Influenza Virus-Infected Epithelial Cells Present Viral Antigens to Antigen-Specific CD8⁺ Cytotoxic T Lymphocytes

Abstract

We have investigated the mechanisms involved in the clearance of viral infection at the epithelium level by analyzing the activity of influenza virus-specific cytotoxic T lymphocytes (CTL) against virus-infected CMT-93 intestinal epithelial cells. Epithelial cells infected with live influenza virus effectively present viral antigens and were lysed by both homotypic and heterotypic influenza virus-specific CD8⁺ T cells. These results shed new light on the control of viral infection through the elimination of virus-infected epithelial cells by virus-specific CTL and demonstrate that CMT-93 cells furnish an appropriate model for in vitro evaluation of CTL activity against virus-infected epithelial cells.

Although mucosal vaccines are thought to provide antibody- and cell-mediated immunity at the portal entries of pathogens, there is no direct evidence that viral infection of epithelia can be controlled by the elimination of virus-infected epithelial cells in vivo. Thus, in vitro systems of interaction between virus-specific cytotoxic T lymphocytes (CTL) and virus-infected epithelial cells would provide interesting tools for the evaluation of potential protective cell-mediated immunity at the mucosal level. However, controversial results as to the ability of epithelial cells infected with intracellular pathogens to express viral proteins and to serve as targets for antigen-specific CTL have been reported. In fact, intestinal epithelial cells infected with Salmonella failed to present Salmonella-expressed transgenes to specific CTL (4). One CTL clone was reported to lyse epithelial cells derived from skin and thymuses of transgenic mice expressing the E6 oncogene of human papillomavirus type 16 (10); however, others have suggested that such CTL activity could result from cross- or alloreactivity (7). Here we report that the replication of influenza virus within epithelial cells is required for an effective presentation of viral antigens to homotypic as well as heterotypic CTL via major histocompatibility complex (MHC) class I molecules.

To determine whether viral antigens could be expressed by CMT-93 epithelial cells, influenza virus-infected cells were stained with virus-specific guinea pig antiserum. A large majority (82%) of CMT-93 cells were stained with this virus-specific antiserum after a 20-min exposure to live influenza virus followed by 2 h of incubation at 37°C (Fig. 1a). Virus-infected epithelial cells were next tested for their ability to present viral antigens to influenza virus-specific CTL effectors generated from splenocytes of C57BL/6 mice (H-2^b) infected with the influenza virus type A, strain PR34 (H1N1). These specific CTL effector cells lysed CMT-93 epithelial cells infected with influenza virus (Fig. 1b) but not uninfected CMT-93 cells. Virus-specific CTL also killed MHC-matched virus-infected EL-4 (H-2^b) target cells (Fig. 1c) but not the MHC-mismatched mastocytoma P815 (H-2^d) cells (Fig. 1d). Thus, the CTL activity of influenza-specific effectors was specifically directed against MHC-matched target cells infected with live influenza virus.

FIG. 1.

(a) Expression and presentation of the influenza virus antigen to antigen-specific CTL by CMT-93 epithelial cell line. Egg-grown influenza virus-infected epithelial cells were incubated with guinea pig antiserum against Udorn virus, followed by fluorescein isothiocyanate-labeled goat anti-guinea pig immunoglobulin (Sigma). All controls, including infected cells stained with normal guinea pig serum, showed histograms similar to the one for uninfected cells. (b to d) Antigen-specific MHC-restricted CTL were generated in a 7-day culture of splenocytes isolated from C57BL/6 infected (E+) with influenza type A, strain PR/8/34, serotype H1N1 or uninfected (E−) C57BL/6. PR/8/34-infected (T+) and uninfected (T−) target cells with distinct MHC haplotypes were used. Data from each target cell type are shown separately: CMT-93 (H-2^b) (b), EL-4 (H-2^b) (c), and P815 (H-2^d) (d).

The ability of virus-specific CTL to facilitate recovery from acute influenza virus infection (8, 9) and other viral infections (8, 12) might depend upon their reactivity toward target cells expressing heterotypic viral proteins. In this regard, viral nucleoproteins which are conserved gene products among different serotypes were shown to be responsible for heterotypic immunity (20–22, 24). We found that epithelial cells infected with influenza virus type A/Udorn (H3N2) were lysed by CTL effector cells generated from mice infected with the heterologous serotype H1N1 (Fig. 2a). This observation supports theories of the role of heterotypic virus-specific CTL in the clearance of virus infections at the epithelium level (5). Mechanisms involved in the processing and presentation of viral antigen by epithelial cells might differ from those that are involved following infection with invading bacteria. Indeed, CMT-93 cells fail to process and present fusion protein Crl-OVA to specific CTL after infection with Salmonella typhimurium expressing these genes (4), while Salmonella-infected P815 cells are recognized and lysed by specific CTL (11).

FIG. 2.

(a) Recognition of influenza virus-infected CMT-93 epithelial cells by heterotypic CTL. CMT-93 epithelial cells infected with the influenza virus, strain Udorn, serotype H3N2, were susceptible target cells for CTL induced by infection with strain PR/8/34 (serotype H1N1). The susceptibility was comparable to that of standard EL-4 target cells for heterotypic CTL. To demonstrate MHC restriction of CTL activity, P815 target cells with a distinct MHC haplotype (H-2^d) were used. (b) MHC class I-restricted presentation of viral antigen to CTL by infected CMT-93 epithelial cells. Infected CMT-93 epithelial target cells were incubated with monoclonal antibodies specific for MHC class I (H-2d^b and H-2k^b) or MHC class II (I-a^b) for 30 min at 37°C before the addition of untreated and anti-CD8-treated effector cells as indicated. (c) Presentation of viral antigen to CD8 but not CD4 CTL by infected CMT-93 epithelial cells. The effector cells were treated with monoclonal antibody specific for either CD8 (3.155) or CD4 (GK1.5) plus complement before being added to a cytotoxic reaction. (d) Failure of processing and presentation of formalin-inactivated influenza virus to antigen-specific CTL by CMT-93 epithelial cells. CMT-93 epithelial cells were incubated with different preparations of inactivated virus as indicated. Infected and virus-unrelated protein (OVA)-treated cells were used as positive and negative controls, respectively. HK, Hong Kong virus.

We next examined the phenotype of CTL active against influenza virus-infected epithelial cells and the nature of MHC molecules implicated in that recognition. Monoclonal antibodies specific for H-2k^b MHC class I molecules inhibited the cytotoxic activity of virus-specific CTL (Fig. 2b). In contrast, no inhibition of cytotoxic activity was observed with anti-MHC class II molecules (I-a^b), suggesting that CD8⁺ T cells were effectors of the CTL activity. The involvement of CD8⁺ T cells in cytotoxic activity against virus-infected epithelial cells was further demonstrated by the incubation of effector cells with anti-CD4 and anti-CD8 antibodies. Treatment of effector cells with monoclonal antibody against CD8 (3.155), which is known to inhibit T-cell-mediated cytolysis in the absence of complement (14, 15), significantly blocked antigen-specific CTL activity, and total inhibition was observed when anti-CD8 antibody was used in combination with anti-H-2k^b antibody (Fig. 2b and c). The CTL activity was not affected by incubating effector cells with anti-CD4 antibody (Fig. 2c).

Finally, since it has been reported that CMT-93 epithelial cells can present the OVA 257-264 peptide to CD8⁺ OVA-specific T-cell hybridomas (4), we asked if they could also process nonreplicating influenza virus and present viral antigen to virus-specific CTL (4). Results depicted in Fig. 2d clearly show that infection with replicating virus is required for epithelial cells to present viral antigens effectively to CTL effector, since no CTL activity was observed against epithelial cells incubated with split flu vaccine or any formalin-inactivated strain of influenza virus. Our results could explain why most inactivated virus vaccines usually fail to induce influenza virus-specific CTL responses (1, 2, 13, 23), especially when the antigens are delivered through mucosal routes. Since split viral vaccine and formalin-inactivated virus induced virus-specific antibody responses (13), regulatory CD4⁺ T cells may be preferentially stimulated by nonreplicating virus.

Although the epithelial cell line used in our study originated from intestinal epithelium, this cell line was readily infected with influenza virus, a pathogen of respiratory tract epithelium. Studies in other animal models have shown anal virus shedding and stimulation of the immune response in ferrets after oral or rectal administration of influenza A/Hong Kong/1/68 (H3N2) virus (3). Other investigators have demonstrated that type A influenza virus could be recovered from both the respiratory and the intestinal tract of fowls and could cause subclinical infection (6, 16–19). Although in severe influenza infections respiratory epithelial cells may be desquamated before CTL killing can play a role in host defense, the recognition and killing of infected epithelial cells by virus-specific CTL may be involved in less severe or recurrent infections or after immunization when memory CTL have been generated. Heterotypic immune responses mediated by cross-reactive CTL may be especially important in host protection when an antigenic shift in influenza occurs or after immunization with a previous season’s vaccine.

In conclusion, our study shows that CMT-93 intestinal epithelial cells constitute an attractive model for the evaluation of CTL activity against virus-infected epithelial cells and provide tools for the investigation of immunity to viral infection at the mucosal level.