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

摘要 同种异体皮肤移植是严重烧伤患者的一种救命方法， 由于伤口的严重性，自体移植物或人工替代物不可用。然而，他们的终极 排斥增加了患者的发病率和死亡率并需要再次移植。临床全身使用 免疫抑制虽然在其他器官移植中有用，但不能为该患者群开具处方，因为它们 存在威胁生命的感染的高风险。因此，可以产生捐助者特有的 需要耐受性，同时通过局部免疫调节保留其他保护性免疫功能。我们 最近证明，基于水凝胶的微针（MN）贴片可用于促进 调节性T细胞（TCLs）在皮肤同种异体移植物中，通过局部递送免疫调节剂（CCL22和IL-2）， 移植部位MN还有助于通过皮肤间质液（ISF）监测Treg归巢过程。 采样（Artzi，azzi等人，Advanced Materials，2021）。在这里，我们将开发一个新的家庭的水凝胶基 MN将被化学官能化，以定制免疫调节剂的释放动力学曲线， 最佳Treg募集到同种异体移植物中以维持局部免疫抑制作用并恢复同种异体移植物， 宽容我们将研究顶部CCL22/IL-2掺杂的MN平台在小鼠中的免疫调节作用。 延长长期皮肤同种异体移植，同时提供对生物学过程的机械见解 通过ISF取样在同种异体移植物中驱动局部免疫调节和Treg稳态， 免疫活性和人源化皮肤移植鼠模型。具体来说，我们的目标是定义 分别最大化Treg募集和Treg增殖所需的最佳剂量的CCL 22和IL-2， 评估补充细胞因子如IL-7在支持Treg稳态中的作用（基于我们先前的研究）。 数据，Azzi et al，Science Translational Medicine，2020）。而且我们 将获得对基于MN的治疗后Treg稳定性、功能和同种抗原特异性的机制性见解 使用转基因小鼠模型，该模型允许追踪Tcb的遗传谱系。此外，我们将验证 MN平台用于原位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优点， 细胞因子/趋化因子的治疗组合及其诱导长期同种异体移植物存活的能力。我们 这项研究将为开发更安全的技术提供基础， 最小侵入性的方式，而不损害烧伤患者的免疫系统的调节臂， 提供了连续监测对治疗的反应的机会。

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.