PMC10149821_01
(A) Immunohistochemical staining of hematoxylin-eosin staining (H&E) of nasopharyngeal mucosa were presented with infiltrate of atypical lymphoid cells in the setting of coagulative necrosis and immunohistochemistry showed these neoplastic cells were positive for CD56, CD3, TIA, GranB, Perforin, Ki67 (50%+), CD4 (majority of cells), CD8 (minority of cells) and EBV-encoded RNA, and negative for CD20 and CD5. (B) Radiographic examinations of different disease stage: Up: Hypermetabolism lesions were seen in nasopharyngeal mucosa and cervical and thoracic lymph node on PET image when initially diagnosed. Middle: PET/CT scanning showed complete remission after chemotherapy and nasal radiation. Down: Enhanced CT scanning showed exudative lesion of the right lower lobe of the lung, multiple low-density patches in liver and obvious edema of the right-side iliopsoas muscle. (C) Bone marrow smear showed abnormal phagocytes engulfing normal blood cells (Left) and a blood smear showed heterotypic lymphocytes (Right). (D) Clinical course (Left) and increased EBV-DNA load (Right) of the patient were presented.
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The patient had a six-year history of untreated chronic inflammation of the nasopharyngeal mucosa with lymphadenosis, as seen in  Figure S1A and 1B . Pathologic examination of the neoplasms revealed hyperplastic lymphoid tissue with necrosis ( Figure 1A ). Figure 1A ), supporting a pathological diagnosis of ENKTL, nasal type. The whole-blood EBV-DNA test was negative, and flow cytometry showed a low CD4/CD8 ratio. Full-body positron emission tomography/computed tomography (PET/CT) revealed hypermetabolism in the thickened tissue of the nasopharynx (maximum standardized uptake value 25.1), areas of tumor infiltration in the nasal cavity, oropharynx, and tonsillar tissue, as well as multiple enlarged cervical, hilar, mediastinal, and axillary lymph nodes of varying sizes ( Figure 1B ). Figure 1B ). Follow-up was planned ( Figure 1B ). A contrast-enhanced CT revealed an exudative lesion on the right lower lobe of the lung, multiple low-density patches in the liver, and edema of the iliopsoas muscle on the right side, indicating either infection or tumor recurrence ( Figure 1B ). Figure 1C ). Unfortunately, the patient died a month after receiving anti-PD-1 therapy ( Figure 1D ).
(A) Single-cell transcriptome profiling of PBMCs from the patient showed that two-dimensional UMAP visualization of PBMCs resulted in 6 clusters (Left) and were classified based on different samples (Middle). Finally, the fractions of all cell clusters were presented before and after PD-1 therapy (Right). (B) T-cells were further subdivided into 2 clusters (Left) and classified based on different samples (Right). (C) CD8+ effector T-cells were further subdivided into three clusters (Left) and classified based on different samples (Right). (D) NK cells were further subdivided into three clusters (Left) and classified based on different samples (Right). (E) Top 10 biological processes by GO gene set enrichment analysis in NK cells were shown in bubble plot according to gene ratio.
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Flow cytometry of bone marrow showed that about 3.9% of cells had the NK phenotype, and were strongly positive for CD56, CD7, CD45, CD16 (including a proportion of CD16dim), positive for CD2, and negative for CD3, CD5, CD57, CD4, and CD8 ( Figure S2A ). Flow cytometry of peripheral blood cells showed that the proportion of CD56-positive lymphocytes had increased to 67.7% with abnormal expression of killer inhibitory receptors, no CD158a, CD158e or CD158i, and low CD158b expression (1.5%) ( Figure S2C ). However, contrast-enhanced CT showed that the lesions had decreased in size, but flow cytometry revealed that 7.3% of the NK cells in bone marrow were still abnormal ( Figure S2B ). Using unbiased clustering, we identified six distinct cell types, including T-cells, B-cells, neutrophils, monocytes, granulocyte-monocyte progenitor cells, and erythrocytes ( Figure 2A ). We further classified these cells according to their source to compare cell type changes ( Figure 2A ). Further subclustering identified two main clusters: CD8+ effector T-cells and NK cells ( Figure 2B ). The CD8+ effector T-cells were further divided into three clusters ( Figure 2C ). Figure 2E ). Furthermore, NK cells secreted mainly pro-inflammatory chemokines, including CCL3, CCL4, and CCL5. CellPhoneDB results showed that the receptors for these chemokines on monocytes, neutrophils, and CD8+ effector T-cells were responsible for the interaction between these cells after anti-PD-1 therapy ( Figure 2F ). NK cells were further divided into three regional clusters ( Figure 2D ,  Figure S3B ).
(A) Neutrophils were further subdivided into six clusters (Left) and classified based on different samples (Right). (B) Monocytes were further subdivided into three main clusters (Left) and classified based on different samples (Right). (C) Top 10 biological processes by GO gene set enrichment analysis before (Left) and after (Right) therapy in neutrophils were shown in bubble plot according to gene ratio. (D) The interaction between each cluster by cytokines in the sample before therapy was presented.
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Although there was an overall reduction in the CD8+ effector T-cell count after anti-PD-1 therapy, the constitution of each cluster was similar ( Figure S3A ). Remarkably, these three clusters showed enhanced cytotoxicity according to type of UCell signature score ( Figure S3C ). We observed upregulation of the type I interferon signaling pathway and a marked response to type I interferon by the Gene Ontology (GO) analysis, indicating antiviral and antitumor activity ( Figure S3D ). NK cells were further divided into three regional clusters ( Figure 2D ,  Figure S3B ). NK_1, which appeared to be increased after therapy, was found at the start of differentiation from the pseudo-time analysis result ( Figure S3E ) with an upregulated type I interferon signaling pathway and an enhanced response to type I interferon. NK_2 was upregulated in pathways related to production of ribosomes (e.g. biogenesis of the ribonucleoprotein complex and ribosome). In NK_3, the upregulated pathways were primarily associated with an enhanced neutrophil-associated immune response, which was in contrast with other NK cell clusters ( Figure S3F ). The proportion of neutrophils in the sample was high and was further classified into six clusters ( Figure 3A ,  Figure S4A ). NK cells ( Figure 3C ). Neutrophils_1, accounting for the largest percentage before therapy, showed high expression of genes (e.g. CellPhoneDB results indicated that neutrophils maintain a close relationship with other immune cells by secreting and receiving CXCL8 and could be closely associated with an inflammatory response such as HLH ( Figure 3D ). Furthermore, monocytes were activated before anti-PD-1 therapy and partitioned into three main clusters ( Figure 3B ,  Figure S4B ). CellPhoneDB results indicated that monocytes secreting CXCL8 as an inflammatory mediator interacted with neutrophils via CXCR1 and CXCR2 ( Figure 3D ).