ABSTRACT Antigen-specific CD4 + T cells are essential for effective virus-specific host responses, with recent human challenge studies (in volunteers) establishing their importance for influenza A virus (IAV)-specific immunity. However, while many IAV CD4 + T cell epitopes have been identified, few are known to stimulate immunodominant CD4 + T cell responses. Moreover, much remains unclear concerning the major antigen(s) responded to by the human CD4 + T cells and the extents and magnitudes of these responses. We initiated a systematic screen of immunodominant CD4 + T cell responses to IAV in healthy individuals. Using in vitro expanded-multispecificity IAV-specific T cell lines and individual IAV protein antigens produced by recombinant vaccinia viruses, we found that the internal matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of CD4 + T cell responses. Ten epitopes derived from M1 and NP were definitively characterized. Furthermore, epitope sequence conservation analysis established that immunodominance correlated with an increased frequency of mutations, reflecting the fact that these prominent epitopes are under greater selective pressure. Such evidence that particular CD4 + T cells are important for protection/recovery is of value for the development of novel IAV vaccines and for our understanding of different profiles of susceptibility to these major pathogens. IMPORTANCE Influenza virus causes half a million deaths annually. CD4 + T cell responses have been shown to be important for protection against influenza and for recovery. CD4 + T cell responses are also critical for efficient CD8 + T cell response and antibody response. As immunodominant T cells generally play a more important role, characterizing these immunodominant responses is critical for influenza vaccine development. We show here that the internal matrix protein 1 (M1) and nucleoprotein (NP), rather than the surface proteins reported previously, are the immunodominant targets of CD4 + T cell responses. Interestingly, these immunodominant epitope regions accumulated many mutations over time, which likely indicates increased immune pressure. These findings have significant implications for the design of T cell-based influenza vaccines.
Abstract Immunodominant T cell responses are important for virus clearance. However, the identification of immunodominant T cell peptide+HLA glycoprotein epitopes has been hindered by the extent of HLA polymorphism and the limitations of predictive algorithms. A simple, systematic approach has been used here to screen for new, immunodominant CD8+ T cell epitopes. The analysis targeted healthy HLA-A2+ and HLA-A2- donors. While M158A2 was consistently detected in all individual samples in our study, the response to this epitope was only immunodominant in three out of eight while, for the other five, prominent CD8+ T cell responses tended to focus on various peptides from the influenza nucleoprotein (NP) that were not presented by HLA-A2. In all the tested 7 A2- individuals, immunodominant responses all focused on NP. Importantly, most novel, immunodominant T cell epitopes identified by our study would not have been predicted by the current prediction programs as they are either too long or lacking typical HLA binding motifs. Our data stress the importance of systematic analysis for discovering HLA-dependent, immunodominant CD8+ T cell epitopes derived from viruses and tumors. Focusing on HLA-A2 and predictive algorithms may be too limiting as we seek to develop targeted immunotherapy and vaccine strategies that depend on T cell-mediated immunity.
Differentiation of CD8 + T lymphocytes into effector and memory cells is key for an adequate immune response and relies on complex interplay of pathways that convey signals from the cell surface to the nucleus. In this study, we investigated the proteome of four cytotoxic T‐cell subtypes; naïve, recently activated effector, effector, and memory cells. Cells were fractionated into membrane, cytosol, soluble nuclear, chromatin‐bound, and cytoskeletal compartments. Following LC‐MS/MS analysis, identified peptides were analyzed via MaxQuant. Compartment fractionation and gel‐LC‐MS separation resulted in 2399 proteins identified in total. Comparison between the different subsets resulted in 146 significantly regulated proteins for naïve and effector cells, followed by 116 for activated, and 55 for memory cells. Besides Granzyme B signaling (for activated and/ or effector cells vs. naïve cells), the most prominent changes occurred in the TCA cycle and aspartate degradation. These changes suggest that correct balancing of metabolism is key for differentiation processes. All MS data have been deposited in the ProteomeXchange with identifier PXD001065 ( http://proteomecentral.proteomexchange.org/dataset/PXD001065 ).
The importance of antiviral CD8+ T cell recognition of alternative reading frame (ARF)-derived peptides is uncertain. In this study, we describe an epitope (NS1-ARF21-8) present in a predicted 14-residue peptide encoded by the +1 register of NS1 mRNA in the influenza A virus (IAV). NS1-ARF21-8 elicits a robust, highly functional CD8+ T cell response in IAV-infected BALB/c mice. NS1-ARF21-8 is presented from unspliced NS mRNA, likely from downstream initiation on a Met residue that comprises the P1 position of NS1-ARF21-8 Derived from a 14-residue peptide with no apparent biological function and negligible impacts on IAV infection, infectivity, and pathogenicity, NS1-ARF21-8 provides a clear demonstration of how immunosurveillance exploits natural errors in protein translation to provide antiviral immunity. We further show that IAV infection enhances a model cellular ARF translation, which potentially has important implications for virus-induced autoimmunity.
MHC class I molecules function to display peptides generated from cellular and pathogen gene products for immune surveillance by CD8(+) T cells. Cells typically express approximately 100,000 class I molecules, or approximately 1 per 30,000 cellular proteins. Given "one protein, one peptide" representation, immunosurveillance would be heavily biased toward the most abundant cell proteins. Cells use several mechanisms to prevent this, including the predominant use of defective ribosomal products (DRiPs) to generate peptides from nascent proteins and, as we show here, compartmentalization of DRiP peptide generation to prevent competition from abundant cytosolic peptides. This provides an explanation for the exquisite ability of T cells to recognize peptides generated from otherwise undetected gene products.
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