Lancaster, Jessica N.

ABSTRACT Lung cancer is the leading cause of cancer-related deaths worldwide and, despite treatment advances, immune suppression remains an obstacle to effective therapy. Effector CD4 + T cells (CD4 + Teffs) are critical for antitumor immunity, but their function is often inhibited by regulatory T cells (Tregs), which accumulate in lung tumors and perform suppressive functions through multiple mechanisms. This suppression leads to tumor progression and poor patient outcomes. However, the mechanisms underlying Treg-mediated suppression are not fully understood. Here, we identify the extracellular ATP receptor P2RX7 as a key regulator of Treg function in lung tumors. Using a murine lung cancer model induced by Lewis lung carcinoma cells, we demonstrate that P2RX7 enhances the suppressive capacity of tumor-infiltrating Tregs, promoting tumor growth. In T cell-specific P2RX7-KO mice, reduced Treg infiltration was accompanied by increased CD4 + Teff accumulation and improved tumor control. Treg-specific P2RX7-KO mice exhibit reduced tumor growth, confirming a cell-intrinsic role of P2RX7 in Tregs. Suppression assays revealed that tumor-infiltrating WT Tregs have greater suppressive activity compared to P2RX7-KO Tregs, which failed to inhibit type 1 and Tfh-like responses. This was associated with increased tumor-specific IgG production by lung B cells in P2RX7-KO mice. We also observed that WT Tregs express higher levels of the immunosuppressive surface molecule CTLA-4 when compared to P2RX7-KO Tregs. In summary, we show that P2RX7 expression on Tregs is essential for their suppressive function in lung cancer, and targeting of P2RX7 may constitute a novel strategy to improve lung cancer treatment by alleviating Treg-mediated immune suppression.

Our polyvinyl alcohol feedstock was prepared through carbic anhydride functionalization, UV curing during direct ink writing, and base treatment. The scaffold exhibited mechanical properties similar to pelvic floor tissue.

Abstract 3D printing, also known as additive manufacturing, holds immense potential for rapid prototyping and customized production of functional health‐related devices. With advancements in polymer chemistry and biomedical engineering, polymeric biomaterials have become integral to 3D‐printed biomedical applications. However, there still exists a bottleneck in the compatibility of polymeric biomaterials with different 3D printing methods, as well as intrinsic challenges such as limited printing resolution and rates. Therefore, this review aims to introduce the current state‐of‐the‐art in 3D‐printed functional polymeric health‐related devices. It begins with an overview of the landscape of 3D printing techniques, followed by an examination of commonly used polymeric biomaterials. Subsequently, examples of 3D‐printed biomedical devices are provided and classified into categories such as biosensors, bioactuators, soft robotics, energy storage systems, self‐powered devices, and data science in bioplotting. The emphasis is on exploring the current capabilities of 3D printing in manufacturing polymeric biomaterials into desired geometries that facilitate device functionality and studying the reasons for material choice. Finally, an outlook with challenges and possible improvements in the near future is presented, projecting the contribution of general 3D printing and polymeric biomaterials in the field of healthcare.

As a treatment for the widely spread cardiovascular diseases (CVD), bypass vascular grafts have room for improvement in terms of mechanical property match with native arteries. A 3D‐printed nozzle is presented, featuring unique internal structures, to extrude artificial vascular grafts with a flower‐mimicking geometry. The multilayer‐structured graft wall allows the inner and outer layers to interfere sequentially during lateral expansion, replicating the nonlinear elasticity of native vessels. Both experiment and simulation results verify the necessity and benefit of the flower‐mimicking structure in obtaining the self‐toughening behavior. The gelation study of natural polymers and the utilization of sacrificial phase enables the smooth extrusion of the multiphase conduit, where computer‐assisted image analysis is employed to quantify manufacturing fidelity. The cell viability tests demonstrate the cytocompatibility of the gelatin methacryloyl (GelMA)/sodium alginate grafts, suggesting potential for further clinical research with further developments. This study presents a feasible approach for fabricating bypass vascular grafts and inspires future treatments for CVD.

AbstractWeakened germinal center responses by the aged immune system result in diminished immunity against pathogens and reduced efficacy of vaccines. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have decreased expansion of antigen‐specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with accumulated Tfh cells, even in naïve mice. Despite increased numbers, aged Tfh had reduced expression of master transcription factor BCL6 and increased expression of the ectonucleotidase CD39. In vitro activation revealed that proliferative capacity was maintained in aged CD4+ T cells, but not the costimulatory molecule CD40L. When activated in vitro by aged antigen‐presenting cells, young CD4+ naïve T cells generated reduced numbers of activated cells with upregulated CD40L. To determine the contribution of cell‐extrinsic influences on antigen‐specific Tfh induction, young, antigen‐specific B and CD4+ T cells were adoptively transferred into aged hosts prior to peptide immunization. Transferred cells had reduced expansion and differentiation into germinal center B cell and Tfh and reduced antigen‐specific antibody titers when compared to young hosts. Young CD4+ T cells transferred aged hosts differentiated into Tfh cells with reduced PD‐1 and BCL6 expression, and increased CD39 expression, though they maintained their mitochondrial capacity. These results highlight the role of the lymphoid microenvironment in modulating CD4+ T cell differentiation, which contributes to impaired establishment and maintenance of germinal centers.

AbstractNanoparticles form long‐range micropatterns via self‐assembly or directed self‐assembly with superior mechanical, electrical, optical, magnetic, chemical, and other functional properties for broad applications, such as structural supports, thermal exchangers, optoelectronics, microelectronics, and robotics. The precisely defined particle assembly at the nanoscale with simultaneously scalable patterning at the microscale is indispensable for enabling functionality and improving the performance of devices. This article provides a comprehensive review of nanoparticle assembly formed primarily via the balance of forces at the nanoscale (e.g., van der Waals, colloidal, capillary, convection, and chemical forces) and nanoparticle‐template interactions (e.g., physical confinement, chemical functionalization, additive layer‐upon‐layer). The review commences with a general overview of nanoparticle self‐assembly, with the state‐of‐the‐art literature review and motivation. It subsequently reviews the recent progress in nanoparticle assembly without the presence of surface templates. Manufacturing techniques for surface template fabrication and their influence on nanoparticle assembly efficiency and effectiveness are then explored. The primary focus is the spatial organization and orientational preference of nanoparticles on non‐templated and pre‐templated surfaces in a controlled manner. Moreover, the article discusses broad applications of micropatterned surfaces, encompassing various fields. Finally, the review concludes with a summary of manufacturing methods, their limitations, and future trends in nanoparticle assembly.

Abstract PD-1- and PD-L1-targeted immune checkpoint blockade therapy has shown great clinical potential. Unfortunately, this therapy has been ineffective in a majority of patients bearing solid tumors due to the highly immunosuppressive nature of the tumor microenvironment. As macrophages compose the most abundant myeloid cell subset within the tumor, current macrophage-targeted treatments aim to deplete macrophages within the tumor to reduce immune suppression. However, our goal is to determine whether αPD-1 and αPD-L1 therapy can harness macrophage potential by shifting tumor associated macrophages (TAMs) from a suppressive M2 to a pro-inflammatory M1 phenotype. Our preliminary data has shown that αPD-1/αPD-L1 treatment of mice bearing B16 murine melanoma leads to controlled tumor growth and this was accompanied by a higher M1:M2 ratio and CD8 T-cell abundance. Therefore, we hypothesized that αPD-1/αPD-L1 therapy may drive TAMs towards a pro-inflammatory phenotype and to become better activators of CD8 T-cells. By analyzing antigen presentation machinery on TAMs, we show that in the presence of tumor cells, αPD-L1 blockade upregulates expression of MHC1 and MHC2 on TAMs. Unexpectedly, direct αPD-L1 blockade on TAMs leads to reduced CD80 surface due to mechanisms still to be elucidated. Further, we analyzed co-cultures of αPD-L-treated TAMs loaded with ovalbumin and naïve OT1 cells, which led to greater proliferation of naïve CD8 T-cells compared to their isotype control counterparts. Our observations thus far support the ability of αPD-1/αPD-L1 therapy to induce an M1-phenotype shift among macrophages and also αPD-L1 blockade alone can drive TAMs to become better CD8 T-cell activators. Mayo Clinic David and Margaret Grohne Cancer Immunotherapy and Mayo Clinic Immune Monitoring Core Pilot Awards

Abstract Aging dramatically impacts T cells, contributing to diminished and delayed responses to infection. While the development and maintenance of lymphocytes certainly undergo intrinsic changes with age, the contribution of cell-extrinsic forces, such as the aging tissue microenvironment, has been difficult to uncouple. Macroscopic changes to the lymph nodes have been well described, but the impact of aging on lymph node stromal subsets is less understood. Here, we visualized naïve T cell migration in the aged murine lymph node using live-cell 2-photon microscopy and determined that T cell motility was impaired in regions of increased fibrotic deposition. Using fluorescent reporters for Ccl19 and Cxcl12, homeostatic chemokines important for stromal:lymphocyte crosstalk, we also found that the distribution of stromal cells expressing these chemokines was altered with age, and that migrating naïve T cells were excluded from regions of chemokine expression. Flow cytometric analyses of fibroblastic reticular cells (FRCs) revealed decreased proportions of FRCs within the mesenteric lymph nodes and decreased putative FRC progenitors in the skin-draining lymph nodes. FRC progenitors at both sites had reduced surface expression of lymphotoxin-beta receptor, an important receptor for FRC differentiation and maintenance. We hypothesize that age-associated changes to FRC maturation contribute to increased lymph node fibrosis and altered localization of homeostatic factors. Current studies have indicated that in vitro cultured lymph node fibroblasts are sensitive to TNF-alpha and respond by increasing expression of alpha-smooth muscle actin, suggesting a mechanism by which aging remodels the lymph node microenvironment.

Abstract The aging immune system responds poorly to infection and vaccination, producing reduced numbers of germinal centers, low affinity antibody titers, and reduced B cell memory. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have less expansion of antigen-specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with decreased CD4+ T conventional cells and accumulated Tfh cells and regulatory T cells, even in naïve mice. Despite increased numbers, our results indicate that aged Tfh have decreased metabolic function, which may have repercussions on germinal center responses. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, we adoptively transferred young, antigen-specific B and CD4+ naïve T cells into aged hosts prior to peptide immunization, which resulted in reduced antigen-specific germinal center B cell and Tfh expansion and reduced antibody titers when compared to young hosts. The results from these experiments emphasize the importance of the lymphoid microenvironment in germinal center initiation and maintenance. We hypothesize that age-associated Tfh accumulation may impair antigen-specific Tfh induction and subsequent germinal center responses. Current studies use live-cell 2-photon microscopy to track the impact of age on lymphocyte motility and Tfh:B interactions, in order to determine the relative contributions of aged APCs, non-specific aged Tfh, and aged regulatory T cells to germinal center deficiency. Research supported by the Mayo Clinic Robert and Arlene Kogod Center on Aging Innovation Award UL1R002377