for stimulation with peptide Q the IFN-γ rose 2.2 times, P ≤ 0.001, and for stimulation with peptide T the IFN-γ increased 1.8 times, P ≤ 0.001, which were all statistically significant values; but peptides C and R had no statistically significant change compared with the negative control.
The number of SFCs increased markedly after stimulation with peptides W, Q, T and C; the T cell specifically secreted IFN-γ also increased after stimulation with peptides W, Q and C, compared with the negative control. The SFCs were tested by ELISpot, which only detected the intracellular IFN-γ, whereas ELISA detected the IFN-γ in the supernatant.
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Only when both T cell antigen receptor complex and MHC recognize the antigen in the target cell surface, can T cells kill the target cells. Many reports have shown that T cells can kill target cells through two processes: (1) effector T cells damage the target cell membranes and cause target cell lysis by secreting perforin; (2) effector T cells via FasL-Fas, induce target cells apoptosis.
The ability to kill target cells is the ultimate goal of each reverse immunology approach. The potential of specific CTLs to attack human glioma cell lines was analyzed. The death rate increased by 2.2 ± 0.2 times compared with pCMV, P ≤ 0.0001 (Figure 5A). In addition, for peptides W, T and C the rate increased by 2.2 ± 0.2 times compared with negative peptide, P ≤ 0.0001 (Figure 5A). But the control cell line (A172) was just marginally affected and showed no significant difference (Figure 5B). CTL stimulation with peptides W, Q and T caused approximately 40%–60% specific lysis of U251.
Figure 5. Functional characterization of candidate-epitope-specific CTLs. LDH release assays were used at a ratio of effectors to targets of 50:1. (A) U251 (HLA-A2.1+) cell line and (B) A172 (HLA-A2.1-) cell line. Values are expressed as the mean±standard deviation of LDH release. *P < 0.05, **P < 0.001, ***P < 0.0001 compared with pCMV or the negative peptide control
As preliminary studies showed some promising results, we tried to find out whether the increase in cytotoxicity was related to apoptosis. When transfected with plasmid pCMV-env, the apoptosis of the target cells (U251) stimulated with peptides W, Q and T increased 2.2 ± 0.2 times compared with cells with plasmid pCMV, P ≤ 0.0001 (Figure 6A). For peptides W, T and C, apoptosis increased by 2.2 ± 0.2 times compared with negative peptide, P ≤ 0.0001 (Figure 6A). All showed statistically significant differences. In addition, there were no significant changes in A172 stimulated with any peptides and transfected with any plasmid (Figure 6B). Peptide W- Q- and T-specific CTLs could cause env+ U251 cell apoptosis in a HLA-A* 0201 background, but not env– U251 or A172 cell apoptosis.
Figure 6. Peptides induce apoptosis. The apoptosis rate of the U251 (HLA-A2.1+) cell line was more than that of the A172 (HLA-A2.1-) cell line. Apoptosis assays were used at a ratio of effectors to targets of 50:1. (A) U251 (HLA-A2.1+) cell line and (B) A172 (HLA-A2.1-) cell line. Each data point represents the mean±standard deviation of three independent experiments. *P < 0.05, **P < 0.001, ***P < 0.0001 compared with pCMV or the negative peptide control
In conclusion, peptides W, Q and T were HERV-W env antigenic epitopes, and they have both antigenicity and immunogenicity, so that they could cause strong T cell immune responses. From these results, we suspect that HERV-W env may act as an autoantigen inducing autoimmunity in people with neuropsychiatric disease.
Human leukemia antigen-A*0201-restricted epitopes of human endogenous retrovirus W family envelope (HERV-W env) induce strong cytotoxic T lymphocyte responses
- Received Date: 24 March 2017
- Accepted Date: 20 July 2017
- Published Date: 22 August 2017
Abstract: Human endogenous retrovirus W family (HERV-W) envelope (env) has been reported to be related to several human diseases,including autoimmune disorders,and it could activate innate immunity. However,there are no reports investigating whether human leukemia antigen (HLA)-A*0201+ restriction is involved in the immune response caused by HERV-W env in neuropsychiatric diseases.In the present study,HERV-W env-derived epitopes presented by HLA-A*0201 are described with the potential for use in adoptive immunotherapy.Five peptides displaying HLAA*0201-binding motifs were predicted using SYFEPITHI and BIMAS,and synthesized.A CCK-8 assay showed peptides W,Q and T promoted lymphocyte proliferation.Stimulation of peripheral blood mononuclear cells from HLA-A*0201+ donors with each of these peptides induced peptidespecific CD8+ T cells.High numbers of IFN-γ-secreting T cells were also detectable after several weekly stimulations with W,Q and T.Besides lysis of HERV-W env-loaded target cells,specific apoptosis was also observed.These data demonstrate that human T cells can be sensitized toward HERV-W env peptides (W,Q and T) and,moreover,pose a high killing potential toward HERV-W env-expressing U251 cells.In conclusion,peptides W Q and T,which are HERV-W env antigenic epitopes,have both antigenicity and immunogenicity,and can cause strong T cell immune responses.Our data strengthen the view that HERV-W env should be considered as an autoantigen that can induce autoimmunity in neuropsychiatric diseases,such as multiple sclerosis and schizophrenia.These data might provide an experimental foundation for a HERV-W env peptide vaccine and new insight into the treatment of neuropsychiatric diseases.