MHC-II Expression Research Articles

Neuroinflammation and gliosis in the injured and contralateral retinas after unilateral optic nerve crush

The main purpose of this study is to analyze the effects of unilateral optic nerve crush in the gene expression of pro- and anti-inflammatory mediators, and gliosis markers in injured and contralateral retinas. Retinas from intact, unilaterally optic nerve injured or sham-operated C57BL/6J mice were analyzed 1, 3, 9 and 30 days after the surgery (n = 5/group and time point) and the relative expression of TGF-β1, IL-1β, TNF-α, Iba1, AQP4, GFAP, MHCII, and TSPO was analyzed in injured and contralateral using qPCR. The results indicated that compared with intact retinas, sham-operated animals showed an early (day 1) upregulation of IL-1β, TNF-α and TSPO and a late (day 30) upregulation of TNF-α. In sham-contralateral retinas, TNF-α and TSPO mRNA expression were upregulated and day 30 while GFAP, Iba1, AQP4 and MHCII downregulated at day 9. Compared with sham-operated animals, in retinas affected by optic nerve crush GFAP and TSPO upregulated at day 1 and TNF-α, Iba1, AQP4 and MHCII at day 3. In the crushed-contralateral retinas, TGF-β1, TNF-α, Iba1 and MHCII were upregulated at day 1. TSPO was upregulated up to day 30 whereas TGF-β1 and Iba1 downregulated after day 9. In conclusion, both sham surgery and optic nerve crush changed the profile of inflammatory and gliosis markers in the injured and contralateral retinas, changes that were more pronounced for optic nerve crush when compared to sham.

Protein-rich fraction of Protaetia brevitarsis seulensis larvae induce maturation of dendritic cells and Th1-polarization: A promising adjuvant for anticancer therapy

The objective of this study was to investigate the potential anticancer immunostimulatory properties of a protein-rich fraction obtained from larvae of Protaetia brevitarsis seulensis (PBLP), a highly nutritious edible insect, in CT-26 colon carcinoma-bearing mice. The most abundant essential amino acids (EAA) in PBLP were Val, Ile, and Leu, while the most abundant non-EAA were Pro, Glu, and Gly. PBLP increased the production of Th1-polarized cytokines and the phenotypic maturation of bone marrow-derived dendritic cells (BMDCs). PBLP-treated BMDCs stimulated naïve CD4+ and CD8+ T cells to produce IFN-γ and IL-2 but not IL-5. Significantly, dietary PBLP restored CD86 and MHC-I expression in splenic DCs, and also increased TNF-α+IL-2+IFN-γ+CD107+ polyfunctional CD4+ and CD8+ T cells in the spleen, thereby inhibiting tumor growth in CT-26 tumor-bearing mice. Collectively, PBLP synchronizes DCs and T cells for anticancer immunity, suggesting that it could serve as a potential adjuvant or supplement for cancer treatment.

Vaccination with bacterial ghosts of Streptococcus iniae and Lactococcus garvieae originated from outbreak of marine fish streptococcosis, induce potential protection against the disease in Nile tilapia, Oreochromis niloticus (Linnaeus, 1758)

Streptococcosis is an important bacterial disease affects fresh, brackish and marine fish. The disease caused annual severe economic losses in Egyptian Mari-culture. S. iniae and L. garvieae usually the main causative agents isolated. The presented study conducted to prepare bacterial ghost vaccine (BGV) candidates from isolated strains of marine streptococcosis outbreaks using NaOH chemical approach. Selected strains confirmed as pathogenic for Nile tilapia, therefore the fish selected as an experimental model. In such respect, the re-isolated S. iniae and L. garvieae were used for ghost preparations, BGVs evaluation and fish challenges. Apart of four, three fish groups namely, A, B, C designated for BGVs evaluations, while the fourth one (D) designated as control. Vaccination experiments performed via intra-peritoneal injection with 0.1 mL (1.5 × 108 CFU/mL/fish) of their corresponding BGVs twice with 2 weeks’ interval; however, control fish received 0.1 mL of fish saline instead. Blood, serum, and tissue samples collected from all groups at 2 and 4 weeks post immunization (PI) for estimation of hematological, innate, and specific immune parameters. At the end, all remained fish challenged with appropriated pathogen (s) and the relative percentage of survival (RPS) calculated. Three BGVs generated namely, SiG, in addition to, novel contributions of LgG and SiLgG. Ghosts were corresponding to S. iniae, L. garvieae and their both ghost mixtures, respectively. Fish groups immunized with prepared BGVs revealed variable significant increases in PCV, GLB, PP, SOD, CAT, C5, IL-β1, LZM, specific antibody titers and CD4 expression 2 and 4 weeks PI. MDA decreased in all vaccinated groups that was significantly with group C. Expression of MHC-II showed elevations 2 weeks PI, however, it significantly decreased at 4 weeks. The RPS recorded 90, 88.89 and 95.46% in immunized groups A, B and C, respectively. At all levels tested, obtained results proposed SiG, LgG and SiLgG as innovative vaccine candidates, which can protect cultured fish from being attacked by S. iniae, and/or L. garvieae.

Unleashing T cell anti-tumor immunity: new potential for 5-Nonloxytryptamine as an agent mediating MHC-I upregulation in tumors

BackgroundNew therapies are urgently needed in melanoma, particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors. To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen and identified 5-Nonyloxytryptamine (5-NL), a serotonin agonist, as increasing the ability of T cells to target tumor cells.MethodsThe pharmacological screen utilized lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells and melanoma B16.F10 cells expressing the LCMV gp33 CTL epitope. In vivo tumor growth in C57BL/6 J and NSG mice, in vivo antibody depletion, flow cytometry, immunoblot, CRISPR/Cas9 knockout, histological and RNA-Seq analyses were used to decipher 5-NL’s immunomodulatory effects in vitro and in vivo.Results5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts’ immune system, specifically CD8+ T cells. 5-NL’s pro-immune effects were not directly consequential to T cells. Rather, 5-NL upregulated antigen presenting machinery in melanoma and other tumor cells in vitro and in vivo without increasing PD-L1 expression. Mechanistic studies indicated that 5-NL’s induced MHC-I expression was inhibited by pharmacologically preventing cAMP Response Element-Binding Protein (CREB) phosphorylation. Importantly, 5-NL combined with anti-PD1 therapy showed significant improvement when compared to single anti-PD-1 treatment.ConclusionsThis study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors.

CTLA-4 blockade induces a microglia-Th1 cell partnership that stimulates microglia phagocytosis and anti-tumor function in glioblastoma

The limited efficacy of immunotherapies against glioblastoma underscores the urgency of better understanding immunity in the central nervous system. We found that treatment with αCTLA-4, but not αPD-1, prolonged survival in a mouse model of mesenchymal-like glioblastoma. This effect was lost upon the depletion of CD4+ Tcells but not CD8+ Tcells. αCTLA-4 treatment increased frequencies of intratumoral IFNγ-producing CD4+ Tcells, and IFNγ blockade negated the therapeutic impact of αCTLA-4. The anti-tumor activity of CD4+ Tcells did not require tumor-intrinsic MHC-II expression but rather required conventional dendritic cells as well as MHC-II expression on microglia. CD4+ Tcells interacted directly with microglia, promoting IFNγ-dependent microglia activation and phagocytosis via the AXL/MER tyrosine kinase receptors, which were necessary fortumor suppression. Thus, αCTLA-4 blockade in mesenchymal-like glioblastoma promotes a CD4+ Tcell-microgliacircuit wherein IFNγ triggers microglia activation and phagocytosis and microglia in turn act as antigen-presenting cells fueling the CD4+ Tcell response.

Lentinula Edodes Mycelia extract regulates the function of antigen-presenting cells to activate immune cells and prevent tumor-induced deterioration of immune function

Immune cell activation is essential for cancer rejection; however, the tumor microenvironment leads to deterioration of immune function, which enables cancer cells to survive and proliferate. We previously reported that oral ingestion of Lentinula Edodes Mycelia (L.E.M.) extract enhances the tumor antigen-specific T-cell response and exerts an antitumor effect in a tumor-bearing mouse model. In this study, we focused on antigen-presenting cells (APCs) located upstream of the immune system, induced a T-cell response, then examined the impact of L.E.M. extract on the APCs. L.E.M. extract enhanced the expression of MHC-I, MHC-II, CD86, CD80, and CD40 in bone marrow-derived dendritic cells (DCs) and strongly induced the production of IL-12. L.E.M.-stimulated DCs enhanced IFN-γ production from CD8+ T cells and induced their differentiation into effector cells. Furthermore, L.E.M. extract promoted IL-12 production and suppressed the production of IL-10 and TGF-β by transforming bone marrow-derived macrophages into M1-like macrophages. Furthermore, in a B16F10 melanoma inoculation model, DCs in the spleen were decreased and their activation was suppressed by the presence of cancer; however, ingestion of L.E.M. extract prevented this functional deterioration of DCs. In the spleen of cancer-bearing mice, the number of CD11b− F4/80+ macrophages in a hypoactivated state was also increased, whereas L.E.M. extract suppressed the increase of such macrophages. These findings suggest that L.E.M. extract may exhibit an antitumor immune response by regulating the function of APCs to induce cytotoxic T lymphocytes, as well as by suppressing the decline in antigen-presenting cell activity caused by the presence of cancer.

Adverse Responses following Exposure to Subtoxic Concentrations of Zinc Oxide and Nickle Oxide Nanoparticles in the Raw 264.7 Cells.

The incorporation of engineered nanomaterials (ENMs) in biomedical and consumer products has been growing, leading to increased human exposure. Previous research was largely focused on studying direct ENM toxicity in unrealistic high-exposure settings. This could result in overlooking potential adverse responses at low and subtoxic exposure levels. This study investigated adverse cellular outcomes to subtoxic concentrations of zinc oxide (ZnONPs) or nickel oxide (NiONPs) nanoparticles in the Raw 264.7 cells, a macrophage-like cell model. Exposure to both nanoparticles resulted in a concentration-dependent reduction of cell viability. A subtoxic concentration of 6.25 µg/mL (i.e., no observed adverse effect level) was used in subsequent experiments. Exposure to both nanoparticles at subtoxic levels induced reactive oxygen species generation. Cellular internalization data demonstrated significant uptake of NiONPs, while there was minimal uptake of ZnONPs, suggesting a membrane-driven interaction. Although subtoxic exposure to both nanoparticles was not associated with cell activation (based on the expression of MHC-II and CD86 surface markers), it resulted in the modulation of the lipopolysaccharide-induced inflammatory response (TNFα and IL6), and cells exposed to ZnONPs had reduced cell phagocytic capacity. Furthermore, subtoxic exposure to the nanoparticles distinctly altered the levels of several cellular metabolites involved in cell bioenergetics. These findings suggest that exposure to ENMs at subtoxic levels may not be devoid of adverse health outcomes. This emphasizes the importance of establishing sensitive endpoints of exposure and toxicity beyond conventional toxicological testing.

Intratumoral NKp46+ natural killer cells are spatially distanced from T and MHC-I+ cells with prognostic implications in soft tissue sarcoma.

Soft tissue sarcomas (STS) are rare, heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cells and spatial relationships of TILs with MHC-I expressing cells lacks detailed characterization. Using archived and prospectively collected specimens, we evaluated intratumoral NK cells by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). We assessed spatial localization of NK and T cells by multiplex IF, analyzing the effects of MHC-I expression status on NK and T cell clustering. Both intratumoral NKp46 and CD56dim expression were associated with significantly improved overall survival (P=0.05), while higher infiltrates of CD56bright NK cells predicted a worse prognosis (P=0.05). The presence of intratumoral NK cells was inversely proportional to CD3+ T cells. Spatial analyses showed NK cells preferentially clustering close to other NK cells with sparse CD3+ T and CD8+ T cells in range (P<0.0001). Additionally, CD3+ T and CD8+ T cells showed significantly greater co-localization with MHC-I+ cells, compared to NK cells (P<0.0001). After neoadjuvant radiotherapy, there was greater CD8 clustering, while after neoadjuvant chemotherapy, there was overall lower TIL clustering. Intratumoral NK cells are prognostic in STS and localize closer to MHC-I- cells than T cells. Although both NK and T cells are associated with improved survival in STS, their differential distribution in the TME based on MHC-I expression status may serve as a biomarker for improved immunotherapy treatment selection.

Insight into Cancer Immunity: MHCs, Immune Cells and Commensal Microbiota.

Cancer cells circumvent immune surveillance via diverse strategies. In accordance, a large number of complex studies of the immune system focusing on tumor cell recognition have revealed new insights and strategies developed, largely through major histocompatibility complexes (MHCs). As one of them, tumor-specific MHC-II expression (tsMHC-II) can facilitate immune surveillance to detect tumor antigens, and thereby has been used in immunotherapy, including superior cancer prognosis, clinical sensitivity to immune checkpoint inhibition (ICI) therapy and tumor-bearing rejection in mice. NK cells play a unique role in enhancing innate immune responses, accounting for part of the response including immunosurveillance and immunoregulation. NK cells are also capable of initiating the response of the adaptive immune system to cancer immunotherapy independent of cytotoxic T cells, clearly demonstrating a link between NK cell function and the efficacy of cancer immunotherapies. Eosinophils were shown to feature pleiotropic activities against a variety of solid tumor types, including direct interactions with tumor cells, and accessorily affect immunotherapeutic response through intricating cross-talk with lymphocytes. Additionally, microbial sequencing and reconstitution revealed that commensal microbiota might be involved in the modulation of cancer progression, including positive and negative regulatory bacteria. They may play functional roles in not only mucosal modulation, but also systemic immune responses. Here, we present a panorama of the cancer immune network mediated by MHCI/II molecules, immune cells and commensal microbiota and a discussion of prospective relevant intervening mechanisms involved in cancer immunotherapies.

Chimeric antigen receptor macrophages activated through TLR4 or IFN-γ receptors suppress breast cancer growth by targeting VEGFR2.

Chimeric antigen receptor macrophage (CAR-M) is a promising immunotherapy strategy of anti-tumor due to its high infiltration, direct phagocytosis of tumor cells, immunomodulation of tumor microenvironment (TME) and linkage of innate and adaptive immunity. Here a series of novelly designed CAR-Ms by targeting vascular endothelial growth factor receptor-2 (VEGFR2), which highly expressed in tumor cells and TME, were evaluated. Their activation signals were transduced by Tlr4 or Ifn-γ receptors either alone or in combination, which were designed to mediate M1 polarization of macrophages as the downstream of lipopolysaccharide or Ifn-γ that had been widely reported. Our results showed that VEGFR2-targeting CAR-Ms could be activated under the stimulation of VEGFR2-expressing cells. They exhibited higher expression of CD86, MHCII and TNF-α in vitro and enhanced tumor suppressive abilities in vivo. Implantation of these CAR-Ms into 4T1 breast cancer-bearing mice could obviously inhibit the progression of tumor without significant toxic side effects, especially the group of mmC in which constructed with Tlr4 as the intracellular domain of CAR. In conclusion, this research provides a promising design of CAR that induce macrophages activation by Tlr4 and/or Ifn-γ receptors, and these CAR-Ms could effectively inhibit tumor growth through targeting VEGFR2.

Inflammasome in Skeletal Muscle: NLRP3 Is an Inflammatory Cell Stress Component in Inclusion Body Myositis

Inclusion body myositis (IBM) is a chronic, mostly treatment-resistant, inflammatory myopathy with a pathology that centers around specific interactions between inflammation and protein accumulation. The study aimed to identify the inflammasome as a key event in the complex network of pathomechanisms. Regulation of the inflammasome was assessed in a well-established pro-inflammatory cell culture model using human myoblasts and primary human myotubes. By quantitative PCR, western blot and immunocytochemistry, inflammasome markers including NLRP3 were assessed in muscle cells exposed to the cytokines IL-1β and IFN-γ. The data were corroborated by analysis of muscle biopsies from patients with IBM compared to other myositis subtypes. In the cell culture model of IBM, the NLRP3 inflammasome was significantly overexpressed, as evidenced by western blot (p = 0.03) and quantitative PCR (p &lt; 0.01). Target genes that play a role in inflammasome assembly, T-cell migration, and MHC-I expression (p = 0.009) were highly co-upregulated. NLRP3 was significantly overexpressed in muscle biopsies from IBM samples compared to disease controls (p = 0.049), including other inflammatory myopathies. Due to the extraordinary features of the pathogenesis and the pronounced upregulation of NLRP3 in IBM, the inflammasome could serve as a key molecule that drives the inflammatory cascade as well as protein accumulation in the muscle. These data can be useful for future therapeutic developments.

Pharmacologic inhibition of IL11/STAT3 signaling increases MHC-I expression and T cell infiltration

BackgroundRecent studies have discovered an emerging role of IL11 in various colitis-associated cancers, suggesting that IL11 mainly promotes tumor cell survival and proliferation in regulating tumorigenesis. Herein we aimed to reveal a novel function of IL-11 through STAT3 signaling in regulating tumor immune evasion.MethodsAOM/DSS model in Il11−/− and Apcmin/+/Il11−/− mice were used to detect tumor growth and CD8+ T infiltration. STAT1/3 phosphorylation and MHC-I, CXCL9, H2-K1 and H2-D1 expression were detected in MC38 cells and intestine organoids treated with/without recombinant IL11 to explore effect of IL11/STAT3 signaling, with IL11 mutein used to competitively inhibit IL11 and rescue inhibited STAT1 activation. Correlation between IL11 and CD8+ T infiltration was analyzed using TIMER2.0 website. IL11 expression and survival prognosis was analyzed in clinical data of patient cohort from Nanfang Hospital.ResultsIL11 is highly expressed in CRC and indicates unfavorable prognosis. IL11 knockout increased CD8+ T cell infiltration and reduced intestinal and colon formation. Tumors were significantly suppressed while MHC-I and CXCL9 expression for CD8+ T infiltration were remarkably increased in the tumor tissues of Apcmin/+/Il11−/− mice or Il11−/− mice induced by AOM/DSS. IL11/STAT3 signaling downregulated MHC-I and CXCL9 by inhibiting IFNγ-induced STAT1 phosphorylation. IL11 mutein competitively inhibit IL11 to upregulate CXCL9 and MHC-I in tumor and attenuated tumor growth.ConclusionsThis study ascribes for a new immunomodulatory role for IL11 during tumor development that is amenable to anti-cytokine based therapy of colon cancer.

In Vitro Analysis of the Effect of SARS-CoV-2 Non-VOC and four Variants of Concern on MHC-Class-I Expression on Calu-3 and Caco-2 Cells.

As the MHC-I-pathway is key to antigen presentation to cytotoxic T-cells and, therefore, recognition by the host adaptive immune system, we hypothesized that SARS-CoV-2 including its Variants of Concern (VOCs), influences MHC-I expression on epithelial cell surfaces as an immune evasion strategy. We conducted an in vitro time course experiment with the human airway epithelial cell line Calu-3 and the human colorectal adenocarcinoma cell line Caco-2. Cells were infected with SARS-CoV-2 strains non-VOC/B.1.1, Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, and Delta/B.1.617.2. At 2, 24, 48 and 72 h post-infection we performed RT-qPCR to track viral replication. Simultaneously, we performed intracellular staining with a serum of a double-vaccinated healthy adult containing a high amount of spike protein antibody. In flow cytometry experiments, we differentiated between infected (spike protein positive) and bystander (spike protein negative) cells. To compare their HLA expression levels, cells were stained extracellularly with anti-HLA-A-IgG and anti-HLA-B,C-IgG. While HLA-A expression was stable on infected Calu-3 cells for all variants, it increased to different degrees on bystander cells in samples infected with VOCs Beta, Gamma, Delta, or non-VOC over the time course analyzed. In contrast, HLA-A levels were stable in bystander Calu-3 cells in samples infected with the Alpha variant. The upregulation of MHC-I on spike protein negative bystander cells in Calu-3 cell cultures infected with Beta, Gamma, Delta, and partly non-VOC might suggest that infected cells are still capable of secreting inflammatory cytokines like type-I interferons stimulating the MHC-I expression on bystander cells. In comparison, there was no distinct effect on HLA expression level on Caco-2 cells of any of the VOCs or non-VOC. Further investigations of the full range of immune evasion strategies of SARS-CoV-2 variants are warranted.

136-OR: The MDA5627X Variant Is Associated with Type 1 Diabetes Protection via Attenuated Inflammatory Antiviral Responses

Type 1 Diabetes (T1D) is an autoimmune disease characterized by destruction of pancreatic β cells. Genetics and environmental factors play crucial roles in T1D development. Four single nucleotide polymorphisms in IFIH1, encoding the viral sensor MDA5 have been linked to T1D. MDA5 promotes anti-viral responses and may link genetics with environmental factors to promote or suppress T1D. A stop-gain mutation in IFIH1 (rs35744605) results in a non-functional MDA5 protein (MDA5627X). We hypothesized that MDA5627X protects against T1D by dampening inflammatory anti-viral responses resulting in reduced autoimmunity. We gene edited human induced pluripotent stem cells at rs35744605 using a CRISPR/Cas9 strategy to generate iPSC encoding the stop-gain variant (MDA5627X). Macrophages (Mφ) have been demonstrated to be indispensable for T1D-initiation and regulate anti-viral responses. We differentiated iPSCs into Mφ and studies responses to high molecular weight Poly I:C to mimic viral infection. The outcome measures were Type 1 Interferon (IFN1) production, antigen presentation, and immune activation markers. Treatment of MDA5 intact Mφ with Poly I:C resulted in 60-fold increase in IFN1 production and a 3-fold elevation in human leukocyte antigen Class I (HLA-I) expression. In contrast, treated MDA5627X Mφ failed to increase IFN1 or HLA-I at any Poly I:C dose. While Poly I:C did not increase HLA-I expression, untreated MDA5627X Mφ had elevated HLA-1 compared to MDA5 intact Mφ (P=0.0306). Also, we assessed the expression of CD80, CD86, CD40, and MHC-II, however there were no differences in the expression between Mφ with intact MDA5 or MDA5627X. In contrast, IFNg and LPS treatment elevated expression of CD80, CD86, CD40, and MHC-II in both Mφ populations. In conclusion, our data point to a protective effect of the MDA5627X variant in T1D through decreased inflammatory IFN1 levels and T cell activation by Mφ. Disclosure O.Iwaloye: None. L.H.Armitage: None. A.M.Meacham: None. C.E.Mathews: None. Funding National Institutes of Health (R01DK127497, P01AI042288, UH3DK122638)

IMMU-11. PRECLINICAL HIGH-RISK PEDIATRIC BRAIN TUMOR MODELS FOR IMMUNOTHERAPY: HURDLES AND THE WAY FORWARD

Abstract Despite recent therapeutic advancements in the treatment of pediatric brain tumors, high-risk brain tumors (pHRBT) remain the leading cause of cancer-related deaths in children. In recent years, immunotherapy has become a viable treatment option for several cancers including brain tumors. However, several factors have limited the use of immunotherapy in the treatment of pHRBT. For example, an "immunologically cold" tumor environment has been predominantly implicated in the failure of checkpoint inhibitors (ICIs) as monotherapy in pHRBT. Nevertheless, priming the effects of ICIs with epigenetic modulators through a process called “viral mimicry” that induces the expression of human endogenous retroviruses has the potential to enhance immunotherapy by sparking a T-cell mediated immune response. However, the inadequate understanding of the limitations of the preclinical models has prevented the development of efficient immunotherapy strategies for pHRBT. Although several studies and reviews have compiled some limitations of the available models, a hands-on experience using preclinical models for ICIs testing has not been fully communicated. Here, we share our bedside to bench experience with syngeneic and humanized mouse models of DIPG, ATRT, and medulloblastoma using ICIs therapy in conjunction with an epigenetic alteration. We will present and discuss the limitations of mouse models for the development of immunotherapies for pHRBT. For instance, our results indicated that the baseline levels of MHC-I expression in patients’ DIPG tumors were comparable to matched normal tissue. However, these levels were significantly lower in mouse DIPG tumors. To compensate for this difference, we treated mice with IFN-γ in combination with an epigenetic modulator. Surprisingly, the addition of INF-γ had a negative effect on animal overall survival compared to the treatment with the epigenetic modulator alone. Together, our experience demonstrated the importance of suitability assessment of current models for developing and translating successful immunotherapy strategies to treat pHRBT.

Inhibition of FGFR3 upregulates MHC-I and PD-L1 via TLR3/NF-kB pathway in muscle-invasive bladder cancer.

Improving the potency of immune response is paramount among issues concerning immunotherapy of muscle-invasive bladder cancer (MIBC). On the basis of immune subtypes, we investigated possible molecular mechanisms involved in tumor immune escape in MIBC. According to the 312 immune-related genes, three MIBC immune subtypes were clustered. Cluster 2 subtype is characterized by FGFR3 mutations and has a better clinical prognosis. However, the expression levels of MHC-I and immune checkpoints genes were the lowest, indicating that this subtype is subject to immune escape and has a low response rate to immunotherapy. Bioinformatics analysis and immunofluorescence staining of clinical samples revealed that the FGFR3 is involved in the immune escape in MIBC. Besides, after FGFR3 knockout with siRNA in RT112 and UMUC14 cells, the TLR3/NF-kB pathway was significantly activated and was accompanied by upregulation of MHC-I and PD-L1 gene expression. Furthermore, the use of TLR3 agonists poly(I:C) can further improve the effect. Together, our results suggest that FGFR3 might involve in immunosuppression by inhibition of NF-kB pathway in BC. Considering that TLR3 agonists are currently approved for clinical treatment as immunoadjuvants, our study might provide more insights for improving the efficacy of immunotherapy in MIBC.

Abstract B035: Leveraging therapy-induced senescence for prostate cancer immunotherapy

Abstract Most prostate cancers are immunologically “cold” with poor tumor immunogenicity and a lack of cytotoxic T cells, and as such are generally unresponsive to immune checkpoint blockade (ICB) therapies that can reactivate T cell immunity and lead to durable and curative responses in other treatment-refractory solid tumors. We recently demonstrated that therapeutic induction of a durable growth arrest phenotype known as cellular senescence in the setting of pancreatic cancer can be novel approach to make “cold” tumors “hot” and sensitize them to anti-PD-1 ICB (Ruscetti et al. Cell 2020). This senescence-driven immunological effect is mediated through induction of immunomodulatory cell surface molecules, including MHC-I expression required for antigen presentation to T cells, and activation of the senescence-associated secretory phenotype (SASP), a collection of pleiotropic cytokines, chemokines, and other growth factors that can remodel the surrounding tumor-immune landscape. While this acute SASP activation can promote cytotoxic CD8+ T and Natural Killer (NK) cell anti-tumor immunity in some contexts, chronic SASP can alternatively lead to immune suppression and tumor cell invasion and metastasis that contributes to relapse after therapy. Here we set out to characterize the senescence-inducing capacity of chemotherapies, DNA repair inhibitors, and cell cycle targeting agents implicated in cellular senescence and used in the treatment of castration-resistant prostate cancer (CRPC), and the impact of their subsequent SASPs on immune responses and therapy outcomes. Using a suite of genetically-defined murine prostate cancer cell lines and an in vitro pipeline to rapidly define senescence phenotypes following treatment, we found that many commonly used cancer therapeutics can induce cellular senescence, with chemotherapies, Aurora kinase inhibitors, and CDK inhibitors producing the most robust senescence-induced cell cycle arrest across conditions. Prostate tumor cells harboring compound tumor suppressor losses such as Pten, p53, and Rb1 were more susceptible to therapeutic senescence induction. Strikingly, only a subset of therapies that induce senescence-associated growth arrest produced an inflammatory SASP and induction of activating NK cell ligands and antigen presentation and processing genes necessary for NK and T cell immunity. Moreover, whereas most SASPs induced NK cell activity in vitro, they also polarized macrophages toward a pro-tumorigenic and immune suppressive M2 phenotype that may limit their anti-tumor efficacy. CDK inhibitors produced a SASP that could activate lymphocytes without inducing suppressive myeloid populations in MYC-driven tumors, suggesting their use may be a promising approach to target prostate cancers harboring the currently “undruggable” MYC oncogene. Collectively, our studies demonstrate that induction of senescence and its non-cell autonomous arm, the SASP, could be a unique approach to treat CRPC and create new immunological dependencies that can be potentially harnessed for prostate cancer immunotherapy. Citation Format: Lin Zhou, Katherine C. Murphy, Jarin Snyder, Kelly D. DeMarco, Boyang Ma, Marcus Ruscetti. Leveraging therapy-induced senescence for prostate cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B035.

Regulation of MHC class II and CD86 expression by March-I in immunity and disease.

The expression of MHC-II and CD86 on the surface of antigen-presenting cells (APCs) must be tightly regulated to foster antigen-specific CD4 T-cell activation and to prevent autoimmunity. Surface expression of these proteins is regulated by their dynamic ubiquitination by the E3 ubiquitin ligase March-I. March-I promotes turnover of peptide-MHC-II complexes on resting APCs and termination of March-I expression promotes MHC-II and CD86 surface stability. In this review, we will highlight recent studies examining March-I function in both normal and pathological conditions.

ORACLE-RIPA study: A phase II, randomized, open-label, parallel-group study comparing the immune modulation effect of neoadjuvant ribociclib, palbociclib, and abemaciclib in early ER+/HER2- breast cancer.

TPS632 Background: CDK4/6 inhibitors (CDK4/6i), ribociclib, palbociclib, and abemaciclib, provide similar progression-free survival (PFS) benefits when combined with endocrine therapy in ER+/HER2- metastatic breast cancer (MBC) patients. However, not all CDK4/6i have demonstrated overall-survival (OS) benefits in their respective phase III trials. Both, ribociclib and abemaciclib, have shown significant OS benefits in ER+/HER2- MBC patients, but palbociclib has not. Because of similar benefits of the 3 CDK4/6i on PFS, the OS benefit is likely not a result of differences in their synergistic efficacy of a CDK4/6i with endocrine therapy. CDK4 inhibition on cancer cells can also result in upregulation of MHC-I and MHC-II expression on cell surface and Rb-dependent interferon-gamma secretion. CDK6 inhibition might inhibit T cell and NK cell proliferation causing a negative effect on the tumor microenvironment. CDK6 inhibition can also reduce regulatory T cell numbers and increase cytokine production to enhance cytotoxic T cell function. Thus, we hypothesize that the differential impact of the 3 CDK4/6i on OS might be resulting from different immunomodulation effects. To investigate the functional alterations of the CDK4/6i on the BC microenvironment, the neoadjuvant treatment setting is best to compare the immune modulation effects of CDK4/6i from pre-treatment and post-treatment tumor tissues. Methods: This phase II multicenter, randomized, open-label, parallel-group study evaluates the immunomodulation effects of the 3 different CDK4/6i (ribociclib, palbociclib, or abemaciclib) combined with letrozole in neoadjuvant early ER+/HER2- breast cancer patients. A total of 20 patients will be enrolled into each treatment arm. The major inclusion criteria are female patients aged ≥ 20 years old, having a histologically and/or cytologically confirmed diagnosis of ER+ ( &gt; 10%) and/or PR+, HER2 negative (IHC 0+/1+, or IHC 2+ plus FISH negative) breast cancer, stage II/III, and adequate organ function. The treatment will12 weeks of letrozole plus a CDK4/6i. Premenopausal patients will receive GnRH agonist. Breast tumor biopsy, blood and stool samples will be collected prospectively at the time prior to treatment initiation, at 2 weeks after treatment, and at surgery. A description summary will be utilized to compare the results between the 3 CDK4/6i. The trial results will address questions about differences or similarities of the 3 CDK4/6i within the same study. The study is currently enrolling at 3 medical centers s in Taiwan. Clinical trial information: Pending formal number assignment .

Application of stem cells in guided bone regeneration

Modern medicine allows us to study and develop materials and methods of restorative treatment that would be based on the immunological mechanisms of bone repair. One of the promising directions in guided bone regeneration is the use of mesenchymal stem cells. Interest in MSCs is associated with their ability to regulate the inflammatory process, and directly participate in the formation of new bone structures, thereby providing a physiological repair process. The effector impact of MSCs on the inflammatory process due to their ability to form a specific microenvironment. Low expression of MHC-II and CD80/CD86, the production of PGE2 and NO determines their low immunoconflict, and the production of TGF-b1, IDO and IL-10 has an immunomodulating effect. The ability of MSCs to differentiate into an osteogenic phenotype is accompanied with the synthesis of ALP, BSP and, subsequently, Gla-protein and OPN determine the synthesis of the extracellular matrix and its subsequent mineralization. This process is provided by the action of Runx2, which activates the differentiation of MSCs along the osteogenic pathway. These effects of MSCs were taken as the basis for the development of a new method for the treatment of bone atrophy. To accomplish the task set, a model of bone tissue atrophy and a drug containing MSCs was developed, and an experimental study was conducted to evaluate the effectiveness of the developed methodology. As the main criteria, data from clinical and laboratory studies were taken. Visual changes in the studied area were taken into account, compared with a similar area in the developed model of atrophy, the parameters of the complete blood count (CBC) were evaluated. The performed study allows us to determine the developed treatment method as capable of fully recreating the conditions of bone repair processes, taking into account the optimization of the body’s immune reactions and repair processes, without additional external influence, to obtain predictable and controllable results.