PD-1/CD80+ small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression.

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity.

Ox-LDL-induced CD80+ macrophages expand pro-atherosclerotic NKT cells via CD1d in atherosclerotic mice and hyperlipidemic patients.

Atherosclerosis is an inflammatory disease of blood vessels involving the immune system. Natural killer T (NKT) cells, as crucial components of the innate and acquired immune systems, play critical roles in the development of atherosclerosis. However, the mechanism and clinical relevance of NKT cells in early atherosclerosis are largely unclear. The study investigated the mechanism influencing NKT cell function in apoE deficiency-induced early atherosclerosis. Our findings demonstrated that there were higher populations of NKT cells and interferon-gamma (IFN-γ)-producing NKT cells in the peripheral blood of patients with hyperlipidemia and in the aorta, blood, spleen, and bone marrow of early atherosclerotic mice compared with the control groups. Moreover, we discovered that the infiltration of CD80+ macrophages and CD1d expression on CD80+ macrophages in atherosclerotic mice climbed remarkably. CD1d expression increased in CD80+ macrophages stimulated by oxidized low-density lipoprotein (ox-LDL) ex vivo and in vitro. Ex vivo coculture of macrophages with NKT cells revealed that ox-LDL-induced CD80+ macrophages presented lipid antigen α-Galcer (alpha-galactosylceramide) to NKT cells via CD1d, enabling NKT cells to express more IFN-γ. Furthermore, a greater proportion of CD1d+ monocytes and CD1d+CD80+ monocytes were found in peripheral blood of hyperlipidemic patients compared with that of healthy donors. Positive correlations were found between CD1d+CD80+ monocytes and NKT cells or IFN-γ+ NKT cells in hyperlipidemic patients. Our findings illustrated that CD80+ macrophages stimulated NKT cells to secrete IFN-γ via CD1d-presenting α-Galcer, which may accelerate the progression of early atherosclerosis. Inhibiting lipid antigen presentation by CD80+ macrophages to NKT cells may be a promising immune target for the treatment of early atherosclerosis.NEW & NOTEWORTHY This work proposed the ox-LDL-CD80+ monocyte/macrophage-CD1d-NKT cell-IFN-γ axis in the progression of atherosclerosis. The proinflammatory IFN-γ+ NKT cells are closely related to CD1d+CD80+ monocytes in hyperlipidemic patients. Inhibiting CD80+ macrophages to present lipid antigens to NKT cells through CD1d blocking may be a new therapeutic target for atherosclerosis.

The B7:CD28 family and friends: Unraveling coinhibitory interactions.

Immune responses must be tightly regulated to ensure both optimal protective immunity and tolerance. Costimulatory pathways within the B7:CD28 family provide essential signals for optimal T cell activation and clonal expansion. They provide crucial inhibitory signals that maintain immune homeostasis, control resolution of inflammation, regulate host defense, and promote tolerance to prevent autoimmunity. Tumors and chronic pathogens can exploit these pathways to evade eradication by the immune system. Advances in understanding B7:CD28 pathways have ushered in a new era of immunotherapy with effective drugs to treat cancer, autoimmune diseases, infectious diseases, and transplant rejection. Here, we discuss current understanding of the mechanisms underlying the coinhibitory functions of CTLA-4, PD-1, PD-L1:B7-1 and PD-L2:RGMb interactions and less studied B7 family members, including HHLA2, VISTA, BTNL2, and BTN3A1, as well as their overlapping and unique roles in regulating immune responses, and the therapeutic potential of these insights.

Impaired monocyte-derived dendritic cell phenotype in prostate cancer patients: A phenotypic comparison with healthy donors.

BACKGROUND: Dendritic cells (DCs) play a crucial role in immunity. Research on monocyte-derived DCs (Mo-DCs) cancer vaccines is in progress despite limited success in clinical trials. This study focuses on Mo-DCs generated from prostate cancer (PCA) patients, comparing them with DCs from healthy donors (HD-DCs). METHODS: Mo-DCs were isolated from PCA patient samples, and their phenotype was compared to HD-DCs. Key parameters included monocyte count, CD14 expression, and the levels of maturation markers (HLA-DR, CD80, CD86) were assessed. RESULTS: PCA samples exhibited a significantly lower monocyte count and reduced CD14 expression compared to healthy samples (p ⟨ 0.0001). Additionally, PCA-DCs expressed significantly lower levels of maturation markers, including HLA-DR, CD80, and CD86, when compared to HD-DCs (p = 0.123, p = 0.884, and p = 0.309, respectively). CONCLUSION: The limited success of DC vaccines could be attributed to impaired phenotypic characteristics. These observations suggest that suboptimal characteristics of Mo-DCs generated from cancer patient blood samples might contribute to the limited success of DC vaccines. Consequently, this study underscores the need for alternative strategies to enhance the features of Mo-DCs for more effective cancer immunotherapies.

Clinicopathologic Evaluation of CD80, CD86, and PD-L1 Expressions with Immunohistochemical Methods in Malignant Melanoma Patients.

OBJECTIVE: Diagnostic and prognostic biomarkers for malignant melanoma are crucial for treatment and for developing targeted therapies. Malignant melanoma is a highly immunogenic tumor, and its regression, treatment, and prognostic evaluation are directly related to escape from immune destruction. Therefore, we aimed to determine the expression levels of CD80, CD86, and PD -L1 in malignant melanoma tissue samples by immunohistochemistry and to investigate the possible relationship between these proteins and the clinicopathological features in this study. MATERIAL AND METHODS: Hematoxylin and eosin staining and immunohistochemical staining for CD80, CD86, and PD-L1 were evaluated for clinical data, survival, prognosis, tumor location, malignant melanoma subtypes, tumor size, and prognostic findings. RESULTS: Higher survival rates were observed in patients with lower PD-L1 staining scores in the tumor. The 5-year survival was higher in patients with CD80-positive and CD86-positive biopsies. Mortality was lower in superficial spreading melanoma and Lentigo maligna melanoma types, whereas staining positivity of CD80 and CD86 was higher. Furthermore, a relationship between clinical stage and Breslow thickness ( < 2mm/≥2mm), tumor ulceration, lymph node metastasis, and CD80 and CD86 expression was also identified. CONCLUSION: Our findings suggest that PD-L1, CD80, and CD86 expression are essential in malignant melanoma and could be used as prognostic markers.

cis-B7:CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity.

B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on T cells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ T cells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ T cell survival, migration, and cytokine production. In mouse tumor models, loss of T cell-intrinsic cis-B7:CD28 interactions decreased intratumoral T cells and accelerated tumor growth. Thus, B7 ligands on CD8+ T cells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.

CTLA4 depletes T cell endogenous and trogocytosed B7 ligands via cis-endocytosis.

CD28 and CTLA4 are T cell coreceptors that competitively engage B7 ligands CD80 and CD86 to control adaptive immune responses. While the role of CTLA4 in restraining CD28 costimulatory signaling is well-established, the mechanism has remained unclear. Here, we report that human T cells acquire antigen-presenting-cell (APC)-derived B7 ligands and major histocompatibility complex (MHC) via trogocytosis through CD28:B7 binding. Acquired MHC and B7 enabled T cells to autostimulate, and this process was limited cell-intrinsically by CTLA4, which depletes B7 ligands trogocytosed or endogenously expressed by T cells through cis-endocytosis. Extending this model to the previously proposed extrinsic function of CTLA4 in human regulatory T cells (Treg), we show that blockade of either CD28 or CTLA4 attenuates Treg-mediated depletion of APC B7, indicating that trogocytosis and CTLA4-mediated cis-endocytosis work together to deplete B7 from APCs. Our study establishes CTLA4 as a cell-intrinsic molecular sink that limits B7 availability on the surface of T cells, with implications for CTLA4-targeted therapy.

The Concordant Disruption of B7/CD28 Immune Regulators Predicts the Prognosis of Oral Carcinomas.

Immune modulation is a critical factor in determining the survival of patients with malignancies, including those with oral squamous cell carcinoma (OSCC) and head and neck SCC (HNSCC). Immune escape or stimulation may be driven by the B7/CD28 family and other checkpoint molecules, forming ligand-receptor complexes with immune cells in the tumor microenvironment. Since the members of B7/CD28 can functionally compensate for or counteract each other, the concomitant disruption of multiple members of B7/CD28 in OSCC or HNSCC pathogenesis remains elusive. Transcriptome analysis was performed on 54 OSCC tumors and 28 paired normal oral tissue samples. Upregulation of CD80, CD86, PD-L1, PD-L2, CD276, VTCN1, and CTLA4 and downregulation of L-ICOS in OSCC relative to the control were noted. Concordance in the expression of CD80, CD86, PD-L1, PD-L2, and L-ICOS with CD28 members was observed across tumors. Lower ICOS expression indicated a worse prognosis in late-stage tumors. Moreover, tumors harboring higher PD-L1/ICOS, PD-L2/ICOS, or CD276/ICOS expression ratios had a worse prognosis. The survival of node-positive patients was further worsened in tumors exhibiting higher ratios between PD-L1, PD-L2, or CD276 and ICOS. Alterations in T cell, macrophage, myeloid dendritic cell, and mast cell populations in tumors relative to controls were found. Decreased memory B cells, CD8+ T cells, and Tregs, together with increased resting NK cells and M0 macrophages, occurred in tumors with a worse prognosis. This study confirmed frequent upregulation and eminent co-disruption of B7/CD28 members in OSCC tumors. The ratio between PD-L2 and ICOS is a promising survival predictor in node-positive HNSCC patients.

The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80.

CTLA-4 and PD-1 are key immune checkpoint receptors that are targeted in the treatment of cancer. A recently identified physical interaction between the respective ligands, CD80 and PD-L1, has been shown to block PD-L1/PD-1 binding and to prevent PD-L1 inhibitory functions. Since CTLA-4 is known to capture and degrade its ligands via transendocytosis, we investigated the interplay between CD80 transendocytosis and CD80/PD-L1 interaction. We find that transendocytosis of CD80 results in a time-dependent recovery of PD-L1 availability that correlates with CD80 removal. Moreover, CD80 transendocytosis is highly specific in that only CD80 is internalised, while its heterodimeric PD-L1 partner remains on the plasma membrane of the antigen-presenting cell (APC). CTLA-4 interactions with CD80 do not appear to be inhibited by PD-L1, but efficient removal of CD80 requires an intact CTLA-4 cytoplasmic domain, distinguishing this process from more general trogocytosis and simple CTLA-4 binding to CD80/PD-L1 complexes. These data are consistent with CTLA-4 acting as modulator of PD-L1:PD-1 interactions via control of CD80.

B7-H6 enhances F-actin rearrangement by targeting c-MYC activation to promote medulloblastoma migration and invasion.

Medulloblastoma (MB) is children's most common primary malignant primitive neuro-ectodermal tumor. Group 3 MB showed a higher propensity to metastasis, which is molecularly characterized by c-MYC gene amplification. The activation of c-MYC promotes the remodeling of the F-actin cytoskeleton to enhance metastasis. The B7 homologue 6 (B7-H6) is associated with the manifold essential hallmarks of tumorigenesis. In this study, we will explore whether B7-H6 regulates the reorganization of F-actin by elevating the c-MYC expression to promote metastasis. The Daoy cell line was used to act as the cell model of medulloblastoma. Small interfering RNA and the plasmid were used to downregulate and upregulate the expression of B7-H6 in Daoy cells. Transwell assays with/without the matrigel matrix were used to detect migration and invasion of Daoy cells. Western blots were used to detect the expression of related proteins. Immunofluorescence staining was used to observe the impact of B7-H6 on the c-MYC /F-actin axis. B7-H6 improved migration and invasion in the Daoy cell line. B7-H6 enhanced the rearrangement of F-actin and activated the expression of MMP-9 and MMP-2. B7-H6 promoted the remodeling of F-actin by targeting c-MYC activation to reinforce migration and invasion. B7-H6 acts as a promoter of migration and invasion in medulloblastoma by activating the c-MYC /F-actin axis.

In silico engineering a CD80 variant with increased affinity to CTLA-4 and decreased affinity to CD28 for optimized cancer immunotherapy.

CD80 or cluster of differentiation 80, also known as B7-1, is a member of the immunoglobulin super family, which binds to CTLA-4 and CD28 T cell receptors and induces inhibitory and inductive signals respectively. Although CTLA-4 and CD28 receptors belong to the same protein family, slight differences in their structures leads to CD80 having a higher binding affinity to CTLA-4 (-14.55 kcal/mol) compared with CD28(-12.51 kcal/mol). In this study, we constructed a variant of CD80 protein with increased binding affinity to CTLA-4 and decreased binding affinity to CD28. This variant has no signaling capability, and can act as a cap for these receptors to protect them from natural CD80 proteins existing in the body. The first step was the evolutionary and alanine scanning analysis of CD80 protein to determine conserved regions in this protein. Next, complex alanine scanning technique was employed to determine CD80 protein hotspots in CD80-CTLA-4 and CD80-CD28 protein complexes. This information was fed into a computational model developed in R for in silico mutagenesis and CD80 variant library construction. The 3D structures of variants were modeled using the Swiss model webserver. After modeling the 3D structures, HADDOCK server was employed to build all protein-protein complexes, which contain CTLA-4-CD80 variant complexes, Wild type CD80-CD28 complexes and CD28-CD80 variant complexes. Protein-protein binding free energy was determined using FoldX and the variant number 316 with mutations at 29, 31, 33 positions showed increased binding affinity to CTLA-4 (-21.43 kcal/mol) and decreased binding affinity to CD28 (- 9.54 kcal/mol). Finally, molecular dynamics (MD) simulations confirmed the stability of variant 316. In conclusion, we designed a new CD80 protein variant with potential immunotherapeutic applications.

Identification, binding, and structural characterization of single domain anti-PD-L1 antibodies inhibitory of immune regulatory proteins PD-1 and CD80.

Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment; however, single domain antibodies (VHH) may offer numerous potential benefits. Here, we report the identification and characterization of a diverse panel of 16 novel VHHs specific to PD-L1. The panel of VHHs demonstrate affinities of 0.7 nM to 5.1 µM and were able to completely inhibit PD-1 binding to PD-L1. The binding site for each VHH on PD-L1 was determined using NMR chemical shift perturbation mapping and revealed a common binding surface encompassing the PD-1-binding site. Additionally, we solved crystal structures of two representative VHHs in complex with PD-L1, which revealed unique binding modes. Similar NMR experiments were used to identify the binding site of CD80 on PD-L1, which is another immune response regulatory element and interacts with PD-L1 localized on the same cell surface. CD80 and PD-1 were revealed to share a highly overlapping binding site on PD-L1, with the panel of VHHs identified expected to inhibit CD80 binding. Comparison of the CD80 and PD-1 binding sites on PD-L1 enabled the identification of a potential antibody binding region able to confer specificity for the inhibition of PD-1 binding only, which may offer therapeutic benefits to counteract cancer cell evasion of the immune system.

Breast cancer cells survive chemotherapy by activating targetable immune-modulatory programs characterized by PD-L1 or CD80.

Breast cancer cells must avoid intrinsic and extrinsic cell death to relapse following chemotherapy. Entering senescence enables survival from mitotic catastrophe, apoptosis and nutrient deprivation, but mechanisms of immune evasion are poorly understood. Here we show that breast tumors surviving chemotherapy activate complex programs of immune modulation. Characterization of residual disease revealed distinct tumor cell populations. The first population was characterized by interferon response genes, typified by Cd274, whose expression required chemotherapy to enhance chromatin accessibility, enabling recruitment of IRF1 transcription factor. A second population was characterized by p53 signaling, typified by CD80 expression. Treating mammary tumors with chemotherapy followed by targeting the PD-L1 and/or CD80 axes resulted in marked accumulation of T cells and improved response; however, even combination strategies failed to fully eradicate tumors in the majority of cases. Our findings reveal the challenge of eliminating residual disease populated by senescent cells expressing redundant immune inhibitory pathways and highlight the need for rational immune targeting strategies.

Therapeutic gene editing of T cells to correct CTLA-4 insufficiency.

Heterozygous mutations in CTLA-4 result in an inborn error of immunity with an autoimmune and frequently severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic hematopoietic stem cell transplantation. Here, we designed a homology-directed repair (HDR) gene editing strategy that inserts the CTLA-4 cDNA into the first intron of the CTLA-4 genomic locus in primary human T cells. This resulted in regulated expression of CTLA-4 in CD4+ T cells, and functional studies demonstrated CD80 and CD86 transendocytosis. Gene editing of T cells isolated from three patients with CTLA-4 insufficiency also restored CTLA-4 protein expression and rescued transendocytosis of CD80 and CD86 in vitro. Last, gene-corrected T cells from CTLA-4-/- mice engrafted and prevented lymphoproliferation in an in vivo murine model of CTLA-4 insufficiency. These results demonstrate the feasibility of a therapeutic approach using T cell gene therapy for CTLA-4 insufficiency.

The role of soluble CD80 in patients with soft tissue tumors.

BACKGROUND: Immune checkpoint protein (ICP), which is a central factor group of the immune system, has been reported to have a correlation between the degree of its expression and the prognosis of patients with malignant tumors, and many inhibitors have appeared as therapeutic targets. On the other hand, a soluble form of ICP in circulating blood induced systemic immunosuppression. In this study, we investigated the relationship between the soluble form of CD80 (sCD80) which is a ligand for the inhibitory system CTLA-4, in blood, and clinicopathological parameters in patients with soft tissue tumors. METHODS: A total of 119 patients with primary soft tissue tumors were enrolled in this study. The sCD80 levels were measured by enzyme immunoassay. RESULTS: There were no significant differences in sCD80 levels between benign (34) and soft tissue sarcoma (STS) patients (85). In STS, the high-sCD80 group had significantly lower metastasis-free survival (MS) and lower overall survival (OS) than the low-sCD80 group at 5 years using the log-rank test (OS: high > 404 pg/mL, low ≤ 404 pg/mL, MS: high > 531 pg/ml, low ≤ 531 pg/ml). On multivariate Cox proportional hazard analysis, the high-sCD80 group had significant differences in 5MS and 5OS compared to the low-sCD80 group. CONCLUSIONS: In conclusion, sCD80 may negatively affect systemic immune circumstances, in STS, and may have potential as a therapeutic target.

The MicroRNA-106a/20b Strongly Enhances the Antitumour Immune Responses of Dendritic Cells Pulsed with Glioma Stem Cells by Targeting STAT3.

Background: Evaluate the effect of the miRNA-106a/20b on the efficacy of DCs pulsed with GSCs in activating GSC-specific T cell responses. Methods: We cultured GSCs and prepared GSC antigen lysates by apoptosis. Then, immature DCs were pulsed with GSC antigen lysates in vitro. STAT3 levels in DCs were assessed by Western blotting, and the expression of CD80, CD86, and MHC-II was tested by fluorescence-activated cell sorting. The production and secretion of the cytokines IL-6, IL-12, TNF-α, and IL-10 in DCs induced by GSCs were determined by enzyme-linked immunosorbent assay. Finally, the cytotoxic functions of T cells stimulated by GSC-DC fusion cells transfected with a miR-106a/20b mimic in vitro and the antitumour activity in vivo were detected. Results: We found that the levels of miR-106a/20b were downregulated, but the expression of STAT3 was significantly upregulated. Simultaneously, the inhibition of STAT3 in the fusion cells by STAT3-specific siRNA caused significant upregulation of the expression of CD80, CD86, and MHC-II, and the secretion of the cytokines IL-6 and IL-12 was substantially increased, IL-10 was markedly decreased. These findings revealed that STAT3 is an important regulator of DC maturation. Furthermore, the interactional binding sites between the 3'-untranslated region (3'-UTR) of STAT3 mRNA and miR-106a/20b were predicted by bioinformatics and verified by a dual-luciferase assay. Moreover, the reduction in STAT3 levels in GSC-DCs enhanced the generation of CD8+ T cells and reduced the generation of Foxp3+ regulatory T cells. Meanwhile, the secretion of the T cell cytokine IFN-γ was significantly increased. Further research showed that DCs after miR-106a/20b-mimics transfection could promote the inhibition of GSC proliferation by T cells in vitro and suppress tumour growth in vivo. Conclusions: This study indicted that the miR-106a/20b activation could be one of the important molecular mechanisms leading to enhance antitumour immune responses of GSC-mediated DCs, which downregulated the expression of STAT3 to alleviate its the inhibitory effect.

Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

Anti-CTLA-4 antibodies have pioneered the field of tumour immunotherapy. However, despite impressive clinical response data, the mechanism by which anti-CTLA-4 antibodies work is still controversial. Two major checkpoint antibodies (ipilimumab and tremelimumab) have been trialled clinically. Both have high affinity binding to CTLA-4 and occupy the ligand binding site, however recently it has been suggested that in some settings such antibodies may not block ligand-CTLA-4 interactions. Here we evaluated blocking capabilities of these antibodies in a variety of settings using both soluble and cell bound target proteins. We found that when ligands (CD80 or CD86) were expressed on cells, soluble CTLA-4-Ig bound in line with affinity expectations and that this interaction was effectively disrupted by both ipilimumab and tremelimumab antibodies. Similarly, cellular CTLA-4 binding to soluble ligands was comparably prevented. We further tested the ability of these antibodies to block transendocytosis, whereby CTLA-4 captures ligands from target cells during a cognate cell-cell interaction. Once again ipilimumab and tremelimumab were similar in preventing removal of ligand by transendocytosis. Furthermore, even once transendocytosis was ongoing and cell contact was fully established, the addition of these antibodies could prevent further ligand transfer. Together these data indicate that the above checkpoint inhibitors performed in-line with predictions based on affinity and binding site data and are capable of blocking CTLA-4-ligand interactions in a wide range of settings tested.

Inorganic nanosheets facilitate humoral immunity against medical implant infections by modulating immune co-stimulatory pathways.

Strategies to manipulate immune cell co-inhibitory or co-activating signals have revolutionized immunotherapy. However, certain immunologically cold diseases, such as bacterial biofilm infections of medical implants are hard to target due to the complexity of the immune co-stimulatory pathways involved. Here we show that two-dimensional manganese chalcogenophosphates MnPSe3 (MPS) nanosheets modified with polyvinylpyrrolidone (PVP) are capable of triggering a strong anti-bacterial biofilm humoral immunity in a mouse model of surgical implant infection via modulating antigen presentation and costimulatory molecule expression in the infectious microenvironment (IME). Mechanistically, the PVP-modified MPS (MPS-PVP) damages the structure of the biofilm which results in antigen exposure by generating reactive oxidative species, while changing the balance of immune-inhibitory (IL4I1 and CD206) and co-activator signals (CD40, CD80 and CD69). This leads to amplified APC priming and antigen presentation, resulting in biofilm-specific humoral immune and memory responses. In our work, we demonstrate that pre-surgical neoadjuvant immunotherapy utilizing MPS-PVP successfully mitigates residual and recurrent infections following removal of the infected implants. This study thus offers an alternative to replace antibiotics against hard-to-treat biofilm infections.

Isolation of Decidual Macrophages and Hofbauer Cells from Term Placenta-Comparison of the Expression of CD163 and CD80.

(1) Background: Placental immune cells are playing a very important role in a successful placentation and the prevention of pregnancy complications. Macrophages dominate in number and relevance in the maternal and the fetal part of the placenta. The evidence on the polarization state of fetal and maternal macrophages involved in both, healthy and pregnancy-associated diseases, is limited. There is no representative isolation method for the direct comparison of maternal and fetal macrophages so far. (2) Material and Methods: For the isolation of decidual macrophages and Hofbauer cells from term placenta, fresh tissue was mechanically dissected and digested with trypsin and collagenase A. Afterwards cell enrichment was increased by a Percoll gradient. CD68 is represented as pan-macrophage marker, the surface markers CD80 and CD163 were further investigated. (3) Results: The established method revealed a high cell yield and purity of the isolated macrophages and enabled the comparison between decidual macrophages and Hofbauer cells. No significant difference was observed in the percentage of single CD163+ cells in the distinct macrophage populations, by using FACS and immunofluorescence staining. A slight increase of CD80+ cells could be found in the decidual macrophages. Considering the percentage of CD80+CD163- and CD80-CD163+ cells we could not find differences. Interestingly we found an increased number of double positive cells (CD80+CD163+) in the decidual macrophage population in comparison to Hofbauer cells. (4) Conclusion: In this study we demonstrate that our established isolation method enables the investigation of decidual macrophages and Hofbauer cells in the placenta. It represents a promising method for direct cell comparison, enzyme independently, and unaffected by magnetic beads, to understand the functional subsets of placental macrophages and to identify therapeutic targets of pregnancy associated diseases.

Generation of molecular-targeting helix-loop-helix peptides for inhibition of the interaction between cytotoxic T-lymphocyte-associated protein 4 and B7 in the dog.

Blocking the interaction between CD28 and B7 by cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a potent immune checkpoint that prevents damage to host tissues from excessive immune responses. However, it also significantly diminishes immune responses against cancers and allows cancer cell growth. This study found that recombinant (r) human (h) CTLA-4 specifically binds to canine dendritic cells (DCs) and suppresses the responses of canine T cells to allogeneic DCs. ERY2-4, a peptide targeting rhCTLA-4 selected from a yeast-displayed library of helix-loop-helix (HLH) peptides and improved to have a binding affinity to rhCTLA-4 as strong as that of rhB7, inhibited the binding of rhCTLA-4 to canine DCs. Furthermore, the targeting peptide significantly enhanced the response of canine T cells to allogeneic DCs. These results suggest that the CTLA-4-targeting peptide enhances canine T cell activity by blocking the interaction between canine CTLA-4 on T cells and canine B7 on DCs. This study demonstrates the generation of a new type of immune checkpoint inhibitor, which may be applicable to cancer therapy in dogs.