Tissue-resident memory T (T RM ) cells remain poised in the tissue and mediate robust protection from secondary infection. T RM cells within the intestine and other tissues are heterogeneous in their phenotype and function; however, the contributions of these T RM subsets to secondary infection remain poorly defined. To address the plasticity of intestinal T RM subsets and their role in local and systemic immunity, we generated mice to fate map intestinal CD103 + T RM cells and track their location and function during secondary infection with Yersinia pseudotuberculosis . We found that CD103 + T RM cells remained lodged in the tissue and were poorly reactivated during secondary challenge. CD103 − T RM cells were the primary responders to secondary infection and expanded within the tissue, with limited contribution from circulating memory T cells. The transcriptional profile of CD103 − T RM cells demonstrated maintenance of a gene signature similar to circulating T cells along with increased cytokine production and migratory potential. CD103 − T RM cells also expressed genes associated with T cell receptor (TCR) activation and displayed enhanced TCR-mediated reactivation both in vitro and in vivo compared with their CD103 + counterparts. These studies reveal the limited recall potential of CD103 + T RM subsets and the role of CD103 − T RM cells as central memory–like T cells within peripheral tissues.

IntroductionWe previously demonstrated that insulin secreting cells (ISCs) accelerate healing of chronic wounds, and it is known that mesenchymal stem cells (MSCs) also accelerate wound healing. Here, we report that the combination of both cell types coencapsulated into a synthetic hydrogel dressing accelerates chronic wound healing 3 × faster than control and 2 × faster than each cell type delivered singly. Specifically, insulin released by ISCs activates the PI3/Akt pathway, which is vital to the function and survival of MSCs. MSCs in turn improve the viability and function of ISCs.Materials and MethodsMSCs and/or rat islet tumor RIN-m cells were encapsulated into polyethylene glycol diacrylate hydrogel sheets and applied to 1 cm2 full thickness excisional wounds on the dorsa of genetically diabetic male mice (BKS.Cg-m +/+Leprdb/J) in accordance with protocols approved by the Rutgers IACUC. Encapsulated cell viability was assessed using a LIVE/DEAD® Viability/Cytotoxicity Kit. Akt phosphorylation, insulin, VEGF, and TGF-β1 secretion were assessed by ELISA. Animals were sacrificed on postoperative days 14 and 28 and wound tissue was collected for histological and western blot analysis.ResultsISC:MSC combination groups had the highest levels of every secreted product and phosphorylated Akt, and closed wounds in 14 days, ISC-only or MSC-only groups closed wounds in 28 days, control groups closed wounds in 40 days. Further, ISC:MSC groups healed without intermediate scab or scar.ConclusionsCombining MSCs with ISCs results in a more robust healing response than singly delivered cells, warranting further investigation of coencapsulation for MSC therapies.

Tissue-resident memory (Trm) CD8+ T cells represent a distinct population of memory T cells that are maintained independently of the circulation and are positioned to respond rapidly to reinfection of the tissue. Using the bacterial pathogen Yersinia pseudotuberculosis (Yptb), we identified two distinct intestinal CD8+ Trm populations that are differentiated by their expression of the integrin CD103. Proximity of T cells to areas of inflammation within the intestinal tissue regulated Trm differentiation, with IL-12 leading to increased numbers of CD103− Trm cells. Therefore, we examined the role of the transcription factor STAT4 in the programming of this Trm subset. During infection, both wild-type and Stat4−/− T cells expanded and entered the intestinal tissue at similar rates. Stat4−/− T cells localized to areas of inflammation along with wild-type cells, but failed to express CD103− Trm signature genes. After infection, Stat4−/− CD103+ intestinal Trm cells formed a stable memory population; however, the number of Stat4−/− CD103− Trm cells was significantly reduced relative to wild-type Trm cells. We also observed impaired persistence of Stat4−/− CD103− Trm cells in other tissues with prominent CD103− Trm populations, including the liver, colon, and mesenteric adipose. RNA-seq analysis of wild-type and Stat4−/− CD103− Trm populations was used to identify genes required for the maintenance of this Trm subset. This work has identified STAT4 as a regulator of CD103− Trm differentiation and maintenance across multiple tissues, and these results will help identify strategies to maximize the number and persistence of Trm cells during vaccination and allow us to address the role of Trm heterogeneity in tissue-specific immunity.

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When considering the development pathway for a genetically modified cell therapy product, it is critically important that the product is engineered consistent with its intended human use. For scientists looking to develop and commercialize a new technology, the decision to select a genetic modification method depends on several practical considerations. Whichever path is chosen, the developer must understand the key risks and potential mitigations of the cell engineering approach. The developer should also understand the clinical implications: permanent/memory establishment versus transient expression, and clinical manufacturing considerations when dealing with transplantation of genetically engineered cells. This review covers important topics for mapping out a strategy for developers of new cell-based therapeutics. Biological, technological, manufacturing, and clinical considerations are all presented to map out development lanes for the initiation and risk management of new gene-based cell therapeutic products for human use.

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