Upregulation Of MHC-II Research Articles

388 MHC-I upregulation safeguards neoplastic T cells in the skin against NK cell-mediated eradication in mycosis fungoides

388 MHC-I upregulation safeguards neoplastic T cells in the skin against NK cell-mediated eradication in mycosis fungoides

Differential T cell accumulation within intracranial and subcutaneous melanomas is associated with differences in intratumoral myeloid cells

Patients with metastatic brain melanomas (MBM) experience shorter-lasting survival than patients with extracranial metastases, and this is associated with a higher fraction of dysfunctional CD8 T cells. The goal of this study was to understand the underlying cause of T cell dysfunction in MBM. To accomplish this, we compared murine B16 melanomas implanted intracranially (IC) or subcutaneously (SC). CD8 T cell activation was not altered, but representation in IC tumors was lower. Transferred activated or naïve CD8 T cells accumulated in similar numbers in both tumors, suggesting that the vasculature does not differentially impair T cell presence. Surprisingly, we found no evidence for T cell activation in draining lymph nodes of SC or IC tumor-bearing mice, consistent with the fact that dendritic cells (DC) that had acquired tumor antigen showed an immature phenotype. Instead, T cell activation occurred within both tumors, where the majority of tumor antigen+ myeloid cells were found. While, the numbers of intratumoral DC were comparable, those in IC tumors acquired less tumor antigen, and were alternatively matured based on upregulation of MHCII without upregulation of CD86. Additionally, in IC tumors, the largest population of tumor antigen+ myeloid cells were microglia. However, their presence did not influence either antigen acquisition or the phenotype of other myeloid cell populations. Overall, our data suggest that diminished representation of CD8 T cells in IC tumors is a consequence of alternatively matured DC and/or microglia that induce distinctly activated T cells, which ultimately fail to continue to accumulate inside the tumor.

H4K20me3-Mediated Repression of Inflammatory Genes is a Characteristic and Targetable Vulnerability of Persister Cancer Cells.

Anti-cancer therapies can induce cellular senescence, which is highly stable, or drug-tolerant persistence, which is efficiently reversed upon therapy termination. While approaches to target senescent cells have been extensively studied, further understanding of the processes regulating persistence is needed to develop treatment strategies to suppress persister cell survival. Here, we used mTOR/PI3K inhibition to develop and characterize a model of persistence-associated arrest in human cancer cells of various origins. Persister and senescent cancer cells shared an expanded lysosomal compartment and hypersensitivity to BCL-XL inhibition. However, persister cells lacked other features of senescence, such as loss of lamin B1, senescence-associated β-galactosidase activity, upregulation of MHC-I, and an inflammatory and secretory phenotype (SASP). Genome-wide CRISPR/Cas9 screening for genes required for the survival of persister cells revealed that they are hypersensitive to the inhibition of one-carbon (1C) metabolism, which was validated by the pharmacological inhibition of SHMT, a key enzyme that feeds methyl groups from serine into 1C metabolism. Connecting 1C metabolism with the epigenetic regulation of transcription, the repressive heterochromatic mark H4K20me3 was enriched at the promoters of SASP and interferon response genes in persister cells, while it was absent in proliferative or senescent cells. Moreover, persister cells overexpressed the H4K20 methyltransferases KMT5B/C, and their downregulation unleashed inflammatory programs and compromised the survival of persister cells. In summary, this study defined distinctive features of persister cancer cells, identified actionable vulnerabilities, and provided mechanistic insight into their low inflammatory activity.

IMMUNE AND MOLECULAR CORRELATES OF RESPONSE TO IMMUNOTHERAPY REVEALED BY BRAIN-METASTATIC MELANOMA MODELS.

Despite the promising results of immune checkpoint blockade (ICB) therapy, outcomes for patients with brain metastasis (BrM) remain poor. Identifying resistance mechanisms has been hindered by limited access to patient samples and relevant preclinical models. Here, we developed two mouse melanoma BrM models that recapitulate the disparate responses to ICB seen in patients. We demonstrate that these models capture the cellular and molecular complexity of human disease and reveal key factors shaping the tumor microenvironment and influencing ICB response. BR1-responsive tumor cells express inflammatory programs that polarize microglia into reactive states, eliciting robust T cell recruitment. In contrast, BR3-resistant melanoma cells are enriched in neurological programs and exploit tolerance mechanisms to maintain microglia homeostasis and limit T cell infiltration. In humans, BR1 and BR3 expression signatures correlate positively or negatively with T cell infiltration and BrM patient outcomes, respectively. Our study provides clinically relevant models and uncovers mechanistic insights into BrM ICB responses, offering potential biomarkers and therapeutic targets to improve therapy efficacy.

A-285 MHC-I Upregulation Safeguards Neoplastic T Cells in the Skin Against NK Cell-mediated Eradication in Mycosis Fungoides

A-285 MHC-I Upregulation Safeguards Neoplastic T Cells in the Skin Against NK Cell-mediated Eradication in Mycosis Fungoides

Acidity-Actuated Polymer/Calcium Phosphate Hybrid Nanomotor (PCaPmotor) for Penetrating Drug Delivery and Synergistic Anticancer Immunotherapy.

Tumor acidity-driven nanomotors may offer robust propulsion for tumor-specific penetrating drug delivery. Herein, an acidity-actuated poly(amino acid) calcium phosphate (CaP) hybrid nanomotor (PCaPmotor) was designed, using a mPEG-PAsp-PPhe@THZ531 micelle (Poly@THZ) for CaP mineralization accompanied by αPD-L1 antibody encapsulation. Dissolution of the CaP layer in an acidic tumor environment gave off heat energy to propel the nanomotor to augment the cellular uptake and penetration into deeply seated cancer cells while facilitating αPD-L1 release. THZ531 delivered by the PCaPmotor inhibited CDK12 and its down-streamed phosphorylation of RNAP-II to increase the cancer immunogenicity events such as the DNA damage, cell apoptosis, immunogenic cell death, lysosomal function disturbance, and MHC-I upregulation. THZ531 and αPD-L1 cosupplied by PCaPmotor significantly increased the frequency of DCs maturation and intratumoral infiltration of CTLs, but the two free drugs did not. Consequently, the PCaP@THZ/αPD-L1 nanomotor resulted in synergistic anticancer immunotherapy in mice. This acid-actuated PCaPmotor represented a new paradigm for penetrating drug delivery.

IFNγ mediates the resistance of tumor cells to distinct NK cell subsets

BackgroundImmune checkpoint blockade targeting the adaptive immune system has revolutionized the treatment of cancer. Despite impressive clinical benefits observed, patient subgroups remain non-responsive underscoring the necessity for combinational therapies harnessing...

Irradiated tumour cell-derived microparticles upregulate MHC-I expression in cancer cells via DNA double-strand break repair pathway

Radiotherapy (RT) is used for over 50 % of cancer patients and can promote adaptive immunity against tumour antigens. However, the underlying mechanisms remain unclear. Here, we discovered that RT induces the release of irradiated tumour cell-derived microparticles (RT-MPs), which significantly upregulate MHC-I expression on the membranes of non-irradiated cells, enhancing the recognition and killing of these cells by T cells. Mechanistically, RT-MPs induce DNA double-strand breaks (DSB) in tumour cells, activating the ATM/ATR/CHK1-mediated DNA repair signalling pathway, and upregulating MHC-I expression. Inhibition of ATM/ATR/CHK1 reversed RT-MP-induced upregulation of MHC-I. Furthermore, phosphorylation of STAT1/3 following the activation of ATM/ATR/CHK1 is indispensable for the DSB-dependent upregulation of MHC-I. Therefore, our findings reveal the role of RT-MP-induced DSBs and the subsequent DNA repair signalling pathway in MHC-I expression and provide mechanistic insights into the regulation of MHC-I expression after DSBs.

Abstract LB340: Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice

Abstract FDA approval for chimeric antigen receptor (CAR) T cell therapies targeting hematological cancers has unleashed the potential of utilizing live T cells as therapeutics. However, CAR-mediated targeting of solid tumors is currently limited by a paucity of suitable tumor antigens that are both specific and highly expressed on the tumor cell surface. In this report we explore the possibility of harnessing tumor-specific expression of endogenous retroviruses (ERVs) as a universal target for CAR T cell therapy. We found that gp70, the envelope protein from a murine ERV, is highly expressed on the surface of tumor cell lines derived from a diverse array of murine tumor types. We utilized the B16 melanoma model as a representative poorly inflamed tumor that does not respond to existing immunotherapies to examine efficacy of gp70 CAR T cells. T cells engineered to express a gp70 CAR suppress tumor growth in vivo and recognize B16 tumor cells with improved sensitivity compared to T cells expressing two previously described dominant tumor antigens (gp100 or P15e) due to low expression of MHC-I on B16 tumors. Co-incubation of B16 tumor cells with gp70 CAR T cells results in upregulation of MHC-I on the tumor cell surface suggesting that in addition to killing tumor cells directly, gp70 CAR T cells could facilitate recognition of the tumor by endogenous T cells through local release T cell derived IFNγ. We postulate that utilization of CAR T cells specific for cell surface ERV envelope proteins offers a promising strategy that could be harnessed to achieve local release of T cell derived payloads specifically within the tumor microenvironment. Citation Format: Sophie M. Lehar, Srirupa Roy, Lisa Liao, Haiyin Chen, Yongmei Chen, Mayra Cruz Tleugabulova, Eric Janezic, Azadeh Madjidi, Tamaki Jones, Vincent Javinal, James Ziai, Ira Mellman, Jill Schartner, Andrey Shaw. Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB340.

Abstract 6678: Microbial regulation of anti-tumor immunity in colorectal cancer

Abstract Colorectal cancer (CRC) is the 3rd leading site of cancers and cause of cancer cell death. Although Immunotherapies revolutionized cancer therapy, the majority of CRC patients, particularly with metastatic disease, do not respond to the treatments. This necessitates a better mechanistic understanding of the tumor-immune cell interactions in the context of CRC. Microbiome influences multiple biological aspects of the host including immune responses against cancers. Here, we show that the gut microbiome influences cancer MHC-I and -II expression levels and restricts tumor progression and metastasis, resulting in longer survival in 3 different CRC models and two different mouse strains. We found that the anti-tumor effects are dependent on T cells and B cells. Interestingly, depletion of B cell negated anti-metastatic effects of gut microbiome. Mechanistically, MHC-II expression in cancer cells mediates the anti-tumor effects of the microbiome. MHC-II upregulation on human CRC organoids enhances cancer-immune cell interactions and associated apoptosis, demonstrating an anti-tumor role and significance of MHC-II in human CRC. Thus, gut microbes that enhance MHC-II antigen presentation promotes anti-tumor immunity and may be utilized to enhance efficacy of immunotherapy in metastatic CRC. Citation Format: Charlie Chung, Elif Ozcelik, Jill Habel, Jialin Zhang, Yihan Qin, Libia Garcia, Joseph Merrill, Taemoon Chung, Scott Lyons, Zeli Shen, James G. Fox, Peter Westcott, Semir Beyaz. Microbial regulation of anti-tumor immunity in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6678.

MHC-I upregulation safeguards neoplastic T cells in the skin against NK cell-mediated eradication in mycosis fungoides

Cancer-associated immune dysfunction is a major challenge for effective therapies. The emergence of antibodies targeting tumor cell-surface antigens led to advancements in the treatment of hematopoietic malignancies, particularly blood cancers. Yet their impact is constrained against tumors of hematopoietic origin manifesting in the skin. In this study, we employ a clonality-supervised deep learning methodology to dissect key pathological features implicated in mycosis fungoides, the most common cutaneous T-cell lymphoma. Our investigations unveil the prominence of the IL-32β–major histocompatibility complex (MHC)-I axis as a critical determinant in tumor T-cell immune evasion within the skin microenvironment. In patients’ skin, we find MHC-I to detrimentally impact the functionality of natural killer (NK) cells, diminishing antibody-dependent cellular cytotoxicity and promoting resistance of tumor skin T-cells to cell-surface targeting therapies. Through murine experiments in female mice, we demonstrate that disruption of the MHC-I interaction with NK cell inhibitory Ly49 receptors restores NK cell anti-tumor activity and targeted T-cell lymphoma elimination in vivo. These findings underscore the significance of attenuating the MHC-I-dependent immunosuppressive networks within skin tumors. Overall, our study introduces a strategy to reinvigorate NK cell-mediated anti-tumor responses to overcome treatment resistance to existing cell-surface targeted therapies for skin lymphoma.

LSD1 Inhibition Enhances Antigen Presentation and Co-Stimulation in AML to Promote T Cell-Mediated Anti-Leukemia Immune Responses

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide a curative treatment option for acute myeloid leukemia (AML). Donor T cells play an important role in the curative potential of allo-HSCT by recognizing and eliminating residual leukemic cells, inducing the desirable graft-versus-leukemia (GVL) effect. Evading from this effect of donor T cell immune surveillance through the downregulation of human leukocyte antigen class II (HLA-II) molecules has been shown to contribute to AML relapse after allo-HSCT. Lysine-specific demethylase 1 (LSD1) is an emerging epigenetic therapeutic target in AML due to its role in regulating myeloid differentiation pathways. We hypothesized that LSD1 inhibition has the potential to enhance anti-leukemic immune responses by differentiating AML cells and inducing the acquisition of markers typical for antigen-presenting cells. We have studied these effects by treating human AML cell lines with the LSD1 inhibitor bomedemstat and investigated the expression of antigen-presenting molecules by flow cytometry and quantitative PCR (qPCR). At a concentration of 100 nM, bomedemstat upregulated the expression of CIITA by a log2 fold change of 2.23 ± 0.68 (p < 0.01) in OCI-AML3, 2.17 ± 0.28 (p < 0.01) in HL60 and 1.80 ± 0.67 (p < 0.05) in MOLM-13. To assess if the observed upregulation of CIITA can lead to a re-expression of HLA-DR in these HLA-DR negative cell lines, we then treated these cell lines with 100 nM of bomedemstat with additional IFN-γ stimulation. Expression of HLA-DR and the co-stimulatory molecule CD86 was determined by flow cytometry. Bomedemstat increased the expression of HLA-DR and CD86 across all three cell lines leading to the generation of 26.63 ± 3.07% (p < 0.0001) HLA-DR +/CD86 + cells in OCI-AML3, 4.19 ± 1.68% (p < 0.001) HLA-DR +/CD86 + cells in HL-60 and 14.67 ± 2.03% (p < 0.001) HLA-DR +/CD86 + cells in MOLM-13. We further assessed the effects of bomedemstat treatment on cytokine production by qPCR. Notably, bomedemstat treatment increased the expression of CXCL-10 by a log2 fold change of 2.59 ± 0.66 (p < 0.001) in MOLM-13 and 4.12 ± 0.88 (p < 0.001) in HL-60 at 100 nM concentration. The upregulation of CXCL-10 was also validated using bead-based ELISA assays. To investigate the molecular mechanism of the observed concomitant upregulation of MHC-II molecules and CD86, we then used murine myeloid progenitor cells transformed by overexpression of Hoxa9 and Meis1 (H9M) or MN1. In H9M-transformed cells, bomedemstat concurrently upregulated MHC-II (I-A/I-E) and CD86 even in the absence of IFN-γ stimulation, leading to an increase in MHC-II +/CD86 + cells from 2.52 ± 1.22% at baseline to 29.95 ± 14.11 (p < 0.05). This effect was not observed in MN1-transformed cells. H9M-transformed progenitor cells from Irf8 knockout mice (H9M- Irf8-KO) did not show any upregulation of MHC-II, pointing towards a role for Irf8 in this process. To interrogate whether the concurrent upregulation of MHC-II and CD86 can enhance CD4 + T cell activation, we used ovalbumin-expressing H9M-transformed cells that were co-cultured with OT-II T cells after bomedemstat treatment. We could show that bomedemstat-treatment enhanced the ability of H9M-OVA cells to activate T cells as demonstrated by an enhanced expression of the T cell activation markers CD25 and CD69. Furthermore, bomedemstat also increased the immune killing of OVA-peptide pulsed H9M cells at an effector-to-target (E:T) ratio of 1:1 from 1.12 ± 4.01 to 19.70 ± 6.20% (p < 0.01). It also enhanced the killing of OVA-expressing H9M cells from 5.80 ± 2.95% to 17.70 ± 3.16% (p < 0.05). No significant killing effect was observed for MN1 cells. In conclusion, we demonstrate both phenotypically and functionally that LSD1 inhibition by bomedemstat can enhance the immunogenicity of human and murine AML models. These findings point towards a potential role of LSD1 inhibition as a maintenance therapy after allo-HSCT.

Enterococcus Induces MHC-II Expression in the Colonic Epithelium to Aggravate Graft-Versus-Host Disease

Intestinal domination by Enterococcus, a genus of Gram-positive facultative bacteria, has been associated with an increased risk of GVHD mortality in allo-HCT patients and in preclinical models. However, the immunological mechanisms underlying this association remain unclear. Several preclinical studies have demonstrated that MHC-II expression by intestinal epithelial cells (IECs) is involved in allo-antigen presentation and GVHD and can be modulated by the intestinal microbiota. MHC-II genes are constitutively expressed by professional antigen-presenting cells (APCs) but can be induced in non-professional APCs by IFN-γ. Here we reported that Enterococcus could potentially aggravate GVHD through upregulation of MHC-II expression by IECs in an IFN-γ-dependent manner. Using an MHC-disparate mouse model of GVHD (C57BL/6J into BALB/cJ), we profiled the fecal microbiome of GVHD mice by 16S rRNA sequencing and the intestinal epithelium by flow cytometry on day 7 post-transplant. We found that endogenous Enterococcus relative abundance was positively correlated with MHC-II expression by colonic IECs (CD45- CD31- EpCAM+) during GVHD ( Fig. 1a; p=0.0004; 2 replicates; n=19). To determine whether this phenotype occurs in the absence of systemic inflammation, we utilized gnotobiotic mice treated with either E. faecalis, B. producta (a bacteria genus previously associated with improved GVHD outcomes)or PBS via oral gavage. Monocolonization with E. faecalis was sufficient to upregulate MHC-II expression by colonic IECs in non-transplanted (steady-state) animals compared to PBS-treated germfree controls ( Fig. 1b; p=0.0027; 3 replicates; n=8-12/group). On the contrary, monocolonization by B. producta did not induce MHC-II expression by colonic IECs compared to germfree controls ( p=0.96). We also observed that MHC-II upregulation was promoted by specific species within the genus Enterococcus, such that compared to PBS-treated germfree controls, only E. faecalis ( p=0.0007; 3 replicates; n=8-12/group) was able to induce MHC-II expression by colonic IECs, but not E. durans ( p=0.06) , E. gallinarium ( p=0.98) and E. hirae ( p=0.99). Using a Villin-Cre IFN- γ R fl/flmouse model with genetic ablation of the IFN-γ receptor specifically in epithelial cells, we investigated whether MHC-II expression induced by Enterococcus was dependent on IFN-γ signaling. We colonized Villin-Cre pos and Villin-Cre neg littermate controls with E. faecalis via oral gavage after antibiotics decontamination (ampicillin/enrofloxacin) and profiled the intestinal epithelium. We observed that Villin-Cre posmice exhibited a significantly lower MHC-II expression in the small intestine compared to Villin-Cre negmice on day 3 post- Enterococcus colonization ( p=0.012; 2 replicates; n=7/group), suggesting that IFN-γ signaling is necessary to mediate Enterococcus interaction with the intestinal epithelium. Finally, to explore a potential intervention to reduce intestinal inflammation upon Enterococcus domination, we treated GVHD mice with either butyrate-producing B. producta or E. faecalis. Colonization with B. producta reduced fecal Enterococcus burden on day 7 post-transplant ( p=0.007; 2 replicates; n=8/group) and improved survival in GVHD mice, compared to the E. faecalis-colonized group ( p=0.002; 2 replicates; n=18-19/group). We and others have previously shown that butyrate administration can alleviate GVHD through its effects on epithelial homeostasis. To determine whether butyrate could regulate allo-antigen presentation by the intestinal epithelium, we employed the human colorectal adenocarcinoma HT-29 cell culture. We pre-treated the cells with IFNγ (100U/ml) to induce MHC-II expression, then added butyrate (5mM) to the culture for 24 hours. Butyrate treatment was sufficient to suppress MHC-II expression by HT-29 cells, compared to DMSO-treated controls ( p=0.006; 3 replicates; n=6/group). Altogether, our results suggest that one mechanism by which Enterococcus can aggravate GVHD involves the upregulation of MHC-II expression by colonic IECs, and treatment with butyrate might serve as an intervention to prevent intestinal inflammation. Further identification of the upstream mediators involved will provide additional mechanistic insight into the interaction between Enterococcus and the intestinal epithelium at steady state and during GVHD.

Immune synapse formation promotes lipid peroxidation and MHC-I upregulation in licensed dendritic cells for efficient priming of CD8+ T cells

Antigen cognate dendritic cell (DC)-T cell synaptic interactions drive activation of T cells and instruct DCs. Upon receiving CD4+ T cell help, post-synaptic DCs (psDCs) are licensed to generate CD8+ T cell responses. However, the cellular and molecular mechanisms that enable psDCs licensing remain unclear. Here, we describe that antigen presentation induces an upregulation of MHC-I protein molecules and increased lipid peroxidation on psDCs in vitro and in vivo. We also show that these events mediate DC licensing. In addition, psDC adoptive transfer enhances pathogen-specific CD8+ T responses and protects mice from infection in a CD8+ T cell-dependent manner. Conversely, depletion of psDCs in vivo abrogates antigen-specific CD8+ T cell responses during immunization. Together, our data show that psDCs enable CD8+ T cell responses in vivo during vaccination and reveal crucial molecular events underlying psDC licensing.

The effect of edible bird's nests on the expression of MHC-II and costimulatory molecules of C57BL/6 mouse splenocytes

The glutinous nest that builds by the saliva secretion of swiftlet is recognizable as an edible bird's nest (EBN). It enriched a medicinal value and was regarded as supplementary food that exerts various beneficial health effects, especially immune boosters. This study's objective was to determine the impact of EBN on the expression of MHC-II and costimulatory molecules (CD86 and CD80) related to the initiation of T-cell activation. Both rEBN and pEBN samples were prepared with simulated gastrointestinal digestion for enhancing the bioaccessibility of bioactive compounds. Our result showed that digested EBN samples slightly influence the upregulation of MHC-II, CD86, and CD80 in gene expression of LPS-stimulated Raw 264.7 cells. The concern of endotoxin contamination in EBN samples, which may cause a false-positive result, was measured by quantitative PCR. We found that the inflammatory genes (IL-1β and TNF-α) were not induced by EBN treatments. Moreover, cell surface protein expression in splenocytes treated with EBN was assessed using flow cytometric analysis. Digested EBN samples demonstrated their capacity to promote the elevation of MHC-II, CD86, and CD80 cell surface protein expression. Finally, the digested-EBN-treated splenocytes only exhibited a specific response in the T-cells population. Thus, EBN is a source of the bioactive compound that has been proposed to exert a role in the stimulation of both MHC-II and costimulatory molecules for TCR/pMHC-II interaction leading to T-cell activation.

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.

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.

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.

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+ ( > 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 .

Short chain fatty acids prime colorectal cancer cells to activate antitumor immunity.

Colorectal cancer (CRC) is a leading cause of death worldwide and its growth can either be promoted or inhibited by the metabolic activities of intestinal microbiota. Short chain fatty acids (SCFAs) are microbial metabolites with potent immunoregulatory properties yet there is a poor understanding of how they directly regulate immune modulating pathways within the CRC cells. We used engineered CRC cell lines, primary organoid cultures, orthotopic in vivo models, and patient CRC samples to investigate how SCFA treatment of CRC cells regulates their ability to activate CD8+ T cells. CRC cells treated with SCFAs induced much greater activation of CD8+ T cells than untreated CRC cells. CRCs exhibiting microsatellite instability (MSI) due to inactivation of DNA mismatch repair were much more sensitive to SCFAs and induced much greater CD8+ T cell activation than chromosomally instable (CIN) CRCs with intact DNA repair, indicating a subtype-dependent response to SCFAs. This was due to SCFA-induced DNA damage that triggered upregulation of chemokine, MHCI, and antigen processing or presenting genes. This response was further potentiated by a positive feedback loop between the stimulated CRC cells and activated CD8+ T cells in the tumor microenvironment. The initiating mechanism in the CRCs was inhibition of histone deacetylation by the SCFAs that triggered genetic instability and led to an overall upregulation of genes associated with SCFA signaling and chromatin regulation. Similar gene expression patterns were found in human MSI CRC samples and in orthotopically grown MSI CRCs independent of the amount of SCFA producing bacteria in the intestine. MSI CRCs are widely known to be more immunogenic than CIN CRCs and have a much better prognosis. Our findings indicate that a greater sensitivity to microbially produced SCFAs contributes to the successful activation of CD8+ T cells by MSI CRCs, thereby identifying a mechanism that could be therapeutically targeted to improve antitumor immunity in CIN CRCs.