Lancaster, Jessica N

Abstract In human melanoma, tumor cell-specific MHC class II expression is associated with improved response to programmed death 1 and programmed death-ligand 1 blockade; yet the direct effect of melanoma cell-specific MHC class II expression on anti-melanoma T cell responses remains largely unknown. In the clinically relevant Yale University Mouse Melanoma Exposed to Radiation (YUMMER) cell lines, MHC class II expression was IFN-γ-inducible in a subset of cells. Transduction of melanoma cells with CIITA resulted in uniformly high, constitutive expression of MHC class II. YUMMER.G cells transduced with CIITA vector had increased in vivo tumor growth in wild-type mice compared to melanoma cells transduced with empty vector. YUMMER.G cells transduced with empty or CIITA vectors had the same in vivo tumor growth in RAG−/− mice, indicating that the difference in tumor growth between CIITA- and empty-transduced cells was dependent on an intact adaptive immune system. CIITA-transduced YUMMER.G tumors exhibited an increased frequency and infiltration depth of CD4+ T cells, an increased frequency of T regulatory cells, and a decreased frequency and infiltration depth of CD8+ T cells, compared to empty-transduced tumors. Antibody depletion revealed that CIITA- and empty-transduced YUMMER.G tumors were constrained by CD4+ and CD8+ T cells, but only tumor growth of CIITA-transduced cells was decreased by CD25 blockade, which primarily blocks T regulatory cell function. Overall, this work reveals a potential pro-tumorigenic role of melanoma cell-specific overexpression of MHC class II.

Abstract Macrophages in the tumor microenvironment are known to upregulate PD-L1 expression, thereby suppressing T cells through PD-1 ligation. However, the manner in which PD-L1 expression intrinsically impacts macrophages and their immunomodulatory phenotype is less clear. Clarifying this knowledge gap would yield insight into the mechanisms of immunosuppression within the tumor microenvironment. To characterize the macrophage intrinsic role of PD-L1, we used complementary genetic and pharmacological approaches by analyzing primary murine bone marrow-derived macrophages (BMDMs) with complete genetic PD-L1 deletion and wildtype BMDMs treated with anti-PD-L1 blocking antibodies. Macrophages were evaluated across naïve, pro-inflammatory (M1), and tumor conditioned (TCM) polarization states in vitro. Unlike prior reports, neither genetic deletion nor antibody blockade dramatically altered the expression of macrophage polarization markers or in vitro phagocytic capacity. Both conditions consistently reduced surface levels of the M1-associated costimulatory molecule CD80, prompting further analysis of T cell interacting and antigen presenting proteins, in which we revealed disparate effects of genetic deletion and antibody blockade on the surface levels of MHCI, MHCII, PD-1, and PD-L2. These findings suggest that PD-L1 deletion and antibody-mediated blockade contribute to macrophage immune regulatory profiles in distinct manners. This difference supports a model in which PD-L1 functions in macrophages beyond its canonical role as a ligand for PD-1, influencing antigen presentation and checkpoint molecule levels and playing a broader role in immune regulation in the tumor microenvironment.

The major barrier to clinical translation of αCD137 immunotherapy is separating antitumor efficacy from hepatotoxicity driven by IFN-γ–producing CD8 T cells. We propose a strategy to limit toxicity by promoting contraction of excessively expanded CD8 T cells. We identify CD11c + KLRG1 + effector CD8 T cells (CD11c + T E ) as the primary source of IFN-γ, recirculating between blood and secondary lymphoid organs (SLOs), where they undergo apoptosis during contraction. We show that lymphotoxin β receptor (LTβR) signaling restrains this contraction. Mechanistically, lymphotoxin-expressing B cells activate LTβR in fibroblastic reticular cells (FRCs), suppressing apoptosis of CD11c + T E cells in the spleen and sustaining their systemic circulation and liver infiltration. Pharmacological LTβR blockade abrogates hepatotoxicity by reducing the accumulation of IFN-γ–producing CD11c + T E cells while preserving tumor-specific CD8 T cell responses. These findings identify LTβR as a key regulator of effector CD8 T cell persistence and support the use of LTβR antagonists to improve the safety of αCD137-based immunotherapy.

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, and despite advances in treatment, immune suppression remains an obstacle to effective therapy. Effector CD4+ T cells (CD4+ Teff) are critical for antitumor immunity, but their function is often inhibited by regulatory T cells (Treg), which accumulate in lung tumors and mediate 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. Herein, we identify the extracellular adenosine 5-triphosphate receptor P2RX7 as a key regulator of Treg function in lung tumors. In a murine lung cancer model induced by Lewis lung carcinoma cells, we found that P2RX7 enhanced the suppressive capacity of tumor-infiltrating Tregs, promoting tumor growth. In T cell–specific P2RX7-knockout (P2RX7-KO) mice, reduced Treg infiltration was accompanied by increased CD4+ Teff accumulation and improved tumor control. Treg-specific P2RX7-KO mice exhibited reduced tumor growth, confirming a Treg-intrinsic role of P2RX7. Suppression assays revealed that tumor-infiltrating wild-type Tregs had greater suppressive activity compared with P2RX7-KO Tregs, which failed to inhibit type 1 and follicular helper T–like responses. This was associated with increased tumor-specific IgG production by lung B cells in P2RX7-KO mice. We also observed that wild-type Tregs expressed higher levels of the immunosuppressive molecule CTLA-4 when compared with P2RX7-KO Tregs. Thus, we conclude that P2RX7 expression on Tregs is essential for their suppressive function in lung cancer and targeting P2RX7 may constitute a strategy to improve lung cancer treatment by alleviating Treg-mediated immune suppression.

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.

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.

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.

AbstractPelvic organ prolapse (POP) is a dysfunction that affects a large proportion of women. Current support scaffolds’ lack of biocompatibility, biodegradability, and mechanical compliance are associated with surgical complications including erosion and pain, indicating the urgent need for new tissue scaffolds with customizable functions. A new material that uses polyvinyl alcohol (PVA) as the main ingredient and is chemically tuned to possess suitable mechanical properties and degradation rates for the surgical treatment of POP is developed. Specifically, the thiol‐norbornene “click” chemistry enables the sol‐gel transition of the biomaterial under UV‐light without side‐products. Meanwhile, NaOH treatment further toughens the hydrogel with a higher crosslink density. The PVA‐based biocompatible ink can be printed with UV‐facilitated direct ink writing due to the rapidly UV‐initiated chemical crosslink; in situ image analysis and machine learning methods are applied during this procedure to quantify and improve the printing quality. The cell viability results demonstrate the biocompatibility of the POP scaffolds, suggesting the potential for future animal studies and the possibility of clinical research. This study bridges polymer chemistry and manufacturing engineering with a specific tissue engineering application to solve the common disorder of POP, shedding light on individualized medicine and intelligent systems for biomedical engineering.