Temporal profiling of human lymphoid tissues reveals coordinated defense against viral challenge

nature immunology Article https://doi.org/10.1038/s41590-024-02064-9 Temporal profiling of human lymphoid tissues reveals coordinated defense against viral challenge Received: 14 February 2023 Accepted: 10 December 2024 Published online: 31 January 2025 Check for updates Matthew L. Coates 1,2,5, Nathan Richoz1,2,5, Zewen K. Tuong 1,2,3,5, Georgina S. Bowyer1,2,5, Colin Y. C. Lee 1,2,3, John R. Ferdinand1, Eleanor Gillman 1,2, Mark McClure1,2, Lisa Dratva 3,4, Sarah A. Teichmann David R. Jayne2, Rafael Di Marco Barros1, Benjamin J. Stewart 1,2 & Menna R. Clatworthy 1,2,3 , 3,4 Adaptive immunity is generated in lymphoid organs, but how these structures defend themselves during infection in humans is unknown. The nasal epithelium is a major site of viral entry, with adenoid nasal-associated lymphoid tissue (NALT) generating early adaptive responses. In the present study, using a nasopharyngeal biopsy technique, we investigated longitudinal immune responses in NALT after a viral challenge, using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection as a natural experimental model. In acute infection, infiltrating monocytes formed a subepithelial and perifollicular shield, recruiting neutrophil extracellular trap-forming neutrophils, whereas tissue macrophages expressed pro-repair molecules during convalescence to promote the restoration of tissue integrity. Germinal center B cells expressed antiviral transcripts that inversely correlated with fate-defining transcription factors. Among T cells, tissue-resident memory CD8 T cells alone showed clonal expansion and maintained cytotoxic transcriptional programs into convalescence. Together, our study provides unique insights into how human nasal adaptive immune responses are generated and sustained in the face of viral challenge. Secondary lymphoid organs orchestrate the spatial and temporal arrangement of different immune cell subsets to enable appropriate cellular interactions and the generation of timely pathogen-specific responses. T and B lymphocytes are largely segregated into distinct areas within lymphoid tissues, except within germinal centers (GCs), highly organized structures with light and dark zones, where GC B cells acquire antigen from follicular dendritic cells (FDCs) and present it to PD+CD4+ T follicular helper (TFH) cells1. This interaction leads to iterative rounds of GC B cell proliferation, during which there is somatic hypermutation and the emergence of both memory B cells and antibody-secreting plasmablasts or cells that defend against current and future infection1,2–7. As lymphoid tissues are critical for the generation of adaptive immunity, they need robust defense mechanisms. These processes are well studied in animal models, for example, in mice, subcapsular sinus macrophages contribute to lymph node defense, preventing the spread of lymph-borne viruses and bacteria, the latter via interactions with innate lymphocytes8,9. In contrast, profiling of human lymphoid tissue responses during infection is limited, resulting partly from the challenges of sampling these structures in humans. Department of Medicine, Molecular Immunity Unit, University of Cambridge, Cambridge, UK. 2Cambridge Institute for Therapeutic Immunology and Infectious Diseases, Cambridge, UK. 3Cellular Genetics, Wellcome Sanger Institute, Hinxton, UK. 4Cambridge Stem Cell Institute, Cambridge, UK. 5 These authors contributed equally: Matthew L. Coates, Nathan Richoz, Zewen K. Tuong, Georgina S. Bowyer. e-mail: 1 Nature Immunology | Volume 26 | February 2025 | 215–229 215 Article The nasopharynx contains the pharyngeal tonsils (also known as adenoids), which form part of Waldeyer’s ring of lymphoid tissue and are the major site of nasal-associated lymphoid tissue (NALT). Structurally, they have a surface crypt epithelium with antigen-transporting M cells and underlying B cell follicles and T cell-containing interfollicular areas, which together generate protective antibody-secreting and memory cells after a nasal challenge10,11. Adenoid tissue typically macroscopically involutes in adolescence and the extent to which NALTs contribute to local mucosal adaptive immune responses in adult humans is unclear. CD68+ macrophages have been identified in human adenoid NALT12, but macrophage heterogeneity and contributions to lymphoid tissue defense or repair post-infection in humans are completely unknown. The posterior nasal space can be readily visualized in humans using an endoscope, raising the possibility that endoscopic adenoid NALT biopsies might offer a feasible method to profile adaptive immune responses in secondary lymphoid organs after an upper respiratory tract immune challenge. To test this, we undertook a ‘natural experiment’, sampling adult participants after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection—a virus that uses nasal epithelium as a point of entry and replication13,14—to understand how NALTs defend themselves in the face of local infection to ensure that the cells and infrastructure required to support an ongoing adaptive immune response are maintained. Although SARS-CoV-2 has already been well studied in humans, much of this work has used peripheral blood or epithelial sampling in live participants, which fails to capture lymphoid follicular components, such as GC B cells15–17. Lymphoid tissue sampling in SARS-COV-2 infection has been conducted in children undergoing adenoidectomy, suggesting a robust GC response in pediatric NALT whereas, in contrast, autopsy samples from the spleen and lymph nodes in adults with fatal SARS-CoV-2 infection show minimal or absent GCs18–20. In the present study, we applied single-cell RNA sequencing (scRNA-seq), multi-parameter flow cytometry and confocal imaging to adult NALT biopsies, to understand the cellular molecular adaptations that support, polarize and defend human secondary lymphoid tissue in adults in the face of a viral immune challenge. Results Postnasal space biopsy enables lymphoid tissue profiling We developed a well-tolerated endoscopic technique for collecting NALT biopsies from live humans using topical local anesthesia (Extended Data Fig. 1a,b). 23 participants underwent sampling (aged 19–91 years), including 10 healthy controls, 8 patients with acute COVID-19 (sampled within 1 week of a first positive SARS-CoV-2 reverse transcriptase–polymerase chain reaction (RT–PCR) test) and 5 convalescent patients with COVID-19 (clinically asymptomatic participants sampled 3–5 weeks after the first positive SARS-CoV-2 RT–PCR; Fig. 1a and Supplementary Table 1). Postnasal space biopsies (with additional nasal brushing or curettage in some cases) were collected, alongside Fig. 1 | Experimental overview and cellular landscape of blood and NALT in SARS-CoV-2 infection. a, Schematic of experimental design with number of participants and timing of sampling in each group, sample types (paired NALT and peripheral blood) taken fr (...truncated)