<ol>
	<li>Khurana, A., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+Production+of+Recombinant+CD1d+Protein+in+Insect+Cells.">A Method for Production of Recombinant CD1d Protein in Insect Cells.</a> 12/10/2007) Journal of Visualized Experiments. , .</li></ol>

The authors have nothing to disclose.

interview: glycolipid antigen presentation by cd1d and the therapeutic potential of nkt cell activation

自然杀伤T细胞（NKT细胞）的免疫反应的癌症，调节autioimmune疾病，传染性病原体的间隙，artheriosclerotic斑块的发展的关键因素。在这次采访中，米奇Kronenberg讨论他的实验室的努力去了解通过糖脂抗原激活NKT细胞的机制。这些研究的核心是CD1d - 呈现糖脂NKT细胞的抗原提呈分子。 CD1d四聚体技术的问世，由Kronenberg实验室开发出一种技术，具体糖脂反应性T细胞亚群的分类和识别的关键。米奇解释糖脂受体激动剂作为治疗药物激活NKT细胞在癌症患者中，以及如何CD1d四聚体可以用来评估在体内糖脂受体激动剂治疗后NKT细胞人口的状态。正在进行的临床试验，使用这些受体激动剂的现状进行了讨论，以及米奇的预测，在免疫学领域的地区，将出现在不久的将来的重要性。

Watch this Scientific Journal Video about Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation at JoVE.com

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

自然杀伤T细胞（NKT），癌症autioimmunity监管，清除感染，并发展artheriosclerotic斑块的免疫反应的关键因素。在这次采访中，米奇Kronenberg讨论他的实验室的努力去了解通过糖脂抗原激活NKT细胞的机制。

专访：糖脂抗原提呈CD1d及NKT细胞活化治疗潜力

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

Hello, my name is Mitchell Cronenberg and I'm at the La Jolla Institute for Allergy and Immunology. I'm actually the president and scientific director, but I have a very active research lab and that's what we're gonna be talking about, of course, is one of the technologies we have, which is CD 1D tetramers. Most of the work in my lab concerns CD 1D, reactive T cells.And these cells are unique because they recognize glycolipids rather than peptides. And another branch of my lab is concerned with a completely different topic, the Mucosal immune system and its regulation. CD one molecules Are antigen presenting molecules, and that means that they bind antigens or fragments of proteins or other molecules and and hold them up for T-cell recognition.Now there are really two different kinds of antigen presenting molecules. There are ones that hold or present peptides, and there are ones that hold lipids and glycolipids. And the CD one molecules hold lipids and glycolipids.That's their function and they, they hold up or present these lipids and glycolipids for T cells. And there are actually four different kinds of CD one molecules, CD one, A, B, C, and D.And actually we focus on CD 1D and CD 1D in particular is, is probably the best study in part because there are mouse models. And CD 1D presents glycolipids to a particular kind of T cell called a natural killer T cell, an unusual white blood cell that has features of the innate and adaptive immune response.The glycolipids that are presented can be self glycolipids, although those are not very well characterized at this point, or they can be microbial glycolipids. And the glycolipids that we know that are presented come from two sources. One are what are called FMS bacteria, which are probably the most abundant bacteria in the biosphere.They live in the ocean, they live in plants, they live in soil. However, they don't make many people sick. We're very used to living with these bacteria, I suppose.And the second category that we know of, a different type of glycolipid called a dyl glycerol containing glycolipids. Those are found in Borrelia borre, which cause Lyme disease and probably in many other pathogens. And we're just uncovering now what the different types of bacteria are that have these glycolipids.So we have what are called glyco singal lipids from SMS and dyl glycerol lipids from the Borrelia and probably many others that are truly pathogenic. These lipids get loaded onto CD 1D molecules on dendritic cells, predominantly, although it could be B lymphocytes, ER cells in the liver, and perhaps other cell types. These CD 1D molecules go through lysosomes and endosomes where they probably bind these lipids and then they carry the lipid out to the cell surface for recognition by T cells, including the natural Killer T cells that I mentioned earlier.One of the things that happens If you are exposed to a bacteria that has glycolipids is that the NK T cells get activated very rapidly. Conventional T cells. In other words, peptide reactive T cells typically will take three or four days to get a good immune response going, especially if they're not antigen experience.In other words, if they, they're naive. By contrast, the NK T cells can be activated within a matter of hours and they'll start producing cytokines like gamma interferon, important for bacterial clearance in a very, very short period of time. And in that way, they resemble aspects of the innate immune response from model.From studying model antigens. We know that CD 1D goes into late endosomes and lysosomes, very acidic intracellular compartments where the glycolipid antigens are loaded into CD one, and they're in fact special transfer proteins inside the antigen presenting cells like the dendritic cells that help to get the lipids loaded into the CD 1D molecule with these fona bacteria, borre bacteria and others were dealing with extracellular bacteria. So the lipid antigens must be somehow transported into the cell, probably into a lysosome loaded into the CD one molecule.And then there's an immediate activation of the NKT cell and all of this must take place within, within a few hours in the sinusoids, in the Liv of the liver, in the spleen, perhaps in the bone marrow And other sites where NKT cells are found. The tetrameric structure Is really key to unambiguously defining these natural killer T cells, which have these innate properties that distinguish them from other T cells. The other markers that are available are really too ambiguous, but this, the tetramers allow you to identify the cells based on their antigen receptor specificity, which is the key fact that defines them in a way, the making of tetramers goes back a long way.It was invented by Mark Davis and John Alman, and the idea was that the T-cell receptor has of low affinity, and if you want to define cells or identify cells based on their specificity, the peptides that they recognize or the complexes of peptides and antigen presenting molecules don't have enough affinity, enough binding strength to allow you to identify the cell. And so what Mark Davis and John Allman invented was this tetramer technology, a way of making te tremors in some cases OCRs of the antigen presenting molecule plus the molecule they present, which basically increases the affinity or the affinity of the interaction. So you can see the cell that's specific for flu peptide and a particular antigen presenting molecule or whatever the antigen is that you're interested in.Very important technology because it allows you, for example, to see how effective a vaccine regimen might be. What we did starting back in the late nineties, originally I a paper published in 2000 that's been cited about 500 times by now. What we did is just adopt that technology to lipids.So we made soluble CD one molecules and we, by methods, I'll describe them momentarily, we were able to mize them, in other words, mize them, add them with specific glycolipids, and in that way we could use them as reagents to assay for specific T-cell receptors. In other words, those that would see the Glycolipid we're interested in plus CD 1D. The Tetris can be used In any situation in which you want to analyze the number of NKT cells and their activation state and their function.And in our lab it could be from any one of a number of different projects. For example, if you want to ask, does a particular mutant and a signaling pathway affect NK T cell development, we can do that with our tetramers, ask about the number of NKT cells. And then there are other markers, other cell surface proteins we can look at that will help us to assess if the NKT cells are mature or if there are activated using the tetramers we, after an antigenic stimulation, we can also ask, not only are they activated, but what cytokines are they making.So we can combine tetramer staining with, for example, intracellular cytokine staining and ask what percentage of the NKT cells are making gamma interferon, what percentage you're making IL four, what percentage you're making, both with multi parameter flow cytometry, we can, we can ask a variety of questions about cytokine production directly ex vivo by these cells. We can also, we can also carry out further experiments. For example, we can use the tetramers to purify NK T cells for culture in vitro to ask about how they interact with other cell types, or even for transfer to a recipient animal, for example, which might lack NK T cells for genetic reasons.We wanna restore that function. We can do that and ask and ask about where the cells home and how they function. Finally, I know in, in the case of human NKT cells, people have actually used the tetramers to expand cell lines in vitro to grow up cells for analytical purposes And potentially even for therapeutic purposes.Well, we use a Bao virus expression system for generating CD 1D tetramers. So the process starts with a, a common DNA construct containing promoter and expression of the BID two microglobulin and the CD 1D heavy chain. So this, this CD one molecule is a heterodimer of two polypeptides.So we have this two, two promoter expression system, and we infect insect tissue culture cells with the vao virus viruses carefully titrated as our channel explained. And then we infect the cells and we harvest from the tissue culture medium, the, the protein that's expressed by the cells. The CD 1D heavy chain is engineered, especially for tetramer production.There are two things, or rather three things I should say, that we do. One is we remove the transmembrane piece. So we, we have, we're expressing a soluble protein.Secondly, we've added a hexa, his tag so that the protein can be purified by metal ion chromatography. And third is we have what's called a beer, a site for enzymatic biotin. And this is the innovation.It was very important when Davis and Alman first invented the tetramer production technology. So after we harvest the protein, it's, it is purified on the nickel comp by nickel column chromatography there. Sometimes we do size exclusion chromatography after that.And then the protein can be enzymatically biotinylated incubated, and that once it's enzymatically biotinylated, then when incubated with strep ab, it will form tetramers. But the soluble CD one protein that's ated can be incubated basically overnight in a solution of a glycolipid of interest. That very, it's very important that the glycolipid not be dissolved in DMSO because which is typical for glycolipids as a diluent or solvent, because CD one's very sensitive to that.So we typically use a, a buffer that contains a small amount of tween and PBS. And that buffer is, is, is very effective for dissolving most of our glycolipids. And for loading into CD 1D, which seems to occur spontaneously, we use streptavidin from any one of a variety of manufacturers.And it coupled to pe, fico, eryn or a PC phycocyanin dyes, either one, seems to work very well and gives us a very good staining. So overall, really the procedure is not too different from the well characterized procedures for making MHC Class one tetramers, except we don't do, we, we don't use bacterial expression, we use bacteria virus expression, and of course we Have lipid antigens rather than peptides. One point I should stress Is that humans have NK T cells and they have the same specificity as the mouse NK T cells.So the specificity has been for at least 60 million years since we diverged from the rodents, and in fact, we make human CD 1D tetramers by the same techniques that you, you can see in the video that's been made here. And, but in fact, human NK T cells work very well with mouse tetramers and vice versa. So we don't really make the human tetramers as often just because we can use our mouse tetramers for human work.So there are many mouse models in which NK T cells have been shown to be critical determinants of the outcome of the immune response. And this ranges from the response to cancers regulation of autoimmune disease, clearance of infectious agents, and even the development of a sclerotic plaques. That's a chronic inflammatory process that occurs in in the arteries.There's some, some question about the importance of these cells in humans where there are a little less abundant, there have been many reports indicating that they are altered in autoimmune states, and also there are reports that they're very enriched in the lungs of asthma patients. Although these reports are the one report in the England Journal of Medicine, there's some controversy about that. But there are a number of studies in humans suggesting these cells could be important.In terms of clinical trials, the, the stimulation of NKT cells has been tested in four phase one trials for advanced cancer patients. And there's some evidence of efficacy, of course, these people are very sick and for the studies are ongoing in several different types of cancers. And there are people who are interested in using NK T cell media therapy also in autoimmune disease, although that has not gotten off the ground yet.Tetramers may play a role in these studies principally in terms of analyzing what happened to the NKT cells after the agonist therapy. So the idea typically is to use glycolipid agonists or in some cases even glycolipid antagonists, which other groups are developing, depending upon the response and the condition. And then to assess what happens, what happened to the NK T cells, which one would do normally with with tetramers.The other potential use would be in the in vitro expansion of human NKT cells. It's quite, it's quite feasible and quite possible to grow large numbers of human NKT cells in vitro by, for example, antigenic stimulation, which could be done with tetramers or it could be done with cells having the right glycolipids. And in addition to using the tetramer or the cells, you need to add cytokine.But under those conditions, one can expand great numbers of human NK T cells. And another is at least one group in Boston interested in that kind of therapy as opposed to The glycolipid based therapy. Another portion of my laboratory is involved In studying mucosal immunity.Most immunologists focused on the lymphocytes or white blood cells in the lymph node and the spleen. And yet perhaps the majority that are found in our bodies are actually found in the intestinal mucosa, a site where we also have between 10th to the 14th and 10th to the 15th bacteria probably. So we are mostly sterile from a microbial point of view, but not in our intestine.And also in the intestine we have a very large collection of white blood cells. These cells have unique properties and actually they're somewhat understudied. So we have some basic science investigations of these cells.But we also study a process of what I would call immune dysregulation, the intestine leading to inflammatory bowel disease, which is in, these are basically immune mediate diseases of either the small or large intestine, and they go by the names Crohn's disease or ulcerative colitis. And although we do some human work, most of our work is in mouse models. We have what I consider to be very good mouse models for the dysregulation of the mucosal immune system that leads to Crohn's disease like symptoms.We're studying that process and ways also to interfere with that process, ultimately with the hope of developing better therapies for Patients who suffer from these immune-mediated diseases. So in conclusion, We might think a little bit about where is immunology going over the next 10 years or so, and it's really difficult to see 10 years ahead. Surprising as that may sound.I, I do think the mucosal immune system is one of the major unexplored areas of immunology actually. Although more and more people and more and more of the high profile groups are getting interested in mucosal immunology studies. I think another area that's gonna have tremendous impacts is genetics, and particularly human genetics as we migrate or advance, I should say, to the 1, 000 sequence of a genome of a human genome, it's really gonna be possible to do studies on individuals and to assess how DNA variability affects the immune response.Right now, most of the best investigations of basic pathways involve rodents. Rodents, particularly mice are really critical for our studies. But I think in the future when we consider complex autoimmune phenomenon or other things that might be going on in individuals, we'll be able to do this based upon human genetic polymorphisms.We'll be able to assess the polymorphisms and then use that information to try to predict how that will affect immune responses and eventually move towards what I would, what I would call and what others are calling a personalized immune therapies or per, in a broader sense, personalized medicine. So genetics is gonna have a tremendous impact. We already have great tools in mouse genetics.We can knock out any gene we want. We can knock out the expression of genes in particular cell types. We can do amazing things.But I think in the future we're gonna be looking at understanding the immune response and immunity from the point of view of Human responses.

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La Jolla Institute for Allergy and Immunology.  自然杀伤T细胞（NKT），癌症autioimmunity监管，清除感染，并发展artheriosclerotic斑块的免疫反应的关键因素。在这次采访中，米奇Kronenberg讨论他的实验室的努力去了解通过糖脂抗原激活NKT细胞的机制。

专访：糖脂抗原提呈CD1d及NKT细胞活化治疗潜力

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