Redefining Clinical Hyperprogression: The Incidence, Clinical Implications, and Risk Factors of Hyperprogression in Non-Small Cell Lung Cancer Treated with Immunotherapy.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) may be associated with hyperprogressive disease (HPD). However, there is currently no standardized definition of HPD, with its risk factors and clinical implications remaining unclear. We investigated HPD in lung cancer patients undergoing immunotherapy, aiming to redefine HPD, identify risk factors, and assess its impact on survival. METHODS: Clinical and radiologic data from 121 non-small cell lung cancer (NSCLC) patients with 136 immunotherapy cases were reviewed retrospectively. Three HPD definitions (Champiat et al., HPDc; Saâda-Bouzid et al., HPDs; and Ferrara et al., HPDf) were employed. Additionally, all new measurable lesions on the post-treatment CT scan were incorporated in measuring the sum of longest diameters (SLD) to define modified HPD (mHPD). RESULTS: Among the 121 patients, 4 (3.3%) had HPDc, 11 (9.1%) had HPDs, and none had HPDf. Adding all new measurable lesions increased HPD incidence by 5%-10% across definitions. Multivariate analysis revealed significantly lower progression-free survival (PFS) and overall survival (OS) for patients with HPDc (HR 5.25, P = .001; HR 3.75, P = .015) and HPDs (HR 3.74, P < .001; HR 3.46, P < .001) compared to those without. Patients with mHPD showed similarly poor survival outcomes as HPD patients. Liver metastasis at diagnosis was associated with HPDs, and a high tumor burden correlated with HPDc. CONCLUSIONS: The incidence and risk factors of HPD varied with different definitions, but mHPD identified more cases with poor outcomes. This comprehensive approach may enhance the identification of at-risk patients and lead to a better understanding of HPD in lung cancer during immunotherapy.

Personalized, tumor-informed, circulating tumor DNA assay for detecting minimal residual disease in non-small cell lung cancer patients receiving curative treatments.

BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as a prognostic and predictive biomarker for detection of minimal residual disease (MRD), monitoring treatment response, and early detection of recurrence in cancer patients. In this study, we explored the utility of ctDNA-based MRD detection to predict recurrence in a real-world cohort of primarily early-stage non-small cell lung cancer (NSCLC) patients treated with curative intent. METHODS: Longitudinal plasma samples were collected post curative-intent treatment from 36 patients with stage I-IV NSCLC. A personalized, tumor-informed assay was used to detect and quantify ctDNA in plasma samples. RESULTS: Of the 24 patients with plasma samples available during the MRD window (within 6 months of curative surgery and before adjuvant therapy), ctDNA was detectable in two patients. Patients with ctDNA-positivity during the MRD window were 15 times more likely to recur compared to ctDNA-negative patients (HR: 15.0, 95% CI: 1.0-253.0, p = 0.010). At any time post-curative intent treatment, ctDNA-positivity was associated with significantly poorer recurrence-free survival compared to persistently ctDNA-negative patients (p < 0.0001). CONCLUSION: Our real-world data indicate that longitudinal, personalized, tumor-informed ctDNA monitoring is a valuable tool in patients with NSCLC receiving curative treatment to identify patients at high risk for recurrence.

Deep response to a combination of mTOR inhibitor temsirolimus and dual immunotherapy of nivolumab/ipilimumab in poorly differentiated thyroid carcinoma with PTEN mutation: a case report and literature review.

Treating advanced thyroid cancer presents challenges due to its resistance to various treatment modalities, thereby limiting therapeutic options. To our knowledge, this study is the first to report the efficacy of temsirolimus in conjunction with dual immunotherapy of nivolumab/ipilimumab to treat heavily treated advanced PDTC. A 50-year-old female initially presented with a rapidly enlarging mass on her right neck. Subsequent diagnosis revealed poorly differentiated thyroid carcinoma, leading to a total thyroidectomy followed by post-operative radioablation therapy. After four years, an examination for persistent cough revealed a recurrence of the disease within multiple mediastinal nodes. Genetic analysis of blood samples uncovered somatic mutations in the tumor, specifically involving PTEN and TP53. The disease progressed despite palliative radiation, lenvatinib, and nivolumab/ipilimumab therapy. Consequently, temsirolimus, functioning as an mTOR inhibitor, was introduced as an adjunct to the nivolumab/ipilimumab regimen. This combination approach yielded remarkable clinical improvement and disease control for a duration of approximately six months. Temsirolimus likely suppressed the aberrantly activated PI3K/AKT/mTOR signaling pathway, facilitated by the PTEN genetic alteration, thus engendering an effective treatment response. This synergy between targeted agents and immunotherapy presents a promising therapeutic strategy for advanced PDTC patients with limited treatment alternatives. In previous clinical trials, mTOR inhibitors have demonstrated the ability to maintain stable disease (SD) in 65% to 74% for advanced thyroid cancer patients, including those with PDTC. When combined with other targeted therapies, the observed SD or partial response rates range from 80% to 97%. Many of these trials primarily involved differentiated thyroid carcinoma, with diverse genetic mutations. Thyroid cancer patients with alterations in the PI3K/mTOR/Akt appeared to benefit most from mTOR inhibitors. However, no clear association between the efficacy of mTOR inhibitors and specific histologies or genetic mutations has been established. Future studies are warranted to elucidate these associations.

Targeting the activin receptor 1C on CD4+ T cells for cancer immunotherapy.

Activins, members of the TGF-beta superfamily, have been isolated and identified in the endocrine system, but have not been substantially investigated in the context of the immune system and endocrine-unrelated cancers. Here, we demonstrated that tumor-bearing mice had elevated systemic activin levels, which correlated directly with tumor burden. Likewise, cancer patients have elevated plasma activin levels compared to healthy controls. We observed that both tumor and immune cells could be sources of activins. Importantly, our in vitro studies suggest that activins promote differentiation of naïve CD4+ cells into Foxp3-expressing induced regulatory T cells (Tregs), particularly when TGF-beta was limited in the culture medium. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that activin receptor 1c (ActRIC) was uniquely expressed on Tregs and that both ActRIC and ActRIIB (activin receptor 2b) were highly upregulated during iTreg differentiation. ActRIC-deficient naïve CD4+ cells were found to be defective in iTreg generation both in vitro and in vivo. Treg suppression assays were also performed, and ActRIC deficiency did not change the function or stability of iTregs. Mice lacking ActRIC or mice treated with monoclonal anti-ActRIC antibody were more resistant to tumor progression than wild-type controls. This phenotype was correlated with reduced expression of Foxp3 in CD4+ cells in the tumor microenvironment. In light of the information presented above, blocking activin-ActRIC signaling is a promising and disease-specific strategy to impede the accumulation of immunosuppressive iTregs in cancer. Therefore, it is a potential candidate for cancer immunotherapy.

Survival Outcomes After Double-Lung Transplantation for Refractory Lung-Limited Cancers and Incidence of Post-Transplant Lung Cancer.

BACKGROUND To evaluate the role of double-lung transplantation (DLT) for lung cancer, the survival outcomes of patients who underwent DLT for lung cancer and the incidence of de novo lung cancer after DLT were assessed. MATERIAL AND METHODS Data from all cases reported in the literature were pooled for analysis and additional data were collected from the Organ Procurement Transplantation Network (OPTN) registry. Recurrence-free survival (RFS), overall survival (OS), and cancer-specific survival (CSS) of patients who underwent DLT for lung cancer were determined. Moreover, the incidence of de novo lung cancer and associated OS in lung transplant recipients were examined. RESULTS Of the 20 cases series and 15 cases from the OPTN registry, the 5-year RFS was 55.0% and 66.7% and the 5-year OS was 55.0% and 26.7%, respectively, and the median CSS was 48.0 (range, 2.0-144.0) and 27.7 (range, 0.2-66.6) months, respectively. In the OPTN data, the incidence of post-transplant lung cancer in patients who underwent DLT for the non-cancerous disease was 0.8% and the 5-year OS was 47.3%. CONCLUSIONS In conclusion, our integrated analysis of the case series and the OPTN registry demonstrated promising survival outcomes for patients with refractory bilateral lung cancer who underwent DLT. Although there are limitations to consider, the results of this study underscore the potential benefits of DLT in managing refractory lung-limited lung cancer.

A Comprehensive Landscape of De Novo Malignancy After Double Lung Transplantation.

Although the association between post-transplant malignancy (PTM) and immunosuppressive therapy after organ transplantation has been studied, an integrated review of PTM after lung transplantation is lacking. We investigated the incidence and types of de novo PTM and its impact on survival following double lung transplantation (DLT). The incidence and type of PTM as well as the annual and cumulative risks of each malignancy after DLT were analyzed. The overall survival (OS) of recipients with or without PTM was compared by the Kaplan-Meier survival method and landmark analysis. There were 5,629 cases (23.52%) with 27 types of PTMs and incidences and OS varied according to the types of PTMs. The recipients with PTM showed a significantly longer OS than those without PTM (p < 0.001). However, while the recipients with PTM showed significantly better OS at 3, and 5 years (p < 0.001, p = 0.007), it was worse at the 10-year landmark time (p = 0.013). And the single PTM group showed a worse OS rate than the multiple PTM group (p < 0.001). This comprehensive report on PTM following DLT can help understand the risks and timing of PTM to improve the implementation of screening and treatment.

Current and future role of double-lung transplantation for bilateral lung cancer.

Technological advances have progressively enhanced the survival rate of lung transplant recipients and expanded its indications for various diseases, including the recent coronavirus disease 2019 (COVID-19). However, according to the International Society for Heart and Lung Transplantation, lung cancer constituted a mere 0.1% of the indications for lung transplantation over the past two decades. This statistic has remained stagnant, and numerous lung cancer patients continue to be excluded from lung transplantation candidacy. Contrary to the general exclusion of lung cancer patients from transplantation, the post-transplant survival rate for these patients is not inferior to that of patients with non-cancerous diseases. Furthermore, lung transplantation may offer curative treatment for patients with bilateral lung cancer whose respiratory insufficiency has advanced independently of cancer progression. This review aims to elucidate and examine the role of double lung transplantation (DLT) in bilateral lung cancer. We summarize the established indications for lung transplantation, appropriate histologic or molecular subtypes of lung cancer for transplantation, technical advances to minimize recurrence, post-DLT survival outcomes for lung cancer patients, and related translational research. We suggest that although DLT for bilateral lung cancer presents challenges, it may be considered a potential treatment option in select circumstances.

Sirolimus-eluting airway stent reduces profibrotic Th17 cells and inhibits laryngotracheal stenosis.

Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4+ T cell-mediated inflammation. However, the role of CD4+ T cells in promoting LTS fibrosis is unknown. The mTOR signaling pathways have been shown to regulate the T cell phenotype. Here we investigated the influence of mTOR signaling in CD4+ T cells on LTS pathogenesis. In this study, human LTS specimens revealed a higher population of CD4+ T cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and Th17 cells. Selective deletion of mTOR in CD4+ cells reduced Th17 cells and attenuated fibrosis, demonstrating CD4+ T cells' pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased Th17 cells. In vitro, Th17 cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of Th17 cells. Collectively, mTOR signaling drove pathologic CD4+ T cell phenotypes in LTS, and targeting mTOR with sirolimus was effective at treating LTS through inhibition of profibrotic Th17 cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.

Design of cell-type-specific hyperstable IL-4 mimetics via modular de novo scaffolds.

The interleukin-4 (IL-4) cytokine plays a critical role in modulating immune homeostasis. Although there is great interest in harnessing this cytokine as a therapeutic in natural or engineered formats, the clinical potential of native IL-4 is limited by its instability and pleiotropic actions. Here, we design IL-4 cytokine mimetics (denoted Neo-4) based on a de novo engineered IL-2 mimetic scaffold and demonstrate that these cytokines can recapitulate physiological functions of IL-4 in cellular and animal models. In contrast with natural IL-4, Neo-4 is hyperstable and signals exclusively through the type I IL-4 receptor complex, providing previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors. Because of their hyperstability, our computationally designed mimetics can directly incorporate into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds. Neo-4 should be broadly useful for interrogating IL-4 biology, and the design workflow will inform targeted cytokine therapeutic development.

Helminth egg derivatives as proregenerative immunotherapies.

The immune system is increasingly recognized as an important regulator of tissue repair. We developed a regenerative immunotherapy from the helminth Schistosoma mansoni soluble egg antigen (SEA) to stimulate production of interleukin (IL)-4 and other type 2-associated cytokines without negative infection-related sequelae. The regenerative SEA (rSEA) applied to a murine muscle injury induced accumulation of IL-4-expressing T helper cells, eosinophils, and regulatory T cells and decreased expression of IL-17A in gamma delta (γδ) T cells, resulting in improved repair and decreased fibrosis. Encapsulation and controlled release of rSEA in a hydrogel further enhanced type 2 immunity and larger volumes of tissue repair. The broad regenerative capacity of rSEA was validated in articular joint and corneal injury models. These results introduce a regenerative immunotherapy approach using natural helminth derivatives.

Development of an induced pluripotent stem cell-specific microRNA assay for detection of residual undifferentiated cells in natural killer cell therapy products.

Most clinically evaluated chimeric antigen receptor (CAR)-based cell therapies are generated from autologous immune cells. However, there are several limitations to autologous cell therapy, including low availability, poor quality of starting cellular material and limited expansion capability. Recently, induced pluripotent stem cell (iPSC)-derived allogeneic cell therapy platforms have gained popularity, as they seek to overcome many of the challenges inherent to current autologous cell therapies. However, teratoma risk associated with residual undifferentiated cells (i.e., iPSCs) in the drug product may restrict potential clinical applications if left unaddressed. To ensure the safety of the final cell therapy product, there is a need to develop quality control assays to detect residual iPSCs. Combining microRNA (miRNA) sequencing data with publicly archived miRNA microarray datasets, we demonstrated that miRNAs belonging to the 300 family (miR-302a-5p, miR-302c-3p and miR-302d-5p) and 500 family (miR-518f-5p and miR-519-3p) were highly expressed in iPSCs (both periperal blood mononuclear cell- and T cell-derived iPSCs) compared with a number of differentiated cell types. We developed and validated a sensitive digital droplet polymerase chain reaction (ddPCR) assay targeting these miRNAs to detect low levels of residual iPSCs in differentiated cell samples. The miRNA ddPCR-based method with primers for miR-302a-5p, miR-302c-3p and miR-302d-5p detected as few as 5, 3 and 10 undifferentiated iPSCs, respectively, in the background of 106 iPSC-derived natural killer (iNK) cells. These results suggest that our method targeting identified iPSC-specific miRNA transcripts is specific and sensitive for the quality assessment of NK cell therapy products derived from iPSCs.

Dilations and degeneracy in network controllability.

Network controllability asserts a perspective that the structure-the location of edges that connect nodes-of the network contains important information about fundamental characteristics of our ability to change the behavior that evolves on these networks. It can be used, for example, to determine the parts of the system that when influenced by outside controlling signals, can ultimately steer the behavior of the entire network. One of the challenges in utilizing the ideas from network controllability on real systems is that there is typically more than one potential solution (often many) suggested by the topology of the graph that perform equally well. Picking a single candidate from this degenerate solution set over others should be properly motivated, however, to-date our understanding of how these different options are related has been limited. In this work, we operationalize the existing notion of a dilation into a framework that provides clarity on the source of this control degeneracy and further elucidates many of the existing results surrounding degeneracy in the literature.

Type 2 immunity induced by bladder extracellular matrix enhances corneal wound healing.

The avascular nature of cornea tissue limits its regenerative potential, which may lead to incomplete healing and formation of scars when damaged. Here, we applied micro- and ultrafine porcine urinary bladder matrix (UBM) particulate to promote type 2 immune responses in cornea wounds. Results demonstrated that UBM particulate substantially reduced corneal haze formation as compared to the saline-treated group. Flow cytometry and gene expression analysis showed that UBM particulate suppressed the differentiation of corneal stromal cells into α-smooth muscle actin-positive (αSMA+) myofibroblasts. UBM treatments up-regulated interleukin-4 (IL-4) produced primarily by eosinophils in the wounded corneas and CD4+ T cells in draining lymph nodes, suggesting a cross-talk between local and peripheral immunity. Gata1-/- mice lacking eosinophils did not respond to UBM treatment and had impaired wound healing. In summary, stimulating type 2 immune responses in the wounded cornea can promote proregenerative environments that lead to improved wound healing for vision restoration.

Bacterial-Driven Inflammation and Mutant BRAF Expression Combine to Promote Murine Colon Tumorigenesis That Is Sensitive to Immune Checkpoint Therapy.

Colorectal cancer is multifaceted, with subtypes defined by genetic, histologic, and immunologic features that are potentially influenced by inflammation, mutagens, and/or microbiota. Colorectal cancers with activating mutations in BRAF are associated with distinct clinical characteristics, although the pathogenesis is not well understood. The Wnt-driven multiple intestinal neoplasia (MinApcΔ716/+) enterotoxigenic Bacteroides fragilis (ETBF) murine model is characterized by IL17-dependent, distal colon adenomas. Herein, we report that the addition of the BRAF V600E mutation to this model results in the emergence of a distinct locus of midcolon tumors. In ETBF-colonized BRAF V600E Lgr5 CreMin (BLM) mice, tumors have similarities to human BRAF V600E tumors, including histology, CpG island DNA hypermethylation, and immune signatures. In comparison to Min ETBF tumors, BLM ETBF tumors are infiltrated by CD8+ T cells, express IFNγ signatures, and are sensitive to anti-PD-L1 treatment. These results provide direct evidence for critical roles of host genetic and microbiota interactions in colorectal cancer pathogenesis and sensitivity to immunotherapy. SIGNIFICANCE: Colorectal cancers with BRAF mutations have distinct characteristics. We present evidence of specific colorectal cancer gene-microbial interactions in which colonization with toxigenic bacteria drives tumorigenesis in BRAF V600E Lgr5 CreMin mice, wherein tumors phenocopy aspects of human BRAF-mutated tumors and have a distinct IFNγ-dominant immune microenvironment uniquely responsive to immune checkpoint blockade.This article is highlighted in the In This Issue feature, p. 1601.

Glutamine Inhibition Reduces Iatrogenic Laryngotracheal Stenosis.

OBJECTIVE/HYPOTHESIS: Glutamine inhibition has been demonstrated an antifibrotic effect in iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts in vitro. We hypothesize that broadly active glutamine antagonist, DON will reduce collagen formation and fibrosis-associated gene expression in iLTS mice. STUDY DESIGN: Prospective controlled animal study. METHODS: iLTS in mice were induced by chemomechanical injury of the trachea using a bleomycin-coated wire brush. PBS or DON (1.3 mg/kg) were administered by intraperitoneal injection (i.p.) every other day. Laryngotracheal complexes were harvested at days 7 and 14 after the initiation of DON treatment for the measurement of lamina propria thickness, trichrome stain, immunofluorescence staining of collagen 1, and fibrosis-associated gene expression. RESULTS: The study demonstrated that DON treatment reduced lamina propria thickness (P = .025) and collagen formation in trichrome stain and immunofluorescence staining of collagen 1. In addition, DON decreased fibrosis-associated gene expression in iLTS mice. At day 7, DON inhibited Col1a1 (P < .0001), Col3a1 (P = .0046), Col5a1 (P < .0001), and Tgfß (P = .023) expression. At day 14, DON reduced Co1a1 (P = .0076) and Tgfß (P = .023) expression. CONCLUSIONS: Broadly active glutamine antagonist, DON, significantly reduces fibrosis in iLTS mice. These results suggest that the concept of glutamine inhibition may be a therapeutic option to reduce fibrosis in the laryngotracheal stenosis. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E2125-E2130, 2021.

IL-17 and immunologically induced senescence regulate response to injury in osteoarthritis.

Senescent cells (SnCs) are implicated in the pathogenesis of age-related diseases including osteoarthritis (OA), in part via expression of a senescence-associated secretory phenotype (SASP) that includes immunologically relevant factors and cytokines. In a model of posttraumatic OA (PTOA), anterior cruciate ligament transection (ACLT) induced a type 17 immune response in the articular compartment and draining inguinal lymph nodes (LNs) that paralleled expression of the senescence marker p16INK4a (Cdkn2a) and p21 (Cdkn1a). Innate lymphoid cells, γδ+ T cells, and CD4+ T cells contributed to IL-17 expression. Intra-articular injection of IL-17-neutralizing antibody reduced joint degeneration and decreased expression of the senescence marker Cdkn1a. Local and systemic senolysis was required to attenuate tissue damage in aged animals and was associated with decreased IL-17 and increased IL-4 expression in the articular joint and draining LNs. In vitro, we found that Th17 cells induced senescence in fibroblasts and that SnCs skewed naive T cells toward Th17 or Th1, depending on the presence of TGF-ß. The SASP profile of the inflammation-induced SnCs included altered Wnt signaling, tissue remodeling, and cell-cycle pathways not previously implicated in senescence. These findings provide molecular targets and mechanisms for senescence induction and therapeutic strategies to support tissue healing in an aged environment.

Interleukin 17 and senescent cells regulate the foreign body response to synthetic material implants in mice and humans.

Medical devices and implants made of synthetic materials can induce an immune-mediated process when implanted in the body called the foreign body response, which results in formation of a fibrous capsule around the implant. To explore the immune and stromal connections underpinning the foreign body response, we analyzed fibrotic capsules surrounding surgically excised human breast implants from 12 individuals. We found increased numbers of interleukin 17 (IL17)-producing γδ+ T cells and CD4+ T helper 17 (TH17) cells as well as senescent stromal cells in the fibrotic capsules. Further analysis in a murine model demonstrated an early innate IL17 response to implanted synthetic material (polycaprolactone) particles that was mediated by innate lymphoid cells and γδ+ T cells. This was followed by a chronic adaptive CD4+ TH17 cell response that was antigen dependent. Synthetic materials with varying chemical and physical properties implanted either in injured muscle or subcutaneously induced similar IL17 responses in mice. Mice deficient in IL17 signaling established that IL17 was required for the fibrotic response to implanted synthetic materials and the development of p16INK4a senescent cells. IL6 produced by senescent cells was sufficient for the induction of IL17 expression in T cells. Treatment with a senolytic agent (navitoclax) that killed senescent cells reduced IL17 expression and fibrosis in the mouse implant model. Discovery of a feed-forward loop between the TH17 immune response and the senescence response to implanted synthetic materials introduces new targets for therapeutic intervention in the foreign body response.

YAP Attenuates CD8 T Cell-Mediated Anti-tumor Response.

YAP is a transcriptional coactivator of the Hippo signaling pathway that has largely been studied for its role in the regulation of organ size during development. Several studies have shown that YAP is upregulated in cancer cells, and more recently in the T regulatory (Treg) subset of CD4+ cells. These observations suggest that the transcriptional co-activator may promote tumor persistence and progression. Here, we report that YAP also plays an immunoinhibitory role in CD8 T cells, especially in activated cytotoxic cells usually found in the tumor microenvironment. Our findings add further rationale for the development and use of pharmacologic inhibitors of YAP to treat cancer.

Multifunctional synthetic Bowman's membrane-stromal biomimetic for corneal reconstruction.

As the outermost layer of the eye, the cornea is vulnerable to physical and chemical trauma, which can result in loss of transparency and lead to corneal blindness. Given the global corneal donor shortage, there is an unmet need for biocompatible corneal substitutes that have high transparency, mechanical integrity and regenerative potentials. Herein we engineered a dual-layered collagen vitrigel containing biomimetic synthetic Bowman's membrane (sBM) and stromal layer (sSL). The sBM supported rapid epithelial cell migration, maturation and multilayer formation, and the sSL containing tissue-derived extracellular matrix (ECM) microparticles presented a biomimetic lamellar ultrastructure mimicking the native corneal stroma. The incorporation of tissue-derived microparticles in sSL layer significantly enhanced the mechanical properties and suturability of the implant without compromising the transparency after vitrification. In vivo performance of the vitrigel in a rabbit anterior lamellar keratoplasty model showed full re-epithelialization within 14 days and integration of the vitrigel with the host tissue stroma by day 30. The migrated epithelial cells formed functional multilayer with limbal stem cell marker p63 K14 expressed in the lower layer, epithelial marker K3 and K12 expressed through the layers and tight junction protein ZO-1 expressed by the multilayers. Corneal fibroblasts migrated into the implants to facilitate host/implant integration and corneal stromal regeneration. In summary, these results suggest that the multi-functional layers of this novel collagen vitrigel exhibited significantly improved biological performance as corneal substitute by harnessing a fast re-epithelialization and stromal regeneration potential.

Interleukin-36γ-producing macrophages drive IL-17-mediated fibrosis.

Biomaterials induce an immune response and mobilization of macrophages, yet identification and phenotypic characterization of functional macrophage subsets in vivo remain limited. We performed single-cell RNA sequencing analysis on macrophages sorted from either a biologic matrix [urinary bladder matrix (UBM)] or synthetic biomaterial [polycaprolactone (PCL)]. Implantation of UBM promotes tissue repair through generation of a tissue environment characterized by a T helper 2 (TH2)/interleukin (IL)-4 immune profile, whereas PCL induces a standard foreign body response characterized by TH17/IL-17 and fibrosis. Unbiased clustering and pseudotime analysis revealed distinct macrophage subsets responsible for antigen presentation, chemoattraction, and phagocytosis, as well as a small population with expression profiles of both dendritic cells and skeletal muscle after UBM implantation. In the PCL tissue environment, we identified a CD9hi+IL-36γ+ macrophage subset that expressed TH17-associated molecules. These macrophages were virtually absent in mice lacking the IL-17 receptor, suggesting that they might be involved in IL-17-dependent immune and autoimmune responses. Identification and comparison of the unique phenotypical and functional macrophage subsets in mouse and human tissue samples suggest broad relevance of the new classification. These distinct macrophage subsets demonstrate previously unrecognized myeloid phenotypes involved in different tissue responses and provide targets for potential therapeutic modulation in tissue repair and pathology.