KRN 7000

KRN7000[(2S,3S.4R)-l-O-(a-D-galactopyranosyl)-2-(N-hexacosanoylamino)-l,3.4-octadecanetriol] is a highly potent synthetic alpha-galactose ceramide originally isolated from an ocean sponge that is widely used in the study of tumors and autoimmune diseases due to its ability to specifically present CD1d on the cell surface and thus activate NKT cells.^[1] KRN7000 is a potent activator of antigen-presenting cells which leads to stimulation of immunoregulatory cells such as T cells, natural killer T cells (NKT), and macrophages.^[3]

In vitro study indicated that KRN7000 can be recognized by NKT-cell clones and can trigger proliferation, cytokine release (IL-4 and IFN-c) and cytotoxic activity. KRN7000 decreased in the percentage of Va24⁺ Vb11⁺T cells expressing CD161, indicating that CD161 does not play a major role in the cytotoxicity mediated by KRN7000-activated Va24⁺ Vb11⁺ T cells. Moreover, KRN7000-cultured Va24⁺Vb11⁺ T cells strongly expressed CD25 (IL-2Ra), it can be expected that cytokines targeting the IL-2R will have potent effects on the expansion of Va24⁺ Vb11⁺ T cells.^[2]

In vivo study demonstrated that KRN7000 prevented GVHD onset in F1 mice inoculated with haploidentical C57 splenocytes with no apparent toxicity. This treatment results in the maintenance of a healthy profile and body weight and also prevents other clinical signs of GVHD. In vivo treatment with KRN7000 resulted in stimulation of the NK1.1⁺T cell subset, which is implicated in the suppression of immune responses directed against autoantigens and induction of tumor immunity. KRN7000 treatment in vivo also led to increased production of IL-4. Which suggests that KRN7000 treatment in vivo might prevent the onset of GVHD through upregulation of IL-4 production.^[3]

KRN 7000 is a potent synthetic α-galactosylceramide, which, in association with the antigen-presenting CD1d protein, activates NKT immune cells. As these cells have important roles in the rejection of malignant tumors and in the regulation of several autoimmune diseases, KRN 7000 has activity in many diseases, including cancer, lupus, diabetes, malaria, and tuberculosis.

KRN7000 is inherently an extremely hydrophobic molecule, therefore, almost all the methods for solubilizing this material in primarily aqueous media will contain at least some detergent. For cell culture or other in vivo models, it is recommended that the KRN7000 be initially dissolved in a 2:1 mixture of chloroform and methanol. This solution should then be aliquoted into amounts suitable for a day’s use into glass vials. After aliquoting, the chloroform/methanol solvent can be evaporated off under a stream of nitrogen or argon. We recommend turning the tube during this process to try to get the KRN7000 deposited in as thin a film as possible. The thinner the KRN7000 layer is, the easier it will be to reconstitute it. These dry aliquots can then be stored under nitrogen or argon at -20°C for several months or until needed.

On the day of the experiment, use a dried aliquot and reconstitute it using any of the options listed below:

PBS with 0.5% Tween-20. Note: It will be necessary to warm at 37°C and sonicate for 2 hours or more in a water bath sonicator in order to get the material to dissolve. Heating and sonication should be done immediately prior to every use.

5.6% sucrose with 0.75% L-histidine and 0.5% Tween-20. Heating to 80°C for several minutes will be necessary with this solvent system.

100% DMSO (anhydrous). It is critical that the DMSO be completely anhydrous. Heating to 80°C may be necessary to get the KRN7000 to completely dissolve. Please note: while this method will produce a true clear solution in DMSO, the KRN7000 will be very likely to precipitate out as soon as the DMSO is diluted into aqueous media. It is recommended that the aqueous media the DMSO stock solution will be diluted in should contain 10% serum, or BSA and that the DMSO stock solution be diluted no more than 1/100. Depending upon the make-up of the aqueous buffer, there may well still be a precipitate, however the precipitate should eventually disappear with warming and repeated vortexing or sonication in a water bath sonicator. This may take some time and will likely need to be repeated each time an aliquot is thawed for use.

Depending upon the solvent system used and the final concentration of the KRN7000, the results may be either a true clear solution or a somewhat cloudy suspension. The cloudy suspensions are not a problem, and will work fine when treating cells, just make sure they are mixed well immediately before use.

KRN7000是一种高效的合成α-半乳糖鞘氨醇，最初从海绵中分离出来。它能够特异性地在细胞表面呈现CD1d，并激活NKT细胞，因此被广泛用于肿瘤和自身免疫性疾病的研究中。^[1] KRN7000是抗原提呈细胞的强效激活剂，可导致免疫调节细胞如T细胞、自然杀伤T细胞（NKT）和巨噬细胞的刺激。^[3]

体外研究表明，KRN7000可以被NKT细胞克隆所识别，并能够触发增殖、细胞因子释放（IL-4和IFN-c）和细胞毒性活性。 KRN7000降低了表达CD161的Va24 + Vb11 + T细胞百分比，这表明CD161在KRN7000激活的Va24 + Vb11 + T细胞介导的细胞毒性中并不起主要作用。此外，经过KRN7000培养的Va24 + Vb11 + T细胞强烈表达CD25（IL-2Ra），预计针对IL-2R的细胞因子将对Va24+Vb11+T 细胞扩张产生强效影响。 ^[2]

实验结果表明，KRN7000可以预防F1小鼠接种haploidentical C57脾细胞后的GVHD发生，并且没有明显的毒性。这种治疗方法可以保持健康状态和体重，并且还可以预防其他GVHD的临床症状。在活体内使用KRN7000进行治疗会刺激NK1.1+T细胞亚群，该亚群参与抑制针对自身抗原的免疫反应和诱导肿瘤免疫。在活体内使用KRN7000进行治疗还会导致IL-4产生增加，这表明通过上调IL-4产生，在活体内使用KRN7000可能能够预防GVHD的发生。^[3]

KRN 7000是一种强效的合成α-半乳糖鞘氨醇，它与抗原呈递CD1d蛋白结合后可以激活NKT免疫细胞。由于这些细胞在拒绝恶性肿瘤和调节多种自身免疫性疾病方面具有重要作用，因此KRN 7000在许多疾病中都有活性，包括癌症、红斑性 lupus、 糖尿病、 疟疾和结核等。

KRN7000是一种极疏水的分子，因此几乎所有将其溶解在主要水性介质中的方法都会含有至少一些洗涤剂。对于细胞培养或其他体内模型，建议最初将KRN7000溶解在氯仿和甲醇的2:1混合物中。然后应该将这个溶液分装到适量用于一天使用的玻璃瓶中。分装后，可以在氮气或氩气流下蒸发掉氯仿/甲醇溶剂。我们建议在这个过程中转动管子，以尽可能地使KRN7000沉积成薄膜。 KRN7000层越薄，则重新制备它就越容易。 这些干燥样品可以存放在-20°C下数月或直到需要为止。

实验当天，请使用一个干燥样品，并使用以下任何选项重新制备：

PBS与0.5％Tween-20。注意：必须在37°C加温并超声处理2小时或更长时间才能使材料溶解。每次使用前应立即进行加温和超声处理。

这个溶剂系统包含5.6%蔗糖、0.75% L-组氨酸和0.5% Tween-20。需要将其加热到80°C数分钟才能使用。

这是一种100%的无水DMSO。 DMSO必须完全无水才能使用。加热到80°C可能需要使KRN7000完全溶解。请注意：虽然这种方法会在DMSO中产生真正的清晰溶液，但是一旦将DMSO稀释成水介质，KRN7000很可能会沉淀出来。建议将DMSO储备溶液稀释在含有10％血清或BSA的水介质中，并且不要超过1/100地稀释储备溶液。根据水缓冲剂的组成，仍可能存在沉淀物，但随着温暖和重复振荡或声波处理，在水浴声波器中进行操作后应该最终消失。这可能需要一些时间，并且每次解冻用于使用时都需要重复。 根据所使用的溶剂系统和KRN7000的最终浓度，结果可以是真正的清晰溶液或略带浑浊悬浮液。 浑浊悬浮物并不是问题，在处理细胞时工作得很好，请确保立即在使用前充分混合它们。

References:
[1]. Chen Y, et al. KRN7000 Reduces Airway Inflammation via Natural Killer T Cells in Obese Asthmatic Mice. Inflammation. 2021 Oct;44(5):1982-1992.
[2]. Van Der Vliet HJ, et al. Effects of alpha-galactosylceramide (KRN7000), interleukin-12 and interleukin-7 on phenotype and cytokine profile of human Valpha24⁺ Vbeta11⁺ T cells. Immunology. 1999 Dec;98(4):557-63.
[3]. Morecki S, et al. Effect of KRN7000 on induced graft-vs-host disease. Exp Hematol. 2004 Jul;32(7):630-7.