Adrianus Boon

The Magnitude and Specificity of Influenza A Virus-Specific Cytotoxic T-Lymphocyte Responses in Humans Is Related to HLA-A and -B Phenotype

ABSTRACT The repertoire of human cytotoxic T-lymphocytes (CTL) in response to influenza A viruses has been shown to be directed towards multiple epitopes, with a dominant response to the HLA-A2-restricted M158–66 epitope. These studies, however, were performed with peripheral blood mononuclear cells (PBMC) of individuals selected randomly with respect to HLA phenotype or selected for the expression of one HLA allele without considering an influence of other HLA molecules. In addition, little information is available on the influence of HLA makeup on the overall CTL response against influenza viruses. Here, the influenza A virus-specific CTL response was investigated in groups of HLA-A and -B identical individuals. Between groups the individuals shared two or three of the four HLA-A and -B alleles. After in vitro stimulation of PBMC with influenza virus, the highest CTL activity was found in HLA-A2+ donors. A similar pattern was observed for the precursor frequency of virus-specific CTL (CTLp) ex vivo, with a higher CTLp frequency in HLA-A2-positive donors than in HLA-A2-negative donors, which were unable to recognize the immunodominant M158–66 epitope. In addition, CTL activity and frequency of CTLp for the individual influenza virus epitopes were determined. The frequency of CTLp specific for the HLA-B8-restricted epitope NP380–388 was threefold lower in HLA-B27-positive donors than in HLA-B27-negative donors. In addition, the frequency of CTLp specific for the HLA-A1-restricted epitope NP44–52 was threefold higher in HLA-A1-, -A2-, -B8-, and -B35-positive donors than in other donors, which was confirmed by measuring the CTL activity in vitro. These findings indicate that the epitope specificity of the CTL response is related to the phenotype of the other HLA molecules. Furthermore, the magnitude of the influenza virus-specific CTL response seems dependent on the HLA-A and -B phenotypes.

Recognition of Homo- and Heterosubtypic Variants of Influenza A Viruses by Human CD8+ T Lymphocytes

In the present study, the recognition of epitope variants of influenza A viruses by human CTL was investigated. To this end, human CD8+ CTL clones, specific for natural variants of the HLA-B*3501-restricted epitope in the nucleoprotein (NP418–426), were generated. As determined in 51Cr release assays and by flow cytometry with HLA-B*3501-peptide tetrameric complexes, CTL clones were found to be specific for epitopes within one subtype or cross-reactive with heterosubtypic variants of the epitope. Using eight natural variants of the epitope, positions in the 9-mer important for T cell recognition and involved in escape from CTL immunity were identified and visualized using multidimensional scaling. It was shown that positions 4 and 5 in the 9-mer epitope were important determinants of T cell specificity. The in vivo existence of CD8+ cells cross-reactive with homo- and heterosubtypic variants of the epitope was further confirmed using polyclonal T cell populations obtained after stimulation of PBMC with different influenza A viruses. Based on the observed recognition patterns of the clonal and polyclonal T cell populations and serology, it is hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo.

Functional Constraints of Influenza A Virus Epitopes Limit Escape from Cytotoxic T Lymphocytes

ABSTRACT Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M158-66. We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M158-66 epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes.

Preferential HLA Usage in the Influenza Virus-Specific CTL Response

To study whether individual HLA class I alleles are used preferentially or equally in human virus-specific CTL responses, the contribution of individual HLA-A and -B alleles to the human influenza virus-specific CTL response was investigated. To this end, PBMC were obtained from three groups of HLA-A and -B identical blood donors and stimulated with influenza virus. In the virus-specific CD8+ T cell population, the proportion of IFN-γ- and TNF-α-producing cells, restricted by individual HLA-A and -B alleles, was determined using virus-infected C1R cells expressing a single HLA-A or -B allele for restimulation of these cells. In HLA-B*2705- and HLA-B*3501-positive individuals, these alleles were preferentially used in the influenza A virus-specific CTL response, while the contribution of HLA-B*0801 and HLA-A*0101 was minor in these donors. The magnitude of the HLA-B*0801-restricted response was even lower in the presence of HLA-B*2705. C1R cells expressing HLA-B*2705, HLA-A*0101, or HLA-A*0201 were preferentially lysed by virus-specific CD8+ T cells. In contrast, the CTL response to influenza B virus was mainly directed toward HLA-B*0801-restricted epitopes. Thus, the preferential use of HLA alleles depended on the virus studied.

Fluorescent Antigen–Transfected Target Cell Cytotoxic T Lymphocyte Assay for Ex Vivo Detection of Antigen-Specific Cell-Mediated Cytotoxicity

Ex vivo detection of virus-specific cytotoxic T lymphocyte (CTL) responses is limited to the use of methods assessing cytokine production, degranulation, or perforin contents of antigen-specific CD8+ T cells. Generally, their cytotoxic activity is detectable only after cultivation. We describe the fluorescent antigentransfected target cellCTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated by nucleofection with DNA vectors encoding antigengreen fluorescent protein (GFP) fusion proteins. After coculture at various effector : target (E : T) cell ratios, viable and dead GFP-positive cells were quantified by flow cytometry, and antigen-specific target-cell elimination was calculated. The assay was validated with human immunodeficiency virus (HIV) and influenza virusspecific CTL clones and revealed cytotoxicity at lower E : T cell ratios than standard 51Cr-release assays. Moreover, antigen-specific cytotoxicity was detected ex vivo within 1 day in peripheral blood mononuclear cells from HIV-infected individuals. The FATT-CTL assay provides a versatile tool that will advance our understanding of cell-mediated immunity.

Functional profile of human influenza virus-specific cytotoxic T lymphocyte activity is influenced by interleukin-2 concentration and epitope specificity

The ability of influenza A virus‐specific cytotoxic T lymphocytes (CTL) to degranulate and produce cytokines upon antigenic restimulation was studied in four HLA‐A*0101 and HLA‐A*0201 positive subjects. Peripheral blood mononuclear cells of these subjects were stimulated with influenza A virus in the presence of high or low interleukin (IL)‐2 concentrations. CD8+ T cell populations specific for the HLA‐A*0101 restricted epitope NP44‐52 and the HLA‐A*0201 restricted epitope M158‐66 were identified by positive staining with tetramers of peptide major histocompatibility complexes (MHC) (NP‐Tm and M1‐Tm, respectively). Within these populations, the proportion of cells mobilizing CD107a, or expressing interferon (IFN)‐γ and tumour necrosis factor‐(TNF)‐α upon short‐term peptide restimulation was determined by flow cytometry. Independent of IL‐2 concentrations, large subject‐dependent  differences  in  the  mobilization  of  CD107a  and  expression  of  IFN‐γ and TNF‐α by both NP‐ and M1‐specific T cells were observed. In two of the four subjects, the functional profile of NP‐Tm+ and M1‐Tm+ cells differed considerably. Overall, no difference in the proportion of NP‐Tm+ or M1‐Tm+ cells expressing CD107a was observed. The proportion of M1‐Tm+ cells that produced IFN‐γ (P < 0·05) was larger than for NP‐Tm+ cells, independent of IL‐2 concentration. When cultured under IL‐2hi concentrations higher TNF‐α expression was also observed in M1‐Tm+ cells (P < 0·05). The IL‐2 concentration during expansion of virus‐specific cells had a profound effect on the functionality of both M1‐Tm+ and NP‐Tm+ cells.

Binding of DC-SIGN to the Hemagglutinin of Influenza A Viruses Supports Virus Replication in DC-SIGN Expressing Cells

Dendritic cells express lectins receptors, like DC-SIGN, which allow these cells to sense glycans that are present on various bacterial and viral pathogens. Interaction of DC-SIGN with carbohydrate moieties induces maturation of dendritic cells and promotes endocytosis of pathogens which is an important property of these professional antigen presenting cells. Uptake of pathogens by dendritic cells may lead to cross-presentation of antigens or infection of these cells, which ultimately results in activation of virus-specific T cells in draining lymph nodes. Little is known about the interaction of DC-SIGN with influenza A viruses. Here we show that a virus with a non-functional receptor binding site in its hemagglutinin, can replicate in cells expressing DC-SIGN. Also in the absence of sialic acids, which is the receptor for influenza A viruses, these viruses replicate in DC-SIGN expressing cells including human dendritic cells. Furthermore, the efficiency of DC-SIGN mediated infection is dependent on the extent of glycosylation of the viral hemagglutinin.

In vitro effect of bioactive compounds on influenza virus specific B- and t-cell responses

In vitro studies have demonstrated positive effects of bioactive compounds on several functions of the immune system. In the present study, 25 of such compounds were tested for their immune modulating properties on influenza virus specific human B‐ and T‐cell responses in vitro. One of these compounds, N‐acetyl‐l‐cysteine was shown to increase influenza virus specific lymphocyte proliferation and interferon(IFN)‐γ production at a concentration of 1.0 mmol/l. Furthermore, N‐acetyl‐l‐cysteine was found to enhance a specific activity of two influenza specific CD8+ cytotoxic T‐lymphocyte clones directed towards HLA‐A*0201 and HLA‐B*2705 restricted epitopes. A second compound, chlorogenic acid, was shown to enhance antigen specific proliferation of lymphocytes in three out of four donors, at concentrations of 10–50 µmol/l. Neither of the two compounds exhibited a positive effect on the production of influenza virus specific antibodies by human peripheral blood mononuclear cells in vitro.