Monomeric subunit of ProImmune's CD1d tetramer, loaded with KRN7000 (red)
Detect NKT Cells with Tetramers
ProImmune is the only commercial source worldwide for fluorescently labeled human and mouse CD1d tetramers.
CD1d-lipid complexes bind to T cell receptors of NKT cells of a particular specificity (as determined by the lipid ligand used), allowing identification and enumeration of antigen-specific CD1d-restricted NKT cells by flow cytometry.
Our tetramers show excellent brightness and experimental reproducibility. To avoid the difficulties associated with dissolving and loading the most commonly used ligand, alpha-Galactosyl Ceramide* (α-GalCer), our tetramers come pre-loaded. They can also be supplied empty for loading with your ligand of choice.
Natural killer T (NKT) cells are implicated in the regulation of immune responses associated with a broad range of diseases, and seem to be essential for several aspects of immunity. They represent a unique lymphocyte population that co-express NK cell markers and a semi-invariant T cell receptor. When stimulated with CD1d-restricted glycolipid antigen, NKT cells produce large amounts of Th1-type and/or Th2-type cytokines that lead to downstream activation of dendritic cells, NK cells, B cells and T cells. The dysfunction or deficiency of NKT cells has been shown to lead to the development of autoimmune diseases (such as diabetes or atherosclerosis) and cancers, and they have also been implicated in the disease progression of human asthma.
CD1d molecules are non-classical MHC molecules that are characterized as non-polymorphic, conserved among species and possessing narrow, deep, hydrophobic ligand binding pockets. These binding pockets are capable of presenting glycolipids and phospholipids to Natural Killer T (NKT) cells. NKT cells represent a unique lymphocyte population that co-express NK cell markers and a semi-invariant T cell receptor (TCR). They are implicated in the regulation of immune responses associated with a broad range of diseases.
The best characterized CD1d ligand is α-Galactosyl Ceramide* (α-GalCer), originally derived from marine sponge extract. Presentation of α-GalCer by CD1d molecules results in NKT cell recognition and rapid production of large amounts of IFN-gamma and IL-4, bestowing α-GalCer with therapeutic efficacy. More recently, the lysosomal sphingolipid isoglobotrihexosylceramide (iGb3) has been identified as a CD1d ligand. This endogenous sphingolipid is thought to be responsible for NKT cell development.
ProImmune’s fluorescent human CD1d negative control tetramer is mock-loaded with carrier only (no ligand loaded) and will not bind to NKT cells. The use of this negative control reagent in conjunction with a ligand loaded CD1d tetramer will allow low frequency positive populations to be accurately quantified. Note: the negative control CD1d tetramer cannot be loaded with ligand.
Human CD1d Tetramer and Negative Control Tetramer
Human CD1d tetramer pre-loaded with α-GalCer* and negative control tetramer, APC labeled, tested with PBMCs.
Figure 1: 1 x 106 PBMCs were incubated with 1 test (0.5 µl) APC labeled, α-GalCer loaded human CD1d tetramer (left plot), or 1 test (0.5 µl) APC labeled, negative control human CD1d tetramer (right plot) for 30 minutes at 4°C. Following a wash step the cells were incubated at 4°C for 20 minutes with anti-CD3 FITC and anti-CD19 PE in 50 µl total volume. Following two further washes the cells were acquired and analyzed by flow cytometry. Non-specific staining was eliminated from the plot by gating on CD19 negative cells before plotting CD1d tetramer vs CD3.
ProImmune's human CD1d tetramer has also been shown to stain PBMCs from rhesus macaque monkeys - see data from the customer case study.
Mouse CD1d Tetramer
Dr. Markus Skold and Dr. Sam Behar, Harvard Medical School (USA), tested ProImmune's mouse CD1d R-PE-labeled tetramer with splenocytes from a naive B6 mouse depleted of B cells. The tetramer was used empty or loaded with α-GalCer*. Cells were also stained with anti-CD4-Alexa488 and anti-CD3-PerCP monoclonal antibodies and gated on live, CD3 positive cells.
Figure 2: In order to reduce background staining, splenocytes were depleted of B cells using CD19 microbeads (Miltenyi Biotec). The procedure used 3 x 105 cells per stain. Cells were incubated with 2.4G2 monoclonal antibody at 25µg/ml in 50µl per sample, at 4oC for 15 minutes in order to block Fc receptors. Following a wash step, cells were incubated with one test (2µl) of mouse CD1d tetramer for 30 minutes. The cells were then incubated at 4oC for 20 minutes with anti-CD4-Alexa488 and anti-CD3-PerCP monoclonal antibodies in 50µl total volume. Following two further washes, the cells were acquired and analyzed by flow cytometry.
* The alpha-GalCer molecule and derivatives thereof are covered by US Patent No.5,936,076, which is owned by Kirin Pharma. The alpha-GalCer molecule is licensed to Funakoshi Co. Ltd. for research use worldwide.