Mucosal T-Cell Dynamics in Influenza A infections and Cross-Recognition of Avian Influenza
While peripheral blood mononuclear cells (PBMCs) serve as the standard compartment for immune monitoring due to ease of sampling and protocol standardization, they provide only an approximation of localized antiviral immunity, such as mucosal T-cell responses in the upper human airways.
Yet, as Dr. Eva Grüner (LMU Munich) emphasized during the peptides&elephants Scientific Lunchsymposium at the 55th Annual Meeting of the German Society for Immunology (DGfI) in Munich: "This is the key entry point for respiratory infections; cells that act there are also the first line of defense of the body, fighting the disease at the very earliest onset of infection."
This local immune response is crucial as it limits viral replication at the site of entry, reduces pathogen transmission to the lungs, and ultimately helps prevent severe disease as shown in animal models.
Methodological Optimization: Sampling the Upper Airway
Analyzing mucosal T cells presents technical challenges regarding sampling consistency and cell yield. To establish a standardized protocol, Grüner and colleagues optimized a non-invasive, swab-based collection technique alongside nasal fluid suction:
- Sampling Location: Sampling at approximately 6 cm depth, targeting the middle part of the inferior turbinate, yielded higher lymphocyte counts compared to a shallower 3 cm swab.
- Cell Phenotype: Flow cytometry revealed that swab-derived mucosal immune cells are predominantly T cells, with a strong bias toward CD8+ T cells.
- Cryopreservation Limitation: Freezing and thawing mucosal cells caused a marked reduction in cell yield, indicating that fresh cell processing is optimal for functional assays such as IFN-γ ELISpot.
Results
T-Cell Dynamics: Upper Airway versus Peripheral Blood
By following up individuals for up to one year after laboratory-confirmed influenza or SARS-CoV-2 infection, Grüner and colleagues tracked the kinetics of virus-specific T-cell responses across compartments.
For this purpose, they stimulated T cells with peptide pools and analyzed them by ELISpot.
- Correlation at Peak: A correlation in peak IFN-γ T-cell responses was observed between mucosal swabs and PBMCs in recently infected individuals, whereas no correlation was found in healthy uninfected controls.
- Kinetics & Waning: PBMC-derived T cells exhibited a classic expansion pattern, peaking between days 15 and 21 post-infection. While mucosal T-cell kinetics generally mirrored this peak, mucosal T-cell responses declined more rapidly over time compared to systemic PBMC responses.
Broad Cross-Recognition of Avian Influenza (H5N1)
„Last but not least, we wanted to see if those T-cells might promote a certain level of cross-recognition of avian influenza viruses“, Grüner outlined their further investigations.
- Epitope Background: Internal structural proteins like the Nucleoprotein (NP) and Matrix protein (M1) share high amino acid sequence conservation between seasonal (H3N2) and avian (H5N1) viruses, as Grüner pointed out—building a strong contrast to the highly variable surface hemagglutinin (HA), which is the main target of antibody responses.
- T-Cell Cross-Recognition: Mucosal T cells from individuals recently infected with seasonal H3N2 showed comparable IFN-γ response levels when stimulated with either H3N2 or H5N1 peptide pools, demonstrating robust cross-recognition. Participants with previous SARS-CoV-2 infection also displayed baseline cross-reactivity to H5N1, though at lower levels.
- Divergence from Antibody Responses: Luminex-based profiling of mucosal IgG and IgA antibodies isolated from nasal suction fluid showed strong binding of IgG and lower-level binding of IgA to the seasonal strains H1N1 and H3N2, but minimal cross-binding to avian H5N1 strains.
Key Takeaways
Summarizing the results, Grüner stressed: „Tissue really matters. There is a difference between the upper airway and the blood compartment, and it's worth looking into this in more detail.“
She then highlighted the key takeaways of her talk:
- Predominance of Mucosal CD8+ T Cells: CD8+ T cells predominate the immune cell population in the nasal turbinate mucosa.
- Faster Kinetics in the Mucosa: Mucosal T-cell responses appear to decline faster following infection compared to systemic responses measured in PBMCs, showing distinct compartmental kinetics.
- Broad Cellular Cross-Recognition: Unlike antibody responses, local T-cell responses in this setup demonstrate broad cross-recognition of avian influenza strains (H5N1).
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