{"doi":"10.1002/cyto.a.24802","title":"<scp>OMIP</scp> ‐098: A 26 parameter, 24 color flow cytometry panel for human memory <scp>NK</scp> cell phenotyping","abstract":"This 26-parameter flow cytometry panel has been developed and optimized to analyze NK cell phenotype, using cryopreserved peripheral blood mononuclear cells (PBMCs) from people living with and without human immunodeficiency virus (PLWH, PWOH). Our panel is designed for the analysis of several parameters of total NK cells and memory NK cell subsets including markers of maturation, activation, and proliferation, as well as activating and inhibitory receptors. Other tissues have not been tested (Table 1). Natural killer (NK) cells are components of the innate immune system. They are comparable to T cells, in particular CD8 T cells [1], serving both as cytotoxic effector cells and playing a crucial role in anti-viral and anti-tumor immune responses. However, they differ in recognition, specificity, and memory mechanisms. CD8 T cells use their T cell antigen receptor to recognize the peptide-major histocompatibility complex on the surface of the antigen presenting cells, and subsequently trigger their activation, differentiation, and function. NK cells on the other hand can perform rapid cytolytic and immunomodulatory functions in the absence of prior sensitization. Once activated, NK cells can help clear virus-infected or tumor cells through multiple mechanisms including direct cytotoxicity by releasing perforin, granzyme, immunoregulatory cytokines, and chemokines, or indirectly by influencing adaptive immune responses through their crosstalk with T and dendritic cells [2-6]. NK cell functional activity is tightly regulated by an array of germline encoded activating and inhibitory receptors. A balance between these receptors determines NK cell activation and responses to alterations due to stress, infections, and cancer. After viral infections, individuals exhibit a reconfiguration of the NK cell receptor repertoire, in particular, an up-regulation of the activating receptor NKG2C and a down-regulation of the inhibitory receptor Siglec-7 [7]. We have designed a 24-color high-dimensional flow cytometry panel (Table 2) that allows us to perform a deep phenotypic analysis of human NK cells. These are defined by the expression of the adhesion molecule CD56 (NCAM-1) and the Fc receptor CD16 (FcγRIIIa), which mediates antibody dependent cellular cytotoxicity (ADCC). Peripheral blood NK cells are mostly mature and cytotoxic and are identified from the lineage-negative cells (CD3− CD19− CD33−) as CD56dim. A smaller proportion of NK cells known as immature or early CD56bright (Figure 1A) produces more cytokines and chemokines than mature CD56dim NK cells and are considered their precursors [8]. A third subset is the dysfunctional CD56neg NK cell, a cell subtype that lack the expression of CD56 (Figure 1A), have a reduced cytotoxicity, and expand in chronic HIV, Cytomegalovirus (CMV) and Epstein–Barr virus infections [9, 10]. Despite being considered innate cells, NK cells can also display features of adaptive immunity. Initial studies reported NKG2C+ NK cells expansion in CMV infection [11], while later studies identified NKG2C+ memory NK cells with adaptive features in peripheral blood in the context of several other viral infections [12-15]. These cells were characterized by increased responses to target cells expressing the non-classical human leukocyte antigen E (HLA-E) [12, 13]. Moreover, mounting evidence demonstrates that NK cells have the potential to develop into long-lived and antigen-specific memory cells [16-19]. Our panel design allows us to analyze some of these memory NK cell subsets. The adaptive memory NK cells are characterized by the acquisition of phenotypic markers CD57 and NKG2C (Figure 1A), and decreased expression of the inhibitory receptor NKG2A [20]. The FcεRIγ-deficient memory NK cells (ΔgNK) are identifiable by their NKG2C expression, lack of expression of the transmembrane signaling adaptor FcRγ, and downregulation of SYK kinases (Figure 1A). These ΔgNK cells have been shown to exhibit potent antiviral functio","journal":"Cytometry Part A","year":2023,"id":383804,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9325,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1151171,"name":"Justin Degler","orcid":null,"position":1,"is_corresponding":false},{"id":332537,"name":"Dominic Paquin‐Proulx","orcid":"0000-0003-1407-3414","position":2,"is_corresponding":false},{"id":332556,"name":"Michael A. Eller","orcid":"0000-0003-3905-4877","position":3,"is_corresponding":false},{"id":513811,"name":"Kawthar Machmach","orcid":"0000-0002-6173-1726","position":4,"is_corresponding":false},{"id":347959,"name":"Matthew Creegan","orcid":"0000-0002-4205-466X","position":0,"is_corresponding":true}],"reference_count":47,"raw_metadata":null,"created_at":"2026-07-19T01:17:29.050285Z","pmid":"37807668","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}