B-like natural killer cells related to the epstein-barr virus infection present an adaptive immune phenotype and stronger killing activity in lymphomas Free

Background:

Natural killer (NK) cells play a profound role in the immune system, contributing to antitumor and antiviral defenses. Recent advances in immunology have discovered adaptive NK cells with cytomegalovirus (CMV) infection.In this study, we identified a novel peripheral blood NK cell subset, CD19^dimNKG2C⁺ NK cells (referred to as B-like NK cells) related to Epstein-Barr virus (EBV) infection and with stronger killing activity.

Methods:

We conducted a single-center retrospective analysis to investigate patients with CD19^dimCD56⁺ cells at Sun Yat-sen University Cancer Center from March 1, 2013 to December 30, 2023.

Flow cytometry: Cells were stained with a panel of antibodies to identify T cells, NK cells, and B cells. NK cell activation and inhibitory receptors panel, and intracellular cytokine and killing markers panel were also assessed.

Imaging full-spectrum flow cytometry: We used the FACSDiscover™ S8 from BD Company to verify whether the formation of CD19dimCD56+ cells was related to trogocytosis.

Sequencing: Using fresh PBMC samples to construct scRNAseq and Epi Multiome ATAC + Gene Expression.

EBV infection-related indicators detection: Serum and plasma samples were collected from 100 positive and negative cases, respectively.

Results:

• A retrospective analysis of 85,540 lymphocyte subset raw data wasincluded in this analysis, with CD19^dimCD56⁺ cells detection rate ranging from 0.12% to 27.61% among 64 lymphoma patients. Furthermore, we found that patients who had CD19dimCD56+ cells in their peripheral blood before treatment had a better prognosis.

• Traditional flow cytometry, imaging full-spectrum flow cytometry,and morphological staining confirmed that this group of novel CD19^dimCD56⁺ cells were mature NK cells, only expressing CD19 and being negative for all other B-cell markers. Its formation was not caused by trogocytosis, and the morphology of this group of cells was similar to that of traditional NK cells.

• Compared with traditional NK cells, CD19^dimCD56⁺NK cells exhibited stronger killing activity, manifested by the expression of more cytotoxic indicators such as granzyme B and perforin, the production of more intracellular cytokines IFN-r and TNF-a after stimulation, and a significant increase in the expression of the activating receptor CD159c (also known as NKG2C), while the expression of the inhibitory receptor CD159a (also known as NKG2A) significantly decreased.

• The CD19^dimCD56⁺ NK cells had higher expression of CD57 and CD159c (NKG2C) compared to traditional NK cells, presenting a memory adaptive phenotype related to viral infection, suggesting that this group of novel NK cells had been infected by a virus before. The results of EBV infection-related indicators also showed that the Rta-IgG level of positive cases was significantly lower than that of the negative group, suggesting that the positive cases had been infected with EBV but did not replicate in large quantities. Based on the above results, we refer to this group of novel CD19^dimNKG2C⁺ NK cells as B-like NK cells.

• scRNAseq analysis also confirmed CD19^dimNKG2C⁺ cells were NK cells,not B cells (In the dimensionality reduction diagram, this group of cells and NK cells are clustered together). Similarly, at the genetic level, it also indicated that this group of cells wasan adaptive epigenetic memory NK cell subset, characterized by high transcript levels of KLRC2 and CD3E together with marked downregulation of CD7, KLRC1, and FCER1G.

• Differential gene pathway analysis linked this subset to EBV infection, and EBV-associated lncRNA LINC02446 was correlated with this subset.Trajectory analysis suggested its potential origin from CD56dim NK cells. Gene regulatory network analysis highlighted the role of the transcription factor IKZF2 in its development and function. Cell communication analysis revealed expression of antigen-presenting molecules (HLA-DRB5, HLA-DQA2), suggesting stronger antigen-presenting capacity. Bulk ATAC-seq and Multiome ATAC + Gene Expression showed increased transcriptional accessibility of CD19, KLRC2, and BCL11B, implicating that the corresponding chromatin has undergone opening.

Conclusion: This study untangles the identity of the CD19^dimNKG2C⁺ cells NK cell subset as a cytotoxic adaptive NK population linked to EBV infection. Its presence correlates with favorable prognosis, highlighting its potential role in EBV-related immune memory and offering a novel target for immunotherapy.