Venous leg ulcers (VLUs) are the most common cause of leg ulcers, yet only 44% heal with standard-of-care treatment, highlighting the critical need for better understanding of their cellular pathology. We used both bulk and single-cell RNA sequencing (scRNA-seq) to identify molecular mechanisms and cellular functions contributing to VLU pathophysiology. scRNA-seq of chronic VLUs revealed impairments in immune, lymph endothelial, and endothelial cells, along with underlying signaling pathways. Next, bulk RNA-seq was performed alongside weekly wound assessments over 4 weeks in patients with VLUs receiving standard care, classifying them as healers or nonhealers on the basis of ≥50% closure. Transcriptomes of healing and nonhealing VLUs were compared with those of human acute wounds, revealing marked suppression of inflammatory response, lymphangiogenesis, and angiogenesis in nonhealing VLUs. In contrast, healing VLUs resembled the gene expression signature of physiological, acute wound healing. Reduced inflammatory response underlined the nonhealing VLU signature, associated with impaired leukocyte transmigration and egress, with suppression of the activity of multiple kinases. Bioinformatic analyses pinpointed PTEN (phosphatase and TENsin homolog) as a master regulator of these processes and signaling pathways. Increased PTEN protein expression was confirmed in nonhealing compared with healing VLUs. Furthermore, pharmacological PTEN inhibition enhanced immune response and pro-inflammatory signals, angiogenesis, and lymphangiogenesis, which resulted in accelerated acute wound closure in mice. These findings support PTEN as a key regulator of the nonhealing VLU phenotype controlling multiple processes responsible for impaired wound healing and highlight its potential use as a therapeutic target to promote wound closure in VLUs.

IntroductionEndometrial receptivity is essential for implantation in both natural and ART cycles, yet the cellular and molecular environment of the endometrium during this window remains poorly characterized. While cytokines affecting implantation have been studied, data on immune cell subtypes in the endometrium are limited. The objective of this study was to determine the association between endometrial immune cell profile at the time of transfer and live birth in patients undergoing frozen embryo transfer (FET) using the index cycle.MethodsThis exploratory prospective observational cohort study (IRB#20190139) included 48 patients undergoing a hormone replacement FET cycle between May 2022 and May 2024. After ultrasound-guided FET, the catheter tip was rinsed in IMDM + 10% FBS, centrifuged, and stained for CD45, CD3, CD19, CD4, CD8, γδ TCR, CD25, CD127, CD66b, CD14, CD16, and CD56. The primary outcome was live birth. Secondary outcomes included miscarriage, biochemical pregnancy, and ectopic pregnancy.ResultsElective single embryo transfer was performed for all the patients. There were 24 live births (50%), four miscarriages (8.3%), and three biochemical pregnancies (6.3%). There was no significant difference in demographics of patients that had a live birth compared to those who did not achieve implantation. There was an increased percentage of γδ T cells in the group of live birth compared to non-pregnant group (p=0.019). In contrast, an increased percentage of neutrophils (CD66b+) was noted in patients that did not achieve implantation (p<0.003). Importantly, we found receiver operating characteristic (ROC) curve area under the curve (AUC) of 0.72 with 95% confidence interval (CI) 0.5504 to 0.8989 for γδ T cells and AUC is 0.75 (95% CI 0.5681 to 0.9319) for CD66b+ cells, confirming the overall ability of these two tests to discriminate between patients who will achieve a live birth vs. ones who will have failed implantation.DiscussionOur findings suggest that the uterine immune environment during FET may be associated with implantation outcomes. Characterization of endometrial immune cell profiles could provide insights into biological factors linked to implantation and live birth, although their clinical utility remains to be determined. To our knowledge, this study is among the first to describe associations between immune cell profiles assessed during the index FET cycle and subsequent IVF outcomes, supporting a potential role for endometrial immune composition in pregnancy success.

Although regulatory T cells (Tregs) control autoimmune diseases, the role of evolutionarily older Foxp3+ γδTregs is much less understood. We noted that both lesional and nonlesional skin of patients with alopecia areata (AA), one of the most common autoimmune diseases, contains significantly more Vδ1+/Foxp3+ γδTregs than healthy scalp skin. Therefore, we investigated how human γδTregs impact experimentally induced AA in human scalp skin xenotransplants on SCID/beige mice in vivo. Peripheral autologous human Vδ2+/Foxp3+ γδTregs were expanded and preactivated in vitro and then injected intradermally into scalp skin xenografts before or after induction of AA. These γδTregs reduced the perifollicular lymphocytic infiltrate, restored hair follicle immune privilege, prevented AA onset, and promoted hair regrowth in established AA lesions. In parallel, γδTregs cocultured with organ-cultured, MICA/B-overexpressing human scalp hair follicles suppressed pathogenic CD8+/NKG2D+ T-cell activity and counteracted all AA hallmarks ex vivo-including hair follicle immune privilege collapse, hair follicle dystrophy, and premature IFNγ-induced catagen-through IL-10 and TGF-β1 secretion, contact-dependent inhibition, and adenosine generation through CD39/CD73. These findings in a model human autoimmune disease introduce human γδTregs as clinically important regulatory lymphocytes and invite the use of autologous peripheral Vδ2+/Foxp3+ γδTregs as a cell-based therapy for AA and possibly other CD8+ T cell-dependent autoimmune diseases characterized by immune privilege collapse.

Abstract Introduction Diabetes is a widespread metabolic disorder. One of its most severe complications is diabetic foot ulcers (DFUs)-open wounds characterized by chronic inflammation and delayed healing. DFUs are the leading cause of non-traumatic lower limb amputations globally due to poor repair capacity and increased infection risk. Gamma delta (γδ) T cells are key immune cells involved in the skin’s defense and repair processes, making them an important population to study in the context of DFUs. The activity of γδ T cells is shaped by butyrophilin-like (BTNL) proteins. One member of this family, BTNL2, regulates T cell activation and may be altered by chronic conditions such as diabetes. Methods Here, we utilize an STZ-induced diabetic mouse model to investigate the role of multiple γδ T cell subsets (Vγ1—4) in both diabetic and non-diabetic skin, at steady state and during wound healing. Using GFP γδ T cell reporter mouse, flow cytometry and immunofluorescence, we assess γδ T cell activation and localization. Additionally, we examine BTNL2’s role in modulating γδ T cell activity through in vitro coculture assay. Results Under hyperglycemic conditions, γδ T cell subsets are dysregulated, with an increase in pro-inflammatory Vγ1 cells and a decrease in pro-repair Vγ3 dendritic epidermal T cells (DETCs). Diabetic mice display delayed wound closure six days post-injury, confirmed by H&E and Keratin 6 staining. Using the GFP γδ T cell reporter mice, we observe γδ T cell localization at the wound edge, predominantly Vγ1 cells, alongside with increased BTNL2 expression. Coculture assays reveal that BTNL2 immunomodulates human Vδ1 but not Vïδ2 γδ T cells. Conclusion Overall, our findings highlight the distinct roles of skin γδ T cell subsets and BTNL2 expression during wound healing and provide important clues to design BTNL2-based therapeutics that can modulate the skin T cell immune responses in chronic inflammatory conditions. Funding Source NIH T32 University of Miami “Predoctoral Training in Translational Immunology,” NIH/NINR, R01NR015649-01 NIH/NIDDK, R01DK136241 Topic Categories Mucosal and Regional Immunology (MUC)

The skin covers the entire surface of the body and therefore is the largest organ in humans. The skin has various functions, primarily defence from infections and trauma. With aging, profound changes occur that compromise its key functions, leading to impaired barrier protection and immune responses. This is in part due to the increased low-grade systemic inflammation known as inflammaging, driven by senescent cells, and release of pro-inflammatory cytokines, to which the skin also significantly contributes. As a consequence of inflammaging, the skin’s function is compromized. The cellular and molecular components involved are summarized in this review.

Diabetes Mellitus (DM) is a chronic disorder affecting millions globally, leading to complications like diabetic foot ulcers (DFUs) and impaired wound healing. In the skin, gamma delta T cells (GDTs), a specialized subset of T lymphocytes, regulate inflammation and promote wound repair. We have shown that the expression of the antimicrobial protein, perforin-2 (P-2) in skin GDTs is decreased in DFUs, but the specific roles of GDTs in the DFU microenvironment remain unclear. To investigate, we characterized GDTs in a diabetic mouse model (STZ) and confirmed the role of butyrophilins and butyrophilin-like molecules in their activation. Full-thickness wounds were induced on the dorsal skin of diabetic and nondiabetic three-month-old male and female C57BL/6 and P-2 deficient (-/-) mice, with healing assessed on day 3 post-wounding. Diabetic skin exhibited fewer CD45+CD3+ T cells compared to non-diabetic skin. Circulating Vγ1+ GDTs were significantly higher in diabetic skin, while epidermal Vγ3+ GDTs were significantly lower compared to non-diabetic skin. Wound healing was delayed in diabetic skin, with greater delays in P-2 knockout mice. Co-culture experiments confirmed that epithelial Skint1 induces GDT activation. Our findings underscore the critical role of GDTs in diabetic wound healing, highlight the importance of an intact P-2 GDT cell-mediated response for successful wound closure, and suggest cellular therapeutic targets to improve skin repair. NIH/NINR, R01NR015649-01 NIH/NIDDK, R01DK136241 5T32AI162624-03 Immune Response Regulation: Cellular Mechanisms (IRC)

Is there a difference in endometrial immune cell expression at the time of embryo transfer in fresh vs. frozen transfer cycles? Our study is the first to compare the endometrial immune cell profile at the time of the embryo transfer between fresh and frozen cycles. Endometrial receptivity plays a crucial role in implantation success for both natural conception and assisted reproductive technology cycles; however, limited information is available about the endometrial environment during the actual implantation window. Although some data suggest a difference in pregnancy rates between fresh and frozen embryo transfers, the underlying mechanisms remain unclear. This study aims to address this gap by analyzing the endometrial milieu using an innovative approach that examines immune cells collected at the time of embryo transfer in both fresh and frozen transfer cycles. This study was approved by Institutional Review Board (# 49174). The study was designed as a prospective observational cohort study at a single academic fertility center. Fifty-two participants with embryos available for transfer were recruited, from which 42 underwent frozen embryo transfer (FET) and 10 underwent fresh embryo transfer (FrET). IVF protocols for FrET consisted of antagonist protocols with gonadotropin dose adjusted based on ovarian reserve, while FET cycles used hormone replacement protocols. All embryo transfers were performed under transabdominal ultrasound guidance. Upon completion of transfer and catheter verification, the transfer catheter tip was rinsed in IMDM medium containing 10% FBS. After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD56, CD14, and CD66b. The samples were acquired on Sony SP6800 Spectral Analyzer. Mann-Whitney U test was used to test statistical differences between groups. A statistically significant difference in the immune milieu was observed in patients undergoing FET compared to FrET cycles. In patients that received a FrET, there was a significantly higher expression of total immune cells (CD45+), 20.50%, compared to FET cycles, 6.60%, within all live cells (p = 0.0008). In addition, FrET cycles had an increase in the endometrial expression of total T cells (CD3+), 17.75%, compared to 2.70% T cells in the endometrium from patients undergoing FET cycles (p = 0.0051). Furthermore, patients undergoing FET cycles, had significantly greater endometrial expression of CD8+ T cells compared to FrET cycles, 20.5% vs. 2.30%, respectively (p = 0.0058). Finally, FET cycles were noted to have increased endometrial expression of B cells (CD19+) and NK cells (CD56+), while the endometrium for patients undergoing FrET cycles demonstrated greater GDT+ and T regulatory cells (CD4+CD25+CD127-), these differences did not reach statistical significance. There were similar endometrial expressions of T cells (CD4+), macrophages (CD14+), and neutrophils (CD66b+) among both types of cycles. Limitations of our study include the small sample size undergoing FrET. In addition, we are planning to compare the immune cell profiles between the two groups in relation to cycle outcomes. Furthermore, multicolor flow cytometry may provide additional immune cell composition of the immune milieu during the window of implantation. The endometrial milieu in general, and the immune environment in particular, differ between fresh and frozen cycles, and this could play a critical role in implantation. Identifying cycle specific immune cell profiles may reveal factors that optimize implantation, paving the way for diagnostic and therapeutic innovations to improve IVF outcomes. No

Chronic wounds such as diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) are a growing public health burden, contributing to significant morbidity, healthcare costs, and mortality. Current management strategies are hampered by the inability to accurately predict healing trajectories or identify early treatment failures. Here, we present a novel, non-invasive proteomic approach that leverages routinely discarded wound dressings to profile the wound microenvironment across soluble, cellular, and extracellular vesicle (EV) compartments. Using a cohort of patients with DFU and VLU (n = 16), we performed spatially resolved, longitudinal sampling over 4 weeks and identified distinct proteomic signatures associated with healing versus non-healing outcomes. Mass spectrometry revealed more than 1000 unique proteins from viable cells and EVs as well as 489 proteins identified from soluble wound fluid. Our methodology captured immune cell phenotypes, enabling insight into tissue-specific immune responses without the need for biopsy. Longitudinal surveillance revealed patient-specific protein trajectories that correlated with wound size changes, supporting the feasibility of biomarker-based monitoring. Notably, differences in keratinocyte activation markers, acute-phase proteins, and EV-associated stress proteins distinguished healing from non-healing wounds. These findings demonstrate the utility of discarded dressings as a rich, untapped source of diagnostic and prognostic biomarkers. This platform has strong potential to inform future "smart" wound care technologies, including dressings capable of real-time biosensing or therapeutic EV delivery. Larger validation studies will be essential to translate these findings into clinical tools for precision wound care.

Butyrophilin-like 2 (BTNL2), a member of the immunoglobulin (Ig) superfamily, regulates immune responses and maintains immune tolerance. Yet, it remains largely unknown how BTNL2 interacts with gamma/delta (GDT) T cells, key immune surveillance cells in epithelial tissues. Since GDT cells have recently surfaced as important pathogenic T cells in alopecia areata (AA), which results from collapse of the hair follicle’s (HF) physiological immune privilege (IP), we have asked here how BTNL2 influences HF interactions with GDT cells. By immunofluorescent microscopy, we found BTNL2 to be predominately expressed in the outer root sheath keratinocytes of healthy human scalp HFs. Human scalp skin VD1 GDT and CD8 T cells were co-cultured with autologous stressed and non-stressed HFs ex vivo. BTNL2 expression was significantly lower in stressed than in non-stressed HFs (p < 0.01), correlating with increased expression of NKG2D and IFNg in VD1 GDT (p < 0.05) (stressed HFs overexpress MICA and CD1d). In stressed HFs, cytotoxicity by VD1 GDT and CD8+ T cells was heightened, leading to HF dystrophy, as indicated by melanin clumping and premature catagen induction—hallmarks of AA-like immune-mediated HF damage. Interrogating immune-privileged HFs as an instructive model mini-organ, our study introduces BTNL2 expression on HF keratinocytes as a novel player in controlling autoaggressive dermal GDT cell responses in human skin and as a novel candidate target in future AA management. Team Science Funding Program, University of Miami Mucosal and Regional Immunology (MUC)

Hidradenitis suppurativa tunnel structures lined with epithelium within the dermis are unique features of advanced disease stages that significantly impair patients' QOL. The presence of hidradenitis suppurativa tunnels is associated with a decreased likelihood of achieving a clinical response, even when receiving biological therapy. The cellular and molecular mechanisms underlying tunnel formation and pathology are only partially understood, which hampers the development of more effective targeted therapies. Tunnels create a unique microenvironment that drives a vicious cycle of hidradenitis suppurativa inflammation, with tunnel keratinocytes exhibiting an activated phenotype characterized by distinct gene expression signatures. In this review, we summarize the current literature and discuss aspects of the pathophysiology of tunnels, including the role of hair follicle epidermal stem cells in tunnel formation, potential role of fibroblast-mediated epithelial-mesenchymal transition, role of dermal papilla fibroblasts, and aberrant proinflammatory repair response contributing to the observed fibrosis and scarring. Finally, tunnel structures are characterized by unique microbial dysbiosis and an overabundance of Gram-negative anaerobes that are not targeted by current therapeutics. In addition to outlining the possible mechanisms of tunnel formation, we provide perspectives on the translation of current knowledge into more effective treatment approaches for patients with hidradenitis suppurativa tunnels.

Diabetic foot ulcers (DFUs) are a common and debilitating complication of diabetes, and amputations from non-healing ulcers carry high morbidity and mortality. A critical need exists for biomarkers that can identify healing potential early and guide targeted interventions. To address this, we applied an integrated multi-omics approach across four patient cohorts comprising 51 DFUs (29 Healing, 22 Non-healing). Bulk RNA-sequencing revealed marked activation of Th1 and Th2 pathways (activation z-score +4.8, p = 3.8×10-15), and immune cell deconvolution predicted higher proportions of T cell populations in Healers. Spatial proteomics in a second cohort identified elevated CD3+ T cell density and selective enrichment of PD-1 and PD-L1 expression in vascular niches of the papillary dermis in Healers (p < 0.001). Flow cytometry in a third cohort further demonstrated higher proportions of CD3+PD-1+ and CD3+PD-L1+ T cells in Healers compared with Non-healers. Single-cell RNA-sequencing from a fourth cohort showed upregulation of PD-1 and PD-L1 within CD4+ T cells from Healers. Complementary immunofluorescence and serological profiling confirmed that both PD-1 and PD-L1 are elevated in tissue and circulating serum of healing DFUs, supporting their potential use as systemic biomarkers. Taken together, vascular-enriched PD-1/PD-L1 signaling and T cell activation were observed in association with healing DFUs, supporting PD-1/PD-L1 as candidate biomarkers in both tissue and blood with potential translational relevance for predicting DFU outcomes and informing precision therapies.

Immune cells undergo metabolic reprogramming to meet the demands associated with immune responses. The effects of aging on these pathways and on the metabolic phenotype of the immune cells participating in antibody responses to vaccines are still largely unknown. Here we used a vaccine for SARS-CoV-2 that utilizes the cellular heat shock chaperone glycoprotein 96 (gp96), engineered to co-express SARS-CoV-2 Spike (spike) protein (gp96-Ig-S). Results show that this vaccine induces comparable B cell primary responses in young and old mice at later time points, but a significantly lesser secondary response in old as compared to young mice, with the antibodies generated in the secondary response being also of lower avidity. This occurs because aging changes the B cell metabolic phenotype and induces hyper-metabolic B cells that are associated with higher intrinsic inflammation and decreased protective antibody responses. However, the gp96-Ig-S vaccine was found to be effective in significantly reducing the metabolic/inflammatory status of B cells from old mice, suggesting the possibility that targeting metabolic pathways may improve immune function in old mice that do not respond adequately to the vaccine.