Local immunomodulation using a microneedle patch for the management of skin transplant

摘要 同种异体皮肤移植是治疗严重烧伤患者的一种救命方法 由于伤口的严重性，不能使用自体移植或人工替代品。然而，他们的终极目标 排斥反应增加了患者的发病率和死亡率，需要重新移植。临床使用的全身性 免疫抑制，虽然在其他器官移植中有用，但不能开给这个患者队列，因为他们 存在着威胁生命的感染的高风险。因此，能够产生针对捐赠者的新战略 在通过局部免疫调节保护其他保护性免疫功能的同时，需要耐受性。我们 最近发现，基于水凝胶的微针(MN)贴片可用于促进肿瘤细胞的增殖 通过局部递送免疫调节剂(CCL22和IL-2)在同种异体皮肤移植中的调节性T细胞(Tregs) 移植地点。MNS还通过皮肤间质液(ISF)促进Treg归巢过程的监测。 抽样(Artzi、Azzi等人，《先进材料》，2021年)。在这里，我们将开发一种基于水凝胶的新型家族 MNS将被化学功能化，以定制免疫调节剂的释放动力学曲线，以识别 在同种异体移植物中最佳的Treg募集，以维持局部的免疫抑制效应并恢复同种异体移植。 宽容。我们将研究顶端CCL22/IL-2掺杂的MN平台在体内的免疫调节作用 延长长期同种异体皮肤移植，同时提供对生物过程的机械洞察力 通过ISF取样促进异体移植物局部免疫调节和Treg动态平衡 一种免疫活性和人性化的皮肤移植小鼠模型。具体而言，我们的目标是定义 CCL22和IL-2的最佳剂量需要分别最大化Treg的募集和Treg的增殖，并 评估辅助细胞因子，如IL-7在支持Treg动态平衡中的作用(基于我们以前的 数据，Azzi等人，科学转化医学，2020)，当与我们的MN平台联合交付时。此外，我们 将从机制上深入了解MN治疗后Treg的稳定性、功能和同种异体抗原特异性 使用转基因小鼠模型，可以追踪Tregs的遗传谱系。此外，我们还将验证 MN-Platform现场监测治疗反应的诊断能力。最后，我们将测试 MN平台在人源化小鼠模型中的治疗和诊断价值 细胞因子/趋化因子的治疗组合及其诱导同种异体移植长期存活的能力。我们的 这项研究将为开发更安全的技术以提高当地皮肤移植的存活率提供基础， 微创方式，同时不损害烧伤患者免疫系统的调节臂 提供持续监测治疗反应的机会。

ABSTRACT Skin allograft transplantation is a lifesaving approach for the management of severe burn patients when skin autografts or artificial substitutes are not available due to the severity of the wound. However, their ultimate rejection increases patient morbidity and mortality and requires regrafting. Clinically-used systemic immunosuppression, while useful in other organ transplants, cannot be prescribed for this patient cohort as they present a high risk of life-threatening infections. Therefore, new strategies that can generate donor-specific tolerance, while preserving other protective immune functions through local immunoregulation, are needed. We recently demonstrated that hydrogel-based microneedle (MN) patches can be used to promote proliferation of regulatory T cells (Tregs) in skin allografts, by locally delivering immunomodulators (CCL22 and IL-2) at the transplant site. The MNs also facilitate the monitoring of the Treg homing process via skin interstitial fluid (ISF) sampling (Artzi, azzi, et al, Advanced Materials, 2021). Here, we will develop a novel family of hydrogel-based MNs that will be chemically functionalized to tailor the release kinetic profile of the immunomodulators to identify the optimal Treg recruitment to the allograft to sustain a local, immunosuppressive effect and to restore allo- tolerance. We will study the immunomodulatory role of the top CCL22/IL-2-doped MN platform in prolonging long-term skin allograft while providing mechanistic insight into the biological processes driving local immune regulation and Treg homeostasis in the allograft via ISF sampling in both an immunocompetent- and a humanized-skin transplant murine model. Specifically, we aim to define the optimal dose of CCL22 and IL-2 needed to maximize Treg recruitment and Treg proliferation respectively, and to assess the role of supplementary cytokines such as IL-7 in supporting Treg homeostasis (based on our previous data, Azzi et al, Science Translational Medicine, 2020) when co-delivered with our MN platform. Moreover, we will gain mechanistic insight into Treg stability, function and alloantigen specificity following MN-based therapy using a transgenic mouse model, which allows tracing the genetic lineage of Tregs. Also, we will validate the diagnostic ability of the MN-platform for in situ monitoring of the response to therapy. Finally, we will test the therapeutic and diagnostic merit of the MN platform in a humanized mouse model when delivering the optimal therapeutic combination of cytokines/chemokines and its capacity to induce long-term allograft survival. Our study will provide the foundation to develop safer technologies for enhancing skin transplant survival in a local, minimally-invasive way without compromising the regulatory arm of the immune system of burn patients while offering an opportunity to continuously monitor the response to the therapy.