ProImmune is the leading 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.
Engkilde, K. et al. (2010). “Prevention of diabetes in NOD mice by repeated exposures to a contact allergen inducing a sub-clinical dermatitis.” PLoS One. 11;5(5):e10591.[PubMedID:20485668]
Figure 1. Repeated application of PPD inhibits the development of diabetes in NOD mice. The figure shows a Kaplan-Meier curve of the diabetes incidence for four groups of mice. DNCB-rep, PPD-rep and Water, refers to the groups of mice that were repeatedly exposed to DNCB, PPD or water every other week. PPD-induc refers to the group of mice that was only exposed to PPD in the fourth week of life. The NOD mice exposed to PPD repeatedly, displayed a cumulative diabetes incidence of 47%, in contrast to 93% of the water treated group (P=0.004). The log-rank test for all the shown curves gives a significance of P=0.008.
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 pre-loaded with α-GalCer* and negative control tetramer, APC labeled, tested with PBMCs.
Figure 2. 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.
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 3. 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.
Nur, H. et al. (2013). “Preclinical Evaluation of Invariant Natural Killer T Cells in the 5T33 Multiple Myeloma Model” PLoS ONE 2013 8(5): e65075.tps://www.ncbi.nlm.nih.gov/pubmed/23741460
Figure 4. Representative FACS analysis of murine iNKT cells from blood, BM, spleen and liver in naive and 5T33MM mice. Live cells (7-AAD negative) were stained with a-GalCer/CD1d tetramer which specifically binds to Va14 of the invariant TCR and with TCR-b. Double positive iNKT cells were gated. The percentages are indicated in each plot.
Article kindly contributed by Prof. Derek G. Doherty, Discipline of Immunology, School of Medicine, Trinity College Dublin, Ireland
Most T lymphocytes are thought to display specificity for peptide fragments of protein antigens presented by major histocompatibility complex (MHC) molecules. However, many studies have demonstrated T cell reactivity against lipid and glycolipid antigens presented by the MHC-like antigen-presenting molecule CD1. They include lipid components of microorganisms as well as self-antigens, many of which are made in response to cell stress or infection, therefore, detection of lipid-reactive T cells may be important for understanding disease pathology and immunity. Indeed, many studies have highlighted the importance of invariant natural killer T (iNKT) cells in protection against infection, cancer and autoimmune disease and cellular therapies using iNKT cells are showing promise in clinical trials in cancer patients. iNKT cells recognize lipids presented by CD1d, one of four human CD1 isotypes known to present antigens to T cells. They have been extensively studied because they can easily be activated using the xenogeneic glycolipid, α-galactosylceramide (α-GalCer) and detected using antibodies specific for their invariant T cell receptor (TCR) α-chains (Vα24Jα18 in humans). We have recently shown that circulating iNKT cells are depleted from patients with oesophageal adenocarcinoma (OAC) and may protect against the disease. But it is now apparent that humans have many other CD1d-restricted T cells, termed type 2 NKT cells, that recognize other lipids using non-invariant TCRs. Furthermore, murine studies have provided evidence that type 2 NKT cells can regulate tumour immunity.
To examine the presence of type 2 NKT cells in OAC patients, my PhD student Ashanty Melo and I used ProImmune’s CD1d tetramers loaded with various glycolipids to detect CD1d-restricted T cells using flow cytometry. Initially we optimized glycolipid loading using α-GalCer and tested if the loaded tetramer could detect lines of expanded iNKT cells. We loaded the tetramers with a 12 molar excess of vortexed, heated and sonicated α-GalCer, diluted to 0.2 mg/mL in PBS containing Tween-20 or saponin, for 18 hours at room temperature and found that 0.25 µL of original tetramer was sufficient to detect iNKT cells using flow cytometry (Figure 5). Importantly, the same amount of negative control tetramer, provided by ProImmune, did not stain any cells (not shown).
Figure 5. Detection of iNKT cells using α-GalCer-loaded CD1d tetramers. The left panel shows a flow cytometry dot plot showing expression of CD3 and the Vα24/α18 TCR by expanded human iNKT cells. The right hand plots show detection of the same cells using 0.025, 0.1 and 0.25µl of the α-GalCer-loaded CD1d tetramer.
We next attempted to repeat these experiments using other glycolipids that are known or predicted to bind to CD1d and activate T cells. However, optimization was more difficult because we did not have cell lines known to express TCRs specific for the lipid/CD1d complexes. We used peripheral blood mononuclear cells (PBMC) from healthy donors and since some γδ T cells of the Vδ1 subtype are known to recognize glycolipids presented by CD1d, we also tested our loaded tetramers with expanded lines of Vδ1 T cells. Importantly, 0.25 µL of CD1d tetramer, the amount optimized to detect iNKT cells, failed to detect type 2 NKT cells. We found that 1µL of CD1d tetramer loaded with similar molar ratios of sulfatide, lysosulfatide, cardiolipin or tetramyristoyl cardiolipin detected T cells within PBMC and/or Vδ1 T cell lines, whereas similar amounts of the negative control tetramer or empty tetramer did not (Figure 6).
Figure 6. Detection of type 2 NKT cells using glycolipid-loaded CD1d tetramers. PBMC (top panels) or sorted and expanded human Vδ1 T cells (bottom panels) were stained with an anti-CD3 antibody and 1ul of CD1d tetramer, unloaded or loaded with the indicated glycolipids. Tetramer positive cells were then enumerated by flow cytometry. FMO, fluorescence-minus-one control staining no tetramer.
We next enumerated the above type 2 NKT cell populations in blood from OAC patients and healthy control subjects and found that T cells reactive against sulfatide and tetramyristoyl cardiolipin were present in significantly greater numbers in the OAC patients, in whom they accounted for up to 10% of total T cells and up to 80% of Vδ1 T cells. Having identified novel type 2 NKT cells in patients with OAC, we were then able to characterize their functional responses to CD1d-positive cells presenting the glycolipids, and have found evidence that they suppress the anti-tumour responses of iNKT cells. Thus, CD1d tetramers can be used to identify novel T cell populations that may influence immunity against cancer and infectious pathogens.
