In a recent study published in the journal Nature, researchers used nasopharyngeal swabs and single-cell ribonucleic acid (RNA) sequencing (scRNAseq) to characterize the populations of immune cells in upper respiratory tracts to understand the implications of the immune memory in upper airways in respiratory diseases such as coronavirus disease 2019 (COVID-19).
Study: Immunological memory diversity in the human upper airway. Image Credit: Sebastian Kaulitzki / Shutterstock
Background
Blood sample analyses are routinely used to understand the clinical and physiological aspects of health and disease. While these analyses have helped answer many of the questions about immune responses and diseases, they miss some of the vital information that can be found at the site of the infection.
The primary infection site for many human pathogens is the upper respiratory tract. However, studying immunological memory, which is vital to infection- and vaccine-derived protective immunity, in the upper respiratory tract has been challenging. Much remains unknown about the T and B cell immune memory development, maintenance, residency, and abundance in the upper respiratory tracts and germinal centers of secondary lymphoid organs.
About the study
In the present study, the researchers first examined the feasibility of various swabbing and sampling protocols in analyzing the populations of immune cells in the upper respiratory tracts. For this, they enrolled 30 healthy adults in the study and obtained nasopharyngeal swab samples from them every month for a year. These swabs were assessed using spectral flow cytometry.
Based on the findings that nasopharyngeal swabs yielded viable immune cell populations, the researchers obtained nasopharyngeal swabs from 12 donors who had completed their primary vaccinations against COVID-19. Of these 12 donors, seven had contracted breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections.
The samples from the donors were used to prepare single-cell suspensions, which were then used for analysis through cellular indexing of transcriptomes and epitopes by sequencing or CITE-seq and scRNAseq to understand the diversity of immune cells in the upper respiratory tracts.
Sequencing of the variable (V), diversity (D), and joining (J) gene segments of the B and T cell receptors was also conducted to detect the distinct immune cell types and the antigen-specific memory cells. The researchers also aimed to identify several populations of B cells, such as antibody-secreting cells, memory B cells, and resident memory B cells.
Based on the detection of germinal center lymphocytes from the nasopharyngeal swabs, the researchers hypothesized that functional germinal centers might be present in the adenoids of adults, and nasopharyngeal swabs can sample these germinal center lymphocytes directly from the adenoids through the crypts, which are channel-like grooves in the fenestrated, single-layer epithelium of adenoids.
To confirm this hypothesis, they visualized the sampling site through nasal endoscopy. They also collected nasal swabs from the adenoids and the epithelium of the nasal cavity, closer to the center of the inferior nasal turbinate of healthy individuals.
The cells collected through endoscopy were further analyzed using spectral flow cytometry and immunophenotyping. They also compared these results to cells obtained from biopsy samples of adenoids.
Results
The study found that nasopharyngeal swabs provided close to 5 million viable immune cells, of which a third expressed cluster of differentiation (CD)45, a receptor-linked protein tyrosine phosphatase found in all leukocytes. Of the cells not expressing CD45, the primary types were epithelial cells.
The other immune cells detected through the nasopharyngeal swabs were B cells, cytotoxic and helper T cells, and resident memory T cells. All the immune cell populations detected in the upper respiratory tract were found to be stable over time.
The CITE-seq and scRNAseq analysis of nasopharyngeal swabs from the COVID-19 vaccinated donors revealed a variety of immune cell populations, including regulatory T cells, CD4+ or T helper cells, CD8+ or cytotoxic T cells, resident memory T cells, and memory B cells.
Swabs from the upper respiratory tracts of individuals who had previous SARS-CoV-2 infections revealed SARS-CoV-2-specific B and T cell populations, including follicular helper T cells and differentiated B and T cells.
The detection of germinal center T follicular helper cells and germinal center B cells from the nasopharyngeal swab samples indicated the presence of functional adenoid tissue in adults. Adenoids are secondary lymphoid glands that contribute to immunity in early childhood and slowly shrink and disappear through late childhood and into adulthood. However, these findings were surprising as they indicated that the adenoid tissue still retained some function in adulthood.
Conclusions
Overall, the findings revealed that the upper respiratory tract was host to a rich diversity of stable immune cells. The development and persistence of stable, virus-specific immune memory cells provided insights into the immune responses at the primary infection site and highlighted the role of the mucosal barrier tissue in the upper respiratory tracts in mounting a robust immune defense against various pathogens.
Journal reference:
- Ramirez, S. I., Faraji, F., Benjamin, H. L., Lopez, P. G., Goodwin, B., Stacey, H. D., Sutton, H. J., Hastie, K. M., Saphire, E. O., Kim, H. J., Mashoof, S., Yan, C. H., DeConde, A. S., Levi, G., & Crotty, S. (2024). Immunological memory diversity in the human upper airway. Nature. DOI:10.1038/s41586024077488, https://www.nature.com/articles/s41586-024-07748-8