Search

Abstract: Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder associated with autosomal recessive variants in genes required for perforin-mediated lymphocyte cytotoxicity. A rapid diagnosis is crucial for successful treatment. Although defective cytotoxic T lymphocyte (CTL) function causes pathogenesis, quantification of natural killer (NK)-cell exocytosis triggered by K562 target cells currently represents a standard diagnostic procedure for primary HLH. We have prospectively evaluated different lymphocyte exocytosis assays in 213 patients referred for evaluation for suspected HLH and related hyperinflammatory syndromes. A total of 138 patients received a molecular diagnosis consistent with primary HLH. Assessment of Fc receptor-triggered NK-cell and T-cell receptor (TCR)-triggered CTL exocytosis displayed higher sensitivity and improved specificity for the diagnosis of primary HLH than routine K562 cell-based assays, with these assays combined providing a sensitivity of 100% and specificity of 98.3%. By comparison, NK-cell exocytosis after K562 target cell stimulation displayed a higher interindividual variability, in part explained by differences in NK-cell differentiation or large functional reductions after shipment. We thus recommend combined analysis of TCR-triggered CTL and Fc receptor-triggered NK-cell exocytosis for the diagnosis of patients with suspected familial HLH or atypical manifestations of congenital defects in lymphocyte exocytosis., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution.)

Heterozygous GATA2 mutation is associated with immunodeficiency, lymphedema, and myelodysplastic syndrome. Disease presentation is variable, often coinciding with loss of circulating dendritic cells, monocytes, B cells, and natural killer (NK) cells. Nonetheless, in a proportion of patients carrying GATA2 mutation, NK cells persist. We found that peripheral blood NK cells in symptomatic patients uniformly lacked expression of the transcription factor promyelocytic leukemia zinc finger (PLZF), as well as expression of intracellular signaling proteins FcεRγ, spleen tyrosine kinase (SYK), and EWS/FLI1-Activated Transcript 2 (EAT-2) in a variegated manner. Moreover, consistent with an adaptive identity, NK cells from patients with GATA2 mutation displayed altered expression of cytotoxic granule constituents and produced interferon-γ upon Fc-receptor engagement but not following combined interleukin-12 (IL-12) and IL-18 stimulation. Canonical, PLZF-expressing NK cells were retained in asymptomatic carriers of GATA2 mutation. Developmentally, GATA-binding protein-2 (GATA-2) was expressed in hematopoietic stem cells, but not in NK-cell progenitors, CD3 - CD56 bright , canonical, or adaptive CD3 - CD56 dim NK cells. Peripheral blood NK cells from individuals with GATA2 mutation proliferated normally in vitro, whereas lineage-negative progenitors displayed impaired NK-cell differentiation. In summary, adaptive NK cells can persist in patients with GATA2 mutation, even after NK-cell progenitors expire. Moreover, our data suggest that adaptive NK cells are more long-lived than canonical, immunoregulatory NK cells.

Natural killer (NK) cells have long been considered short-lived effectors of innate immunity. However, recent animal models and human studies suggest that subsets of NK cells have adaptive features. We investigate clonal relationships of various NK-cell subsets, including the adaptive population, by taking advantage of naturally occurring X-linked somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs) from patients with paroxysmal nocturnal hemoglobinuria (PNH). The affected HSPCs and their progeny lack expression of glycosylphosphatidylinositol (GPI) anchors on their cell surface, allowing quantification of PIGA -mutant (GPI-negative) HSPC-derived peripheral blood cell populations. The fraction of GPI-negative cells within the CD56 dim NK cells was markedly lower than that of neutrophils and the CD56 bright NK-cell compartments. This discrepancy was most prominent within the adaptive CD56 dim NK-cell population lacking PLZF expression. The functional properties of these adaptive NK cells were similar in PNH patients and healthy individuals. Our findings support the existence of a long-lived, adaptive NK-cell population maintained independently from GPI pos CD56 dim .

Cytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through the release of perforin-containing lytic granules. In the present study, we first performed probability-state modeling of differentiation and lytic granule markers on CD8(+) T cells to enable the comparison of bona fide CTLs with NK cells. Analysis identified CD57(bright) expression as a reliable phenotype of granule marker-containing CTLs. We then compared CD3(+)CD8(+)CD57(bright) CTLs with NK cells. Healthy adult peripheral blood CD3(+)CD8(+)CD57(bright) CTLs expressed more granzyme B but less perforin than CD3(-)CD56(dim) NK cells. On stimulation, such CTLs degranulated more readily than other T-cell subsets, but had a propensity to degranulate that was similar to NK cells. Remarkably, the CTLs produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistiocytosis, CTL and NK cell degranulation were similarly impaired. Therefore, cytotoxic lymphocyte subsets have similar requirements for Munc13-4, syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3(+)CD8(+)CD57(bright) CTLs and NK cells and suggest that analysis of CD57(bright) CTL function may prove useful in the diagnosis of primary immunodeficiencies including familial hemophagocytic lymphohistiocytosis.

An early step in immunosurveillance by cytotoxic lymphocytes is leukocyte functional antigen (LFA)-1-dependent adhesion to target cells, which is promoted by inside-out signals from receptors such as the T cell receptor and a variety of natural killer (NK) cell activating receptors. Inside-out signals induce a conformational change in LFA-1, resulting in an extension of the extracellular domain of the receptor. Here, we have evaluated several mAbs that specifically detect the extended conformation of LFA-1 and detail a protocol for flow cytometric quantification of β2-integrin activation in human peripheral blood cytotoxic T cells and NK cells in response to target cell recognition. By comparison to the markers of degranulation and chemokine synthesis, e.g. surface CD107a expression and intracellular MIP-1β expression, respectively, evaluation of LFA-1 conformational changes represent a sensitive and rapid parameter of primary cytotoxic lymphocyte activation that can facilitate isolation of viable cells. Potentially, combined with other read-outs of cytotoxic lymphocyte function, this technique is applicable to the assessment of various clinical conditions, including for the diagnosis of primary immunodeficiency syndromes and for evaluating the efficacy of tumor targeting by donor lymphocytes., (Copyright © 2011 Elsevier B.V. All rights reserved.)