After influenza A vaccination, antibodies are generated against the viral surface protein hemagglutinin (HA). However, HA undergoes rapid antigenic variation, requiring frequent vaccine updates and limiting effectiveness. As a result, emerging influenza strains may lead to pandemics.
Memory T cells may provide more durable protection, as they recognize internal viral proteins that mutate more slowly and can therefore respond to diverse strains.
Among memory T cells, tissue-resident cells (TRM) are of particular interest. They form directly at the site of infection and act immediately upon reinfection. In influenza A, lung TRM cells protect against multiple viral strains.
CD4 TRM perform several key functions:
- contribute to viral clearance;
- directly kill infected cells;
- support the formation of CD8 TRM;
- coordinate local B cell responses.
CD4 TRM in the lungs differ functionally depending on the viral antigen they recognize.
The upper respiratory tract represents the first site of viral entry. It contains a complex immune network including lymph nodes, nasal-associated lymphoid tissue, and mucosal immune cells. Protective CD4 TRM can form in this region, but their role in influenza A has remained largely unexplored.
Researchers in Sweden investigated antigen-specific CD4 TRM in the upper respiratory tract of mice after influenza A infection and in healthy humans. They demonstrated that such cells form in the upper airways and provide protection against multiple influenza strains.
CD4 TRM Form and Persist Long-Term in the Nasal Epithelium
Conversion of effector T cells into CD4 TRM in the nasal mucosa requires antigen recognition, unlike CD8 TRM, which can form in response to inflammation alone.
Antigen-specific CD4 TRM forms in nasal tissues at frequencies comparable to the lungs, but are less frequent in the nasal-associated lymphoid tissue. These cells localize along the epithelium and express CD103, distinguishing them from lung CD4 TRM. CD103 is a surface protein that anchors T cells in mucosal tissues.
Nearly all cells in both tissues express CD11a, a marker associated with long-term TRM maintenance.
Antigen-specific CD4 TRM appear in nasal tissues as early as 10 days post-infection, followed by a decline in their numbers. At later time points (60 days post-infection), they show low proliferative activity but persist long term – up to 120 days.
Functional Activity of CD4 TRM in the Upper Respiratory Tract
A defining feature of memory T cells, including CD4 TRM, is their rapid and efficient response to reinfection. Upon antigen recognition, they become reactivated and produce cytokines that directly affect infected cells and recruit additional immune cells.
CD4 TRM in the upper respiratory tract specifically respond to influenza A antigens by producing IFN-γ and IL-2 upon stimulation with viral peptides. This response is antigen-specific and not driven by nonspecific inflammation.
However, nasal CD4 TRM produce cytokines at lower levels compared to lung CD4 TRM.
CD4 TRM Provide Protection Against Reinfection
In mouse models, animals were first infected with one influenza strain and then challenged with a different strain 30 days later. Upon reinfection, CD4 TRM in the upper respiratory tract rapidly expanded and became activated. This increase was not due to the expansion of naïve T cells.
Depletion of CD4 TRM in the upper airways severely impaired viral clearance, whereas CD8 T-cell depletion had minimal effect, demonstrating the dominant protective role of CD4 TRM. Importantly, protection was mediated specifically by upper airway CD4 TRM rather than lung TRM.
Vaccination can stimulate CD4 TRM formation in nasal tissues and reduce disease severity. In mouse models, vaccination reduced mortality and weight loss but did not affect viral load in either the lungs or upper airways.
Diversity of CD4 TRM in Nasal Tissues and Lungs
CD4 TRM populations in nasal tissues and lungs are heterogeneous, with Th1 and cytotoxic T cell subsets predominating. Influenza infection does not significantly alter the overall composition of TRM subsets.
A key distinction is the enrichment of Th17 CD4 TRM in nasal tissues, whereas they are nearly absent in the lungs.
Antigen specificity influences differentiation:
- NP-specific cells (recognizing nucleoprotein) in nasal tissues are primarily Th1 and Th17;
- HA-specific cells (recognizing hemagglutinin) include a broader range, including cytotoxic and Tfh CD4 TRM.
Lung CD4 TRM exhibit a more activated phenotype, with increased expression of genes involved in migration (Ccr2) and effector function.
In contrast, nasal CD4 TRM display a profile associated with mucosal barrier function and immune regulation, including increased expression of chemokine receptors Cxcr6 and Ccr8, as well as immunosuppressive genes.
CXCR6–CXCL16 Axis Regulates CD4 TRM Formation
The chemokine receptor CXCR6 is highly expressed on CD4 TRM in nasal tissues but is nearly absent on circulating T cells.
Loss of CXCR6 reduces CD4 TRM numbers in both nasal tissues and lungs, leading to accumulation of T cells in blood and lymphoid organs, indicating impaired tissue homing and retention.
The ligand CXCL16 is expressed in nasal tissues, and some CD4 TRM localize near CXCL16-expressing cells. Blocking CXCL16 reduces accumulation of antigen-specific TRM in the nasal cavity and, to a lesser extent, in the lungs.
Thus, the CXCR6–CXCL16 axis is essential for recruitment of effector CD4 T cells to the respiratory tract and their differentiation into TRM.
Human Nasal CD4 TRM Mirror Findings in Mice
In healthy adults, nasal tissues also contain CD4 TRM enriched for influenza-specific Th17 cells. These cells express tissue-residency markers, including CD103 and CXCR6, similar to those observed in mouse models.
Human nasal CD4 TRM include influenza-specific cells but differ functionally: they produce less IFN-γ and exhibit increased IL-17A production upon stimulation.
Th17 CD4 TRM Reduce Viral Load and Tissue Damage
Th17 cells recruit neutrophils and B cells, facilitate IgA transport to mucosal surfaces, and provide antibody-independent protection during influenza infection. They also contribute to tissue repair by producing anti-apoptotic cytokines that limit tissue damage.
Th17 CD4 TRM form in nasal tissues and can produce IL-17A even without restimulation, but only after infection.
Absence of Th17 CD4 TRM results in increased viral load and greater tissue damage during reinfection, accompanied by increased apoptosis in mucosal tissues. Blocking IL-17 does not reproduce this effect, indicating the presence of IL-17–independent protective mechanisms.
These findings demonstrate that Th17 CD4 TRM in nasal tissues limit viral replication and reduce tissue damage, playing a critical protective role during influenza A infection.
Reference
Nasal CD4+ tissue-resident memory T cells provide cross-protective immunity to influenza
