Generation of Robust Resident Memory T cells in Barrier Tissues through Skin Vaccination

此R01申请建议继续进行过去五年由变革R01资助的工作 （"疫苗接种以产生组织驻留记忆T细胞"） 就这样重新来过）。上一个资助期使Kupper博士和Clark博士能够从根本上 深入了解TRM在皮肤和其他组织中的免疫生物学，包括小鼠模型和人类。采取 过去五年来，这两个联合主要研究者发表的大量工作共同证实了TRM在以下方面的重要性： 保护性免疫，在2015年由Clark博士发表的委托评论中进行了总结（在《科学》杂志上 转化医学）和Kupper博士（在自然医学）。本申请建立在令人兴奋的 初步数据显示，组织特异性印迹在T细胞活化后很早就发生了， 在皮肤引流和肺引流淋巴结（LN）中活化的T细胞的基因表达谱令人惊讶地 类似（但与肠道引流LN非常不同）。这可能解释了为什么用复制缺陷的 痘病毒（MVA）载体通过表皮破坏（以前称为"皮肤划痕"，或艾德/ss）不仅产生 皮肤TRM以及肺TRM具有非常高的效率。这表明，艾德/ss皮肤免疫可能是一种 这是一种在肺部产生保护性免疫力的高效方法，可以对抗流感等肺部病原体。这 假设将在目标1中得到检验。常规接种途径（皮内、皮下和皮下） 肌肉内）在皮肤和肺中产生TRM的效率要低得多。值得注意的是， 对通过这些不同途径递送的相同抗原应答的T细胞的表达谱非常相似。 不同的，有ep/ss和i.m.。是最不同的这些基因表达的差异在 甚至30天后在脾TEM中，并且通过产生TRM的显著不同的能力来表现。 TRM和TCM之间基因表达的一些最大差异是与脂质相关的基因 代谢（脂肪酸结合蛋白/FABP、脂肪酶和CD36/LDL受体）; T细胞产生的TRM 缺乏FABP4和5的皮肤不会持续存在。假设EP/SS免疫模式最 有效地产生TRM，TRM依赖于脂肪酸代谢在外周组织中存活， 目标2测试最后，在目标3中，将在使用人体组织的转化研究中检验相关假设， 血液、皮肤和肺组织从进行肺叶切除术的胸外科患者获得。我们 预测并测试皮肤和肺的T细胞库中的重叠（在CDR3序列水平）， TCRB基因的高通量测序，并将通过基因直接比较人类皮肤和肺TRM 表达、细胞表面表型和功能。以前未被重视的皮肤和 肺归巢T细胞可以解释从结核菌素皮肤试验到"特应性进行曲"的现象， 皮炎之后是哮喘，这对人类疫苗的开发具有重要意义， 通过肺部或皮肤进入宿主的病原体。

This R01 application proposes to continue work funded for the past five years by a Transformative R01 (“Vaccination to generate tissue resident memory T cells”) from the Office of the NIH Director (TR01’s cannot be renewed as such). The previous funding period allowed Drs. Kupper and Clark to make fundamental insights into immunobiology of TRM in skin and other tissues, in both mouse models and humans. Taken together, the body of work published by both co-PI’s over the past five years validates the importance of TRM in protective immunity, is summarized in commissioned reviews published in 2015 by Dr. Clark (in Science Translational Medicine) and Dr. Kupper (in Nature Medicine). The present application builds on exciting preliminary data showing that tissue specific imprinting occurs very early after T cell activation, and that the gene expression profile of T cells activated in skin draining and lung draining lymph nodes (LN) is surprisingly similar (but very different from gut draining LN). This may explain why immunization with a replication deficient poxvirus (MVA) vector through epidermal disruption (formerly “skin scarification”, or ed/ss) generates not only skin TRM but also lung TRM with very high efficiency. This suggests that ed/ss skin immunization may be a highly effective way of generating protective immunity in the lung to pulmonary pathogens like influenza. This hypothesis will be tested in Aim 1. Conventional vaccination routes (intradermal, subcutaneous, and intramuscular) were much less efficient at generating TRM in both skin and lung. Remarkably, the gene expression profile of T cells responding to the same antigen delivered by these different routes was very different, with ep/ss and i.m. being the most different. These differences in gene expression were maintained even 30 days later in splenic TEM, and were manifested by a strikingly different capacity to generate TRM. Some of the greatest differences in gene expression between TRM and TCM are in genes relevant to lipid metabolism (fatty acid binding proteins/FABP, lipases, and CD36/LDL receptor); TRM’s generated from T cells deficient in FABP4 and 5 did not persist in skin. The hypothesis that the ep/ss mode of immunization most efficiently generates TRM that are dependent on fatty acid metabolism for survival in peripheral tissues will be tested in Aim 2. Finally, in Aim 3, related hypotheses will be tested in translational studies using human tissue, with blood, skin, and lung tissue being obtained from thoracic surgery patients undergoing lobectomies. We predict, and will test for, overlap (at the level of CDR3 sequence) in the T cell repertoire of skin and lung by high throughput sequencing of the TCRB gene, and will directly compare human skin and lung TRM by gene expression, cell surface phenotype, and function. The previously unappreciated commonalities in skin and lung homing T cells may explain phenomena from the tuberculin skin test to the “atopic march”, where atopic dermatitis is followed by asthma, and has important implications for human vaccine development against pathogens that enter the host through lung or skin.