10 A multiparameter flow cytometry assay to monitor natural killer cell proliferation and activation in immuno-oncology clinical trials

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A10-A10
Author(s):  
Jennifer Tsau ◽  
Brittney Atzmiller ◽  
David Quinn ◽  
Tanya Mulvey ◽  
Sema Kurtulus ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cell ◽  
Natural Killer ◽  
Nk Cell ◽  
Multiparameter Flow Cytometry ◽  
Parent Population ◽  
Flow Cytometry Assay ◽  
Cd Markers

BackgroundNatural Killer (NK) cells have garnered increasing interest as potential cellular therapies or as targets of biotherapeutic agents due to their ability to kill tumor cells in a non-antigen dependent manner. Hence, measurement of NK cell proliferation and/or activation following treatment can serve as a useful biomarker for assessing the efficacy of immunomodulatory therapies.MethodsWe developed a novel 13-parameter flow cytometry panel incorporating cell differentiation (CD) markers important for identification of NK cell subsets (CD56, CD16), their proliferation (Ki-67), activation (CD25, CD335, NKG2D) and inhibition (CD159a) status. Additionally, CD markers that identify other cellular subsets known to be amenable to cytokine modulation (e.g., CD3 and CD14) were included for concurrent monitoring of T cell proliferation and monocyte activation. Method validation focused on analytical sensitivity, specificity and precision as key criteria of assay performance using peripheral blood mononuclear cells (PBMCs) stimulated with NK cell-activating cytokines and resting PBMCs from healthy donors.ResultsThe assay design allowed for robust quantitation of NK cell, T cell and monocyte functionalities. Lower limit of quantification (LLOQ) of target biomarker population was determined to be 1.0% of the parent population, based upon an analysis of 110 key target populations that displayed a co-efficient of variation (CV) of ≤25% and their frequencies ranged from 0.1% to 97.8% of the parent population. Additionally, ≤25% CV was observed in precision assessments, confirming the repeatability and reproducibility of the assay. Clinical trial utility of the assay was verified on cryopreserved PBMCs from patients with a variety of solid tumor malignancies. In these patients, the assay could clearly identify proliferating and activated NK cells, as well as proliferating T cells and activated monocytes, thus demonstrating its suitability for clinical trial applications.ConclusionsWe developed and validated a novel multiparameter flow cytometry assay that allows for simultaneous measurement of proliferation, activation and inhibitory status of key immune cell subsets. Thus, this assay can help shed light on the mode of efficacy of novel therapeutic agents that modulate the immune system, aimed at treatment of cancer and autoimmune diseases.

Lymphomatoid gastropathy: a distinct clinicopathologic entity of self-limited pseudomalignant NK-cell proliferation

Blood ◽  
2010 ◽  
Vol 116 (25) ◽  
pp. 5631-5637 ◽  
Author(s):  
Kengo Takeuchi ◽  
Masahiro Yokoyama ◽  
Shin Ishizawa ◽  
Yasuhito Terui ◽  
Kimie Nomura ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Natural Killer ◽  
T Cell Receptor ◽  
Nk Cell ◽  
Killer Cell ◽  
Diagnostic Errors ◽  
Cell Receptor ◽  
Clinical Consequences ◽  
Epstein Barr

Abstract Diagnostic errors in distinguishing between malignant and reactive processes can cause serious clinical consequences. We report 10 cases of unrecognized self-limited natural killer–cell proliferation in the stomach, designated as lymphomatoid gastropathy (LyGa). This study included 5 men and 5 women (age, 46-75 years) without any gastric symptoms. Gastroscopy showed elevated lesion(s) (diameter, ∼ 1 cm). Histologically, medium-sized to large atypical cells diffusely infiltrated the lamina propria and, occasionally, the glandular epithelium. The cells were CD2+/−, sCD3−, cCD3+, CD4−, CD5−, CD7+, CD8−, CD16−, CD20−, CD45+, CD56+, CD117−, CD158a−, CD161−, T cell–restricted intracellular antigen-1+, granzyme B+, perforin+, Epstein-Barr early RNA−, T-cell receptor αβ−, and T-cell receptor γδ−. Analysis of the 16 specimens biopsied from 10 patients led to a diagnosis of lymphoma or suspected lymphoma in 11 specimens, gastritis for 1 specimen, adenocarcinoma for 1 specimen, and LyGa or suspected LyGa for 3 specimens. Most lesions underwent self-regression. Three cases relapsed, but none of the patients died. According to conventional histopathologic criteria, LyGa is probably diagnosed as lymphoma, especially as extranodal natural killer/T-cell lymphoma, nasal type. However, LyGa is recognized as a pseudomalignant process because of its clinical characteristics. The concept of LyGa should be well recognized.

KIR-LIGAND Mismatched Natural Killer Cells Effectively Kill Primary Myeloma and Myeloma Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2449-2449
Author(s):  
Frits van Rhee ◽  
Susann M. Szmania ◽  
JuMei Shi ◽  
Michele Cottler-Fox ◽  
Justin W. Dyniewski ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Lines ◽  
Nk Cell ◽  
Allogeneic Transplantation ◽  
Myeloma Cell ◽  
Myeloma Cells

Abstract Recent observations in haplo-identical, T-cell-depleted allogeneic transplantation have focused attention on the rapid and remarkable anti-tumor effect mediated by Killer Inhibitory Receptor (KIR) ligand mismatched natural killer (NK) cells. Most data are confined to the role of KIR-ligand mismatched NK cells for acute leukemia after allogeneic transplantation. In this study, we report both the ability of KIR mismatched NK cells to kill primary myeloma cells, and the effects of interleukin (IL)2 and IL15 on NK cells in vitro. NK cells were prepared from healthy donor PBMCs by depletion of T-lymphocytes using immunomagnetic beads conjugated to anti-CD3 followed by depletion of monocytes using a simple adherence step. After overnight incubation in cytokines (IL2/IL15 as indicated) standard chromium release assays determined the cytotoxicity of NK cells against target cells and 3H-thymidine incorporation assessed NK cell proliferation. KIR-ligand mismatched NK cells incubated overnight in 300 IU/ml IL2 killed primary MM cells from four individuals (76, 64,60,48% lysis) as well as MM lines U266, NCI H929, and the NK sensitive line K562 (95, 86, 76% lysis, respectively) at an E:T ratio of 20:1. NK cells were not cytotoxic towards autologous phytohemagglutinin (PHA) or allogeneic PHA-induced T-cell blasts. Increasing the IL2 concentration during overnight NK cell incubation from 300 to 1000 IU/ml did not significantly enhance the cytotoxicity against U266 myeloma cells or control K562 cells. IL15 was more potent than IL2 in inducing NK cell proliferation. The optimal IL15 concentration was 300 IU/ml (range tested: 0–3000 IU/ml). The use of both cytokines in concert was less effective than the use of IL15 alone, probably due to homology of the IL2 and IL15 b and g receptors. We conclude that T cell and monocyte depletion of PBMC results in a preparation significantly enriched for NK cells (±50%) that effectively kills KIR-ligand mismatched primary myeloma cells and myeloma cell lines. IL15 is the superior cytokine for enhancing NK cell proliferation. The exciting anti-leukemic effects mediated by KIR-ligand mismatched NK cells have thus far only been reported in the haplo-identical transplant setting. In view of our positive in vitro data in MM, we will evaluate in a phase II trial wheter the repeated transfusion of KIR-ligand-mismatched, T-cell depleted NK cells from haplo-identical donors after immunosuppressive treatment with fludarabine and dexamethasone (to avoid rejection of donor NK cells) and tumor reduction with melphalan followed by a delayed auto-transplant can improve outcome in patients who have high risk MM or who have relapsed after a previous auto-transplant. This will be the first clinical application of KIR-ligand-mismatched NK cells in autologous transplantation and the first such trial in myeloma. Figure Figure

Cytoplasmic Expression of CD3ε Heterodimers by Flow Cytometry Rapidly Distinguishes Between Mature T-Cell and Natural Killer–Cell Neoplasms

2020 ◽  
Vol 154 (5) ◽  
pp. 683-691
Author(s):  
Min Shi ◽  
Phuong Nguyen ◽  
Michael M Timm ◽  
Gregory E Otteson ◽  
Pedro Horna ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Natural Killer ◽  
Gene Rearrangement ◽  
Nk Cell ◽  
Molecular Genetic ◽  
Killer Cell ◽  
Cell Receptor ◽  
Molecular Study ◽  
Cytoplasmic Staining

Abstract Objectives Distinguishing between T-cell and natural killer (NK)–cell neoplasms could be difficult given their overlapping immunophenotype. In this study, we investigated whether a flow cytometry assay with cytoplasmic staining for CD3 could be used for this purpose. Methods Flow cytometry immunophenotyping was performed on 19 surface CD3 (sCD3)–negative mature T-cell neoplasms, 10 sCD3-positive mature T-cell neoplasms, 13 mature NK-cell neoplasms, and 19 normal controls. In addition to routine antibody panels (CD2, sCD3, CD4, CD5, CD7, CD8, CD16, CD45, CD56, CD57, CD94, CD158a, CD158b, CD158e, NKG2A TCRγ/δ), cytoplasmic staining for a monoclonal CD3 antibody (clone SK7/Leu-4) was assessed in all cases. A molecular study for T-cell receptor (TCR) gene rearrangement and an immunohistochemical study for TCRβ were performed. Results Our data showed all T-cell neoplasms were uniformly positive for cytoplasmic CD3 (cCD3) regardless of sCD3 expression, whereas 85% of NK-cell neoplasms completely lacked cCD3 expression. The 2 cases with classic NK-cell immunophenotype but partial cCD3 expression showed no molecular genetic features of T-cell lineage by TCR gene rearrangement studies. Conclusions Uniform cCD3 positivity and homogeneous cCD3 negativity highly suggest T-cell and NK lineage, respectively. When partial cCD3 expression is encountered, additional confirmatory studies should be pursued for the most accurate lineage assignment.

scholarly journals 851 Harnessing innate immunity in cancer therapies: the example of natural killer cell engagers

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A892-A892
Author(s):  
Olivier Demaria ◽  
Eric Vivier ◽  
Marie Vetizou ◽  
Audrey Blanchard Alvarez ◽  
Guillaume Habif ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Natural Killer Cell ◽  
Natural Killer ◽  
Immune Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Innate Immune ◽  
Effector Functions ◽  
Cell Responses

BackgroundMost immunomodulatory approaches have focused on enhancing T-cell responses, with immune checkpoint inhibitors, chimeric antigen receptor T cells or bispecific antibodies. Although these therapies have led to exceptional successes, only a minority of cancer patients benefit from these treatments, highlighting the need to identify new cells and molecules that could be exploited in the next generation of immunotherapy. Given the crucial role of innate immune responses in immunity, harnessing these responses opens up new possibilities for tumor control. Antibody engineering provides us with great opportunities to induce synthetic immunity and to optimize the biological functions of innate immune cells, in particular by boosting the capacity of Natural Killer (NK) cells to kill tumor cells directly and to stimulate T-cell responses indirectly.MethodsIn order to leverage the advantages of harnessing NK cell effector functions, we used our Antibody-based NK cell Engager Therapeutics (ANKET) molecular platform1 and designed a new generation of molecules that can engage activating receptors NKp46 and CD16, the IL-2Rβ chain and a tumor antigen in a single tetra-specific molecule (ANKET4). The variant of interleukin-2 (IL-2v) integrated in the ANKET4 molecule is unable to bind the α-subunit of its receptor to limit regulatory T cell activation and IL-2Rα-mediated toxicity.ResultsIn vitro, ANKET4 provides proliferation and activation signals targeted to NK cells and induces primary human NK cell cytolytic activity and the secretion of cytokines and chemokines only after binding to the tumor target. In mouse models of both invasive and solid tumors, ANKET4 induced NK cell proliferation and accumulation at the tumor bed, and had a higher anti-tumor efficacy than approved therapeutic antibodies targeting the same tumor antigen. Mechanistically, transcriptomic analysis and in-vivo studies revealed that the geometry of the ANKET4 molecule including NKp46, CD16 and IL-2 receptor binding moieties on the same molecule was essential for its strong activity which results from a synthetic cooperativity between immunoreceptor tyrosine-based activation motif (ITAM) and cytokine signaling pathways. In non-human primates, CD20-directed ANKET4 resulted in sustained CD20+ B-cell depletion with minimal systemic cytokine release and no clinical sign of toxicity.ConclusionsTetra-specific ANKET4 thus constitutes a technological platform combining the induction of NK cell proliferation and effector functions with a manageable safety profile, supporting its clinical development for next-generation cancer immunotherapies.ReferenceGauthier L, Morel A, Anceriz N, Rossi B, Blanchard-Alvarez A, Grondin G, et al. Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity. Cell 2019;177(7):1701–13 e16.Ethics ApprovalPrimary immune cells were purified from buffy coats from healthy donors obtained from Etablissement Francais du Sang (EFS, Marseille) with written consent from each volunteer.All mouse experiments were performed in accordance with the rules of the Innate Pharma ethics committee and were approved by the Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation – France (APAFIS# 19272 ).All non human-primate procedures were conducted according to European guidelines for animal care and use for scientific purposes (Directive 63-2010, ”Journal Officiel des Communautés Européennes”, L276, September 22, 2010) and according to CEA institutional guidelines. The study was approved by the local ethical committee under the number A18_080 and by the French Administration (APAFIS#20525-2019050616506478 v1)

scholarly journals CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3489
Author(s):  
Takayuki Morimoto ◽  
Tsutomu Nakazawa ◽  
Ryosuke Matsuda ◽  
Fumihiko Nishimura ◽  
Mitsutoshi Nakamura ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Protein Complexes ◽  
Lymphocyte Activation ◽  
Exon 2 ◽  
Effector Cells ◽  
Guide Rna ◽  
Human Natural Killer Cells

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.

542 Expansion of cytotoxic NK Cells from PBMCs using individualized cytokine combination

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A578-A578
Author(s):  
Andreia Maia ◽  
Joana Lerias ◽  
Markus Maeurer ◽  
Mireia Castillo-Martin
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Blood Donors ◽  
Nk Cell ◽  
Tumour Cells ◽  
List Type ◽  
Mhc I ◽  
Cytokine Combination

BackgroundAdoptive immunotherapy relies on the use of T-cells to target tumour cells, through Major Histocompatibility Complex (MHC) Class I recognition(1). However, many tumours display alterations in the MHC-I pathway, a well-described immune evasion mechanism(2). Natural Killer (NK) cells recognize transformed cells independently from the presence of MHC-I and may be a reliable therapeutic option for patients with altered tumour MHC-I expression. The source of NK cells may be autologous or allogeneic and NK cells are also clinically relevant recipients of transgenic receptors (TCRs or antibodies) targeting tumour cells. NK cells have been categorized according to their CD56 and CD16 surface expression into different subpopulations: cytotoxic (CD56+CD16+) and regulatory (CD56brightCD16-)(3). Expanding cytotoxic NK cells is challenging, since the frequency of NK cells is low in peripheral blood(4) and there is also – at this point – not an optimal expansion protocol available.The goal of this project is to determine the best cytokine combination that facilitates expansion of cytotoxic NK cells that either target tumor cells directly or serve as recipients for transgenic receptors.MethodsPeripheral Blood Mononuclear Cells (PBMCs) were extracted using Ficoll methodology from blood donors and cultured in T25 flasks with Cell Genix Medium supplemented with 10% human serum and antibiotics. NK cells were expanded supplemented with feeder cells (ratio 1:1) and different cytokine combinations (1000 U/mL of IL-2, 10 U/ml of IL-12, 180 U/mL of IL-15 and/or 1 U/mL of IL-21) during 20 days. The immunophenotype of expanded NK cells was analyzed at days 0, 5, 10, 15 and 20 by flow cytometry. The cytotoxicity of NK cells was measured by a CD107a Assay or by a Total Cytotoxicity and Apoptosis Assay at days 10 and 20. Thirteen different cytokine combinations were tested.Results4/13 cytokine combinations produced a statistically significant increase of the absolute number of NK cells with a higher percentage of cytotoxic NK cells (figure 1). However, induction of cytotoxicity was not associated with a strong NK cell expansion. The regulatory NK cells subset (CD56brightCD16-) showed the highest percentage of CD107a-expressing cells, more than the CD56+CD16+, the most cytotoxic subpopulation of NK cells.Abstract 542 Figure 1Representative percentage of NK cells in total lymphocytes (A), CD56+CD16+ subpopulation in total NK cells (B), and CD56brightCD16- subpopulation amongst total NK cells (C) at different time points (5, 10, 15 and 20 days) expanded from PBMCs* p-value < 0.05ConclusionsThis work shows that we are able to grow and efficiently expand NK cells from PBMCs with different cytokine combinations leading to clinically relevant NK cell numbers as well as cytotoxic functions. This enables to produce NK cell products for therapy and as recipients for transgenic tumor antigen-specific receptors.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank, the Vivarium Facility and the Flow Cytometry Platform of the Champalimaud Centre for the Unknown.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon.ConsentWritten informed consent was obtained from the blood donors to use their samples for research purposes.ReferencesRosenberg SA, Restifo NP, Yang JC, Morgan RA, Mark E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8(4):299–308.Aptsiauri N, Ruiz-Cabello F, Garrido F. The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses. Curr Opin Immunol 2018;51:123–32.Di Vito C, Mikulak J, Mavilio D. On the way to become a natural killer cell. Front Immunol. 2019;10(August):1–15.Zotto G Del, Antonini F, Pesce S, Moretta F, Moretta L. Comprehensive phenotyping of human PB NK Cells by Flow Cytometry. 2020;1–9.

Natural Killer–Cell Lymphoma Involving the Gynecologic Tract

2000 ◽  
Vol 124 (10) ◽  
pp. 1510-1513 ◽  
Author(s):  
Paulette Mhawech ◽  
L. Jeffrey Medeiros ◽  
Carlos Bueso-Ramos ◽  
Donna M. Coffey ◽  
Alfredo F. Gei ◽  
...  
Keyword(s):  
T Cell ◽  
Natural Killer ◽  
T Cell Receptor ◽  
Nk Cell ◽  
Cell Lymphoma ◽  
Killer Cell ◽  
Cell Receptor ◽  
Lymphoid Cells ◽  
Gene Rearrangements ◽  
Neoplastic Cells

Abstract Non-Hodgkin lymphomas (NHL) can involve the gynecologic tract, most often as a manifestation of systemic involvement, and most cases reported have been of B-cell lineage. We describe 2 women with natural killer (NK)-cell lymphoma involving the gynecologic tract that initially presented with vaginal bleeding. In case 1, the patient had a stage IE nasal-type NK-cell lymphoma involving the cervix. The tumor was composed of medium-sized, irregular lymphoid cells with angioinvasion and necrosis. In case 2, the patient had a stage IV blastoid NK-cell lymphoma/leukemia infiltrating all organs in a hysterectomy and bilateral salpingo-oophorectomy specimen. Subsequent biopsy specimens revealed that the bone marrow and lymph nodes were also involved. The neoplasm was composed of small to medium lymphoid cells with fine nuclear chromatin. Case 1 was assessed immunohistochemically and the neoplastic cells were positive for CD3, CD56, and TIA-1. Case 2 was analyzed using both immunohistochemical and flow cytometry methods. The neoplastic cells were positive for cytoplasmic CD3, CD4, CD7, CD43, CD45, and CD56 and were negative for surface CD3. Both cases were negative for Epstein-Barr virus (EBV) ribonucleic acid (RNA) and molecular studies showed no evidence of T-cell receptor γ chain gene rearrangements. The immunophenotype and absence of T-cell receptor gene rearrangements support NK-cell origin. We report these cases to illustrate that NK-cell lymphomas can involve, and rarely arise in, the gynecologic tract.

scholarly journals Hydroa Vacciniforme and Hydroa Vacciniforme-Like Lymphoproliferative Disorder: A Spectrum of Disease Phenotypes Associated with Ultraviolet Irradiation and Chronic Epstein–Barr Virus Infection

2020 ◽  
Vol 21 (23) ◽  
pp. 9314
Author(s):  
Chien-Chin Chen ◽  
Kung-Chao Chang ◽  
L Jeffrey Medeiros ◽  
Julia Yu-Yun Lee
Keyword(s):  
T Cell ◽  
Natural Killer ◽  
Ultraviolet Irradiation ◽  
Epstein Barr Virus ◽  
Nk Cell ◽  
Current Evidence ◽  
Barr Virus ◽  
Ebv Infection ◽  
Hydroa Vacciniforme ◽  
Epstein Barr

Hydroa vacciniforme (HV) is a rare form of photosensitivity disorder in children and is frequently associated with Epstein–Barr virus (EBV) infection, whereas HV-like lymphoproliferative disorders (HVLPD) describe a spectrum of EBV-associated T-cell or natural killer (NK)-cell lymphoproliferations with HV-like cutaneous manifestations, including EBV-positive HV, atypical HV, and HV-like lymphoma. Classic HV occurs in childhood with papulovesicules on sun-exposed areas, which is usually induced by sunlight and ultraviolet irradiation, and mostly resolves by early adult life. Unlike classic HV, atypical or severe HV manifests itself as recurrent papulovesicular eruptions in sun-exposed and sun-protected areas associated occasionally with facial edema, fever, lymphadenopathy, oculomucosal lesions, gastrointestinal involvement, and hepatosplenomegaly. Notably, atypical or severe HV may progress to EBV-associated systemic T-cell or natural killer (NK)-cell lymphoma after a chronic course. Although rare in the United States and Europe, atypical or severe HV and HV-like lymphoma are predominantly reported in children from Asia and Latin America with high EBV DNA levels, low numbers of NK cells, and T cell clones in the blood. In comparison with the conservative treatment used for patients with classic HV, systemic therapy such as immunomodulatory agents is recommended as the first-line therapy for patients with atypical or severe HV. This review aims to provide an integrated overview of current evidence and knowledge of HV and HVLPD to elucidate the pathophysiology, practical issues, environmental factors, and the impact of EBV infection.

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