Year: 2012
Type: article
Abstract: This panel was developed to characterize the phenotype of human natural killer (NK) cells from cryopreserved peripheral blood mononuclear cells (PBMC) isolated from ACD or EDTA anticoagulated whole blood or apheresis units (Table 1).The application of this panel was to identify changes in NK cell subsets with regard to receptor expression, maturation, homing potential, and activation in the setting of primary HIV-1 natural infection.However, this panel may be applied to a wide variety of disease and normal conditions to characterize NK cells in humans.The performance of this panel was tested on fresh and frozen PBMC as well as using a whole blood lyse no wash procedure. BACKGROUNDNK cells are innate effector cells representing $10% of circulating lymphocytes with more than 2 billion in circulation at any given time (1).NK cells are classically defined by the expression of two cellular markers; CD56, the neural cell adhesion molecule (NCAM) and CD16, the Fcc-receptor IIIa.These markers (CD56/CD16) allow the discrimination of at least three distinct NK cell subsets and their distribution is altered in HIV-1 infection (2).In developing this panel, commonly used conjugates, CD56 PE and CD16 FITC, were replaced with CD56 PE-Cy7 and CD16 Pac Blue, conjugates that better separated the NK cell subsets as well as to allow for commercial reagents to more exotic receptors in the PE and FITC channel (Table 2).CD8, which performs well with any fluorochrome was included using the tandem dye APC-H7 to further define NK subsets.NK cells are noted for the combination of activating and inhibitory receptors that regulate their activity.Most relevant in the HIV-1 research field are the killer cell immunoglobulin-like receptors (KIRs) as a number of genetic associations are observed between certain KIR and HLA combinations with regard to HIV-1 disease progression [reviewed by Bashirova et al. (3)].A very limited selection of antibodies directly conjugated to FITC, APC, and PE, are available for a subset of the KIR receptors with highly variable staining patterns typically observed.The KIR receptors that bind HLA-A, HLA-B, and HLA-C were most interesting and all combinations were procured and tested to identify the best signal-to-noise ratio.The KIR3DL1 demonstrates clear separation of positive cell populations in FITC, PE, and APC but we selected the Alexa Fluor 1 700 conjugate since this conjugate shows sufficient separation between positive and negative populations and is in a less common channel.Similarly, the KIR2DL1/ DS1 (clone HP-MA4) antibody demonstrates clear separation of positive cell populations in FITC and PE but we chose the tandem conjugate PerCP-Cy5.5 because of the highest signal-to-noise ratio.The KIR2DL3/DL3/DS2 antibody is available in FITC, PE, and APC but showed the greatest separation in PE.Based on limited reagent availability and the design of a more generalizable panel, we did not include KIR receptor reagents to discriminate activating forms of these receptors.KIR expression along with CD57 and NKG2A allow for the discrimination of a proposed NK cell differentiation process (4).CD57 (clone HCD57) is available in Alexa
Source: Cytometry Part A
DOI: 10.1002/cyto.a.22033
Authors: Michael A. Eller, Jeffrey R. Currier
Institution: Henry M. Jackson Foundation
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2050544484