|Home | About | Journals | Submit | Contact Us | Français|
T-cell recognition of peptide/MHC is highly specific and is sensitive to very low levels of agonist peptide, however, it is unclear how this is achieved or regulated. Here we show that clustering class I MHC molecules on the cell surface of B-lymphoblasts enhances their recognition by mouse and human T-cells. We increased clustering of MHC I molecules by two methods, cholesterol depletion and direct crosslinking of a dimerizable MHC construct. Imaging showed that both treatments increased the size and intensity of MHC clusters on the cell surface. Enlarged clusters correlated with enhanced lysis and T-effector function. Enhancements were peptide-specific and greatest at low concentrations of peptide. Clustering MHC I enhanced recognition of both strong and weak agonists but not null peptide. Our results indicate that the lateral organization of MHC I on the cell surface can modulate the sensitivity of T-cell recognition of agonist peptide.
Recognition of peptide/MHC molecules by CD8+ cytotoxic T-cells is a fundamental problem for immune function. T-lymphocytes must constantly monitor antigen presenting cells (APCs) of the body to identify epitopes that indicate infection by pathogen or damaged tissue. However, to identify their targets, T-cells must find a specific peptide/MHC in a sea of non-cognate self peptide/MHC on a cell surface. These cognate peptides may be scarce and may also be weakly-activating. It is particularly unclear how weak agonists are distinguished from self peptide/MHC since their affinity for the T-cell Receptor (TcR) may, in some cases, be lower than that of non-activating, antagonist peptides (1).
It has been shown that effector T-cells can respond to cells displaying as little as 1–10 agonist-loaded MHC on the surface (2) (3) (4) (5). These studies have focused on strongly-activating agonist peptides with high affinities for both MHC I and TcR. However, the molecular stoichiometry for binding strong agonists may not hold for weaker agonists. Furthermore, higher concentrations of agonist certainly engender better responses and surface MHC loaded with non-cognate peptides may also contribute to recognition (5). We still lack a comprehensive model to explain the full nature of T-cell recognition of cognate peptide/MHC.
Part of the answer to the problem of a T-cell maintaining high specificity and sensitivity to low levels of antigen may lie in the organization of MHC molecules on the surface of an APC. Biophysical studies have shown that class I MHC molecules are not randomly distributed on the surface of APCs and that disrupting their organization affects recognition of MHC molecules by CTLs. Native class I MHC molecules cluster in the presence of free class I MHC heavy chains (6). Integrins, class II MHC molecules, and the IL-2 receptor are also found in association with class I MHC on the cell surface (7). When these clusters are dissipated with exogenous beta-2-microglobulin, antigen presentation is reduced (8). These studies indicate that the surface organization of class I MHC molecules contributes to their function and suggest that surface MHC organization may participate in T-cell recognition of agonist peptide. Since clusters of class I MHC molecules contain both agonist and non-cognate endogenous peptides, these mixed-peptide bouquets may enhance recognition of agonist peptides complexes contained within them.
Surface organization of class I MHC molecules can be modulated by addition or removal of cholesterol to and from the cell membrane (9); this results in changes in clustering detected by fluorescence resonance energy transfer (FRET) and in reductions in lateral mobility and diffusibility (10). The mechanism of immobilization after cholesterol depletion appears to involve release of the signaling lipid phosphatidylinositol 4,5 bis phosphate, PI(4,5)P2, from the membrane inner leaflet and a consequent reorganization of cell actin (10). Stabilizing the actin membrane skeleton corrals membrane proteins and enhances their clustering on the cell surface (11) (12). Here we show that these cholesterol-depleted cells present peptide/MHC antigen better than control cells and that this enhanced presentation correlates with changes in class I MHC cluster size. Enhanced presentation is peptide-specific and is most effective at low levels of agonist peptide/MHC. We further show that clustering of class I MHC by another mechanism, direct crosslinking of engineered class I MHC molecules, also enhances presentation of peptide/MHC to T-cells, again enhancement is greatest when agonist peptide is scarce. Our results suggest that clustering enhances TcR scanning for and recognition of agonist peptide.
Cells were maintained in RPMI 1640 (MediaTech) + 2mM glutamine, 10% heat inactivated FBS (Hyclone) and 300ug/ml of G418 (Sigma) for plasmid selection, when applicable. JY B-lymphoblasts were a gift from J Strominger, Harvard. T2 (13), T2-Kb (14) and 2C T-cells (15), both naïve and activated, were provided by J Schneck, Dept of Pathology, Johns Hopkins Medical Institutions.
Activated 2C T-cells were generated in the Schneck laboratory by isolating splenocytes, activating with 1μg/ml agonist SIY peptide for four days, and then culturing them with 10U/ml IL-2 (Sigma) for an additional 3–6 days. Naïve CD8+ T-cells were purified by negative selection by MACS. Allogenic, A2-negative T-cells were provided by the JHMI Laboratory of Immunogenetics. Cells from one donor, the senior author, were used for all experiments. Allo T-cells were activated by co-culture with irradiated, JY (A2-positive) cells for 4 days.
JY target cells were acutely depleted of cholesterol by 1 hr incubation with 0.5U/ml cholesterol oxidase (Sigma #C5421) or 10mM methyl-β-cyclodextrin, MCD, (Sigma) for 30 minutes for TIRFM imaging. Chronic depletion was achieved by culturing cells in medium with 10% FBS lacking LDL (Invivogen) for 3 to 14 days. Cell levels of cholesterol were reduced to 50–60% of control by this chronic deprivation. As we noted earlier (10), the effect of cholesterol depletion on lateral mobility was not reversed by growing cells in LDL-containing medium for 12 hours, though it was reversed after 24–36 hours. Actin cytoskeleton was disrupted in JY cells by incubating them with 5μg/ml Cytochalasin D (Sigma) for 30 minutes at 37°C, followed by extensive washing.
T2 cell lines were incubated overnight at 25°C, and then pulsed with peptide in serum-containing medium for 1–2 hours at 37°C. The peptides used were SIY (SIYRYYGL), SIN, (SIINFEKL), and p2CA (LSPFPFDL) (1). After peptide loading, cells were treated for 2 hours with 10nM AP20187 dimerizer from the Ariad Regulated Homodimerization Kit (www.ariad.com) and washed, extensively. When T2 KB-1BP cells were used to stimulate naïve CD8+ 2C T-cells, they were incubated with 0.01nM dimerizer for the duration of the stimulation.
Kb-1BP was cloned into the N3-YFP vector (Clontech) using PCR linkers. Mouse class I H2-Kb heavy chain was tagged with a C-terminal yellow fluorescent protein, YFP, as previously described (16). A single FKBP domain was excised from the pC4-Fv1E plasmid (Ariad) and inserted, inframe and downstream of YFP using XbaI and BamHI sites in both plasmids. Monomeric YFP mutation L221K (17) was added by QuickChange (Stratagene), and the entire open reading frame was sequenced in both orientations to confirm no additional point mutations had been introduced. The HLA-A2-YFP used for JY TIRFM imaging was described earlier (18).
Cells were transfected by electroporation using 10–20μg of plasmid DNA. For JY A2-YFP cells, these cells were selected for expression using 300μg/ml G418, followed by FACS sorting for high expressors. We were unable to create stable clones of T2 expressing Kb-1BP at moderate to high levels. Hence, for all experiments with the construct, T2 were transfected with Kb-1BP and used for assays 24–36 hours later.
T2 Kb-1BP cells were fixed in 4% para-formaldehyde (Electron Microscopy Sciences, C#15710) for 30 minutes on ice. Cells were washed and then plated on poly-L-lysine (Sigma) coated coverslips for 5 minutes, and then mounted with SlowFade© Gold (Invitrogen). Confocal microscope z-stack images were taken on a Zeiss LSM510 Meta with a 63X objective (Zeiss, Thornwood, NY) with appropriate YFP filters, PMT detector and resolution settings. Settings were kept constant for all images.
For TIRFM, live A2-YFP JY cells were plated on poly-L-lysine-coated sapphire coverslips and maintained at 37°C by a Focht Chamber System 2 (FCS2) live cell chamber (Bioptechs, Butler, PA). Images were taken on an Olympus IX-70 microscope (Melville, NY) using 100X 1.65NA objective and a Cooke SensiCamQE CCD camera (Romulus, MI). Detection settings were kept constant between treatments.
Clusters were captured from images using NIH-Image (or ImageJ) above a constant threshold and then analyzed using Origin 7.0 (Microcal), GraphPad 4.0 (Prism) and Excel (Microsoft) to generate distributions for cluster size and intensities. For images of Kb-1BP with or without dimerizer, distributions of cluster mean intensities were compared by using a (Gaussian) student t-test in Graphpad 4.0. Cluster size distributions for treated and untreated were compared by non-parametric t-test assuming equal variances.
For chromium release assays, target cells were incubated with 100μCi 51Cr (Perkin Elmer) for 1hr at 37°C, washed three times, and incubated with effector cells at ranges from 10:1 to 1:4 effector:target, keeping the target cell number fixed at 1×104 cells and the volume of media at 200μl in 96-well round bottom plates. After four hours, cells were centrifuged and 100μl of supernatant was measured for 51Cr using a Beckman 5500 gamma counter. Experiments were conducted in triplicate and compared to spontaneous release of targets alone and maximal release of targets in 1M HCl. Specific lysis was calculated as 100 × (experimental − spontaneous) / (maximal − spontaneous).
Serine esterase release was measured with T-cells mixed at a 10:1 ratio with JY cells for 4 hours. Supernatents were harvested and serine esterases were detected in a colorimetric assay as described (19). To provoke maximal release, effectors were treated with 40ng/ml PMA (Sigma C#13139019) and 2μg/ml calcium ionophore (Sigma C#7522). Percent release was calculated as 100 × (experimental − background) / (total − background). All conditions were performed in triplicate.
FACS analysis was conducted on a FACSCalibur (BD, Franklin Lakes, NJ) using AnnexinV-Cy5 (BD), anti-Kb antibodies, 20.8.4s (20), and Y3 (21) for staining. For the FACS killing assay, cells were mixed at 1×104 targets to 5×104 activated effectors and incubated for 2 hours. Cells were washed and then stained with Annexin-V (AV) for 15 minutes and then washed with AV calcium buffer. Kb-1BP positive cells were gated on FL1 channel for YFP content (~100-fold higher than background autofluorescence) at levels that had comparable surface-Kb levels between treated and untreated cells and then analyzed for their percent Annexin V positive in FL4 channel.
To assay stimulation of naïve cells in terms of IFN-gamma production, CD8+ cells were mixed at 1×105 with an equal amount of stimulator cells in triplicate in 200μl of media for 48 hours. Cells were centrifuged and 50μl of supernatant was assayed for IFN-gamma by ELISA. Experiments were conducted in triplicate, and ELISA was performed using an ELISA kit (BD Cat#551866) following the protocol provided, and using a Molecular Devices plate reader to acquire colorimetric measurements.
Previously we showed that cholesterol depletion reduces lateral mobility of class I MHC (10). As predicted by our model for membrane clustering (11), this change in mobility altered class I MHC clustering. Cells acutely depleted of cholesterol had larger and brighter clusters of HLA-A2 than control cells. This can be seen qualitatively by comparing Figures 1A and 1B and is quantified in 1C and 1D. Clusters of class I MHC molecules at or near the surface of cholesterol-depleted JY cells (Figure 1B) were on average five times as bright as the clusters of class I MHC molecules at the surface of control cells (Figure 1A). The average apparent size of class I MHC clusters increased slightly in cholesterol-depleted cells, from 330 +/− 90 nm to 353 +/− 76 nm, respectively. Although the modal value for the two populations was the same in the range 300–350 nm, there was an excess of larger clusters on the surfaces of cholesterol depleted cells. These increases in clustering were a result of redistribution of class I MHC molecules, as the total intensity of HLA-A2-YFP on the cell surface was not changed.
Increased clustering due to cholesterol depletion enhanced antigen presentation in two different assays of T-cell function. Cholesterol-depleted cells provoked a greater release of serine esterase from effector T-cells than did control cells (Figure 2A). The fraction of granzyme released by CTL stimulated by chronically LDL-deprived cells or by cells treated acutely with cholesterol oxidase was as large as that in a positive control of T-cells stimulated with phorbol ester and more than two-fold that of T-cells responding to control JY cells. Consistent with their enhanced serine esterase release, CTLs killed allo-target cells depleted of cholesterol more efficiently than they killed control target cells. The results of one assay, over a range of effector:target (E:T) ratios are shown in Figure 2B; the lysis of cholesterol-depleted cells was about twice that of control cells at a given E:T. Cholesterol depletion did not change the amount of class I molecules on the cell surface and did not change the rate of E:T conjugation, measured by flow cytometry (data not shown). Enhanced lysis after cholesterol depletion was not due to changes in the osmotic fragility of the target cells, as cholesterol-depleted and control cells were equally sensitive to lysis in hypotonic saline (data not shown).
Restoring class I mobility of cholesterol-depleted cells also restored normal antigen presentation. Our previous results (10) showed that changes in MHC mobility due to cholesterol depletion were mediated through stiffening of the actin cytoskeleton. Consistent with this, disrupting actin filaments restored normal MHC mobility. If enhanced presentation by class I after cholesterol depletion is due to confinement and clustering by actin filaments, we would expect that disrupting actin would reverse the effect of cholesterol depletion. This was the case. When cholesterol-depleted cells were treated with an actin depolymerizer, Cytochalasin D (CytD), for 30 minutes before incubation with effector cells, T-effector response, as measured by serine esterase release, was reduced to control levels (Figure 2C). Control cells treated with CytD alone were as effective at stimulating serine esterase release as untreated cells. Similar effect was seen in CTL-mediated killing by assayed by chromium release; cholesterol-depleted targets produced lower 51Cr release when pretreated with CytD, whose effect is only slowly reversible, than without treatment (data not shown).
The peptides recognized by alloreactive CTL are poorly defined. To better understand the mechanism of enhanced recognition after cholesterol depletion, we investigated peptide-specific recognition and lysis using CTLs from the 2C transgenic mouse. The 2C TcR recognizes the SIY peptide in the context of the mouse class I MHC molecules H2-Kb as a strong agonist, recognizes the p2CA peptide as a weak agonist, and does not respond to the SIN peptide (1). We used a TAP-deficient cell line, T2, expressing H2-Kb, to load H2-Kb specifically, with peptide of our choice at different levels. This allowed us to measure effects of cholesterol depletion for a range of peptides and over a wide range of surface concentrations of peptide/MHC. When target cells were loaded at high agonist peptide (SIY) concentrations (10μM), cholesterol depletion modestly enhanced (10%) cell lysis at a range of E:T ratios. However, when cells were loaded at 1000-fold lower concentrations of agonist peptide (10nM), cholesterol depletion enhanced cell lysis by about 50% (Figure 3A).
The enhancements due to cholesterol depletion as a function of peptide concentration are summarized in Figures 3B–D. Optimal enhancements were seen in the 1–100nM range SIY peptide (Figure 3B). Cholesterol depletion had an even larger effect on the killing of cells loaded with the weak agonist peptide, p2CA, whose affinity for the H2-Kb and 2C TcR is 1000-fold lower than SIY (1). At low concentrations of p2CA, killing was enhanced by over 80% (Figure 3C). At higher concentrations of p2CA, cholesterol depletion did not enhance and actually showed a decrease in killing. Non-specific killing of null peptide, SIN, loaded cells was not enhanced by cholesterol depletion at any concentration; indeed, there was some reduction in basal lysis (Figure 3D). If peptide levels were titrated sufficiently low, or no peptide was added at all (Figure 3C), cholesterol depletion reduced presentation. Overall, cholesterol depletion greatly enhanced presentation at low peptide concentrations of agonist peptide. At other concentrations, effects of cholesterol depletion varied from smaller enhancements to inhibition of presentation.
While cholesterol depletion can be used to cluster MHC, it may have other consequences for the cell (22). Given the correlation between enhanced clustering and enhanced killing, we tested whether aggregating class I MHC independently of cholesterol depletion would enhance recognition by CTLs. We engineered an H2-Kb tagged with a YFP and a single binding domain for an analog of FK506. This construct, Kb-1BP, dimerizes in cells upon the addition of a membrane-permeable, divalent analog of FK506, AP20187 (23). We reasoned that dimerizing class I molecules would stabilize and enhance pre-existing class I MHC clusters (Figure 4A). Confocal images of the upper surface of cells expressing our crosslinkable construct, Kb-1BP showed an increase in cluster size and intensity (Figures 4B–E) after adding dimerizer. Consistent with this result, the avidity of antibody binding to α1/α2 domain of H2-Kb (24) increased after dimerization (Figure 4F). The avidity of a second antibody, Y3, whose binding has only partially been mapped (21), and has a much lower affinity for H2-Kb was unchanged. There was also an unexpected two-fold reduction in overall surface H2-Kb levels (Figure 4G) upon the addition of dimerizer; this probably reflects induced endocytosis of MHC clusters (25).
We measured 2C CTL-mediated killing of target cells transiently expressing Kb-1BP with or without the dimerizer. These cells showed a wide distribution of Kb-1BP surface expression levels, depending on efficiency of transfection and dimerizer concentration. Cells treated with 10nM dimerizer for two hours had half the surface Kb-1BP of untreated cells for any particular YFP expression level. However, to investigate the effects of clustering on lysis of target cells, we needed to compare treated and untreated expressing equal levels of surface H2-Kb. Therefore we used FACS to gate cell populations in terms of YFP fluorescence that would correspond to comparable levels of surface Kb as shown in Figure 4G. We measured apoptosis by staining with Annexin V and compared the difference in Annexin V positive cells after dimerization (Figure 5A). Upon clustering of Kb-1BP, there was an enhancement of T-cell mediated apoptosis by 60–90% at low SIY agonist peptide concentrations (Figure 5B). At high concentrations of agonist, we saw no increase in presentation due to clustering. Again, inducing class I dimerization on cells loaded with the weak agonist, p2CA, increased cell killing at low concentrations and decreased it at high concentrations (Figure 5C), consistent with the data from cholesterol depletion. The dimerizer did not enhance killing of T2-Kb lacking the 1BP domain (data not shown).
The effect of clustering MHC on enhanced recognition by T-cells was not limited to effector cells. We measured activation of naïve CD8+ 2C T-cells in terms of IFN-gamma production stimulated by Kb-1BP under various conditions. Dimerizing the stimulating antigen enhanced agonist recognition after loading with low, nanomolar, concentrations of peptide, while it reduced recognition after loading with high, micromolar, concentrations of peptide (Figure 6).
Class I MHCs are clustered on the cell surface. In order to assess the functional consequences of clustering, we developed two methods to enhance the size of MHC clusters, cholesterol depletion and specific crosslinking of an engineered MHC molecule. Overall, clustering by cholesterol depletion and dimerization produced similar effects in presentation. However, at certain concentrations of peptide, the effective changes differed between treatments. These discrepancies may be due to a difference in the size and nature of clusters generated by the two treatments. They may also be due to technical reasons; we required different assays for the effects of the two treatments, therefore the data in Figure 3 and and55 may not be equivalent. Nevertheless, clustering by either method enhanced recognition of peptide/MHC by T-cells, especially when agonist peptide was scarce.
Both acute and chronic depletion of target cell cholesterol enhanced granzyme, serine esterase, release by activated effector CTL. Enhanced T-cell responses also translated into enhanced lysis of target cells. Allo-reactive T-cells killed APCs with clustered MHC more efficiently than they killed control target cells. These effects were seen at all E:T ratios. Conversely, dispersion of clusters on cholesterol-depleted cells returned their presentation to control levels. As we mentioned earlier, we argue that the effects on class I mobility and clustering by cholesterol depletion are mediated through the actin cytoskeleton (10). Stiffening and loosening the actin cytoskeleton using various actin filament drugs produced the same effects on presentation as predicted.
When we switched to a peptide-specific model of recognition, we could only enhance killing at low concentration of agonist peptide. This is consistent with the idea that allo-recognition involves only a subset of the peptides represented on the surface and that clustering may facilitate T-cell scanning for cognate peptide. If lysis was enhanced at all concentrations of agonist, one could argue that the functional consequences of cholesterol depletion were downstream of agonist recognition. Moreover, if lysis was also enhanced in the case of non-specific peptide or no added peptide, one could argue that recognition by T-cells did not play a role in the effects seen. However, we only saw enhancement at low agonist peptide concentrations, which argues strongly that the changes we found were due to changes in the efficiency of presentation.
Clustering class I MHC by crosslinking engineered H2-Kb molecules confirmed and extended our functional results on clustering of class I MHC molecules by cholesterol depletion. Microscopy shows that dimerizing MHC molecules stabilizes and amalgamates the small clusters of MHC present on APC surfaces.
Different peptides produced different effects when clustered. In the case of cells presenting high concentrations of SIY agonist, we saw weak enhancement of lysis after cholesterol depletion and saw some reduction after dimerizing Kb-1BP. We argue that at high levels of strong agonist, T-cells are efficiently activated; therefore clustering has a small effect on recognition. As we titrated down the levels of agonist peptide, recognition was less efficient, and clustering enhanced it. This was seen even more clearly when cells were loaded with the weak agonist peptide, p2CA, whose recognition by T-cells is inefficient. Clustering class I MHC on cells loaded with low concentrations of p2CA enhanced specific lysis to almost double that of controls while at high concentrations of p2CA, clustering actually inhibited recognition. In most cases, enhancement seemed to peak at nanomolar concentrations and would in some cases drop off at very low concentrations.
Clusters may enhance recognition by providing multiple local copies of some peptide/MHC or by enhancing T-cell residence time on a target cell. Normally, scarce agonist peptides are presented in a sea of self-peptide. Clustering of class I MHC molecules would cause a reduction in their rotational and lateral diffusion as compared to monomers on the plasma membrane. These large, slowly-rotating lattices of class I MHC could enhance recognition by providing T-cells with stable local densities of class I MHC + peptide combinations to sample. Since TcR can dock on a class I MHC in a variety of orientations and tilts (26), cluster of class I may also facilitate proper docking and reduce scan time for TcR sampling. In addition, as some TcRs may exist as clusters as well, engaging clusters from both the T-cell and APC side may provide efficient activation.
Clusters of MHC may also function to provide simultaneous sampling of self- and non-self peptide by multiple TcR in a membrane domain. At high concentrations of strong agonist, the density of signal is more than sufficient and clustering may not be necessary for recognition, as was seen in our results with SIY peptide. However, in the absence of strong agonist at high surface concentrations, the sampling of bouquets of self and agonist may be a way T-cells can differentiate minute class I MHC peptide differences to make dramatically different outcomes for activation. It is important to note that in our peptide-loading model, APCs were loaded in the presence of serum. At high concentrations of peptide with high affinity for MHC, such as SIY or SIN, serum proteins probably had little effect on surface loading of MHC. However at low concentrations of agonist, serum was likely to contribute non-specific, non-activating peptides to the surface display. In the case of weak agonist, p2CA, with weak affinities and short koff for the TcR (1), the role of non-specific peptide may be particularly important for recognition. This may explain why at low concentrations, clustering had such a strong effect on p2CA recognition. It may also explain the negative effects clustering had on presentation of high concentrations of p2CA. Clustering high concentrations of weak agonist might produce a “bland” signal, and may read too similar to a null peptide when presented in the absence of null peptide. This also suggests that the choice of null peptide would play an important role in effective detection of weak agonists as others have suggested for class II MHC (27).
From our results, we would not be surprised to find that professional APCs modulate class I clustering in order to modulate antigen presentation. Others have shown IFN-gamma treatment, known to upregulate class I levels, also reorganizes class I on the cell surface (28) (29). This change could enhance presentation to and activation of naïve T-cells. On the other hand, it may be that dispersion of clusters or their absence on some APCs (6) leads to T-cell anergy rather than activation. It is unclear to what degree cells regulate class I clusters in terms of size and stability. But from their functional importance in presentation, clusters may likely be regulated in an inducible manner.
Taken together, these data suggest that MHC organization plays an important role in modulating T-cell sensitivity for agonist peptide. Clustering MHC may enhance recognition of weak or rare epitopes in cancer and viral vaccine models. One direction others have taken in cancer immunology has been to identify and expand the number of oncogenic epitopes recognized by ex-vivo stimulated tumor infiltrating lymphocytes (30). Clustering class I on these ex-vivo stimulations may expand the repertoire of T-cell clones stimulated thereby enhancing the engendered response.
We thank Jonathan Schneck, Kapil Gupta, Joan Bieler, Dept of Pathology, JHMI, for maintaining and providing 2C cells and many helpful discussions. We thank Antony Rosen, Dept of Rheumatology, JHMI, for help with the granzyme release assay.
1This work was supported by an award from the Center for Alternatives to Animal Testing (JHU) (G.K.G.), and research grants (AI-14584, GM058554) (M.E.) and a training grant (T32 GM07231) to the Department of Biology.
TIRFM – Total Internal Reflection Microscopy
CytD – Cytochalasin D
YFP – Yellow Fluorescent Protein
LDL – low density lipoprotein