Expression of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA is synergistically associated with T cell exclusion, immune checkpoint blockade efficacy and recurrence risk in ER-negative breast cancer.

PURPOSE: Reliable biomarkers to predict the outcome and treatment response of estrogen receptor (ER)-negative breast cancer (BC) are urgently needed. Since immune-related signaling plays an important role in the tumorigenesis of ER-negative BC, we asked whether Notch genes, alone or in combination with other immune genes, can be used to predict the clinical outcome and immune checkpoint blockade (ICB) for this type of cancer. METHODS: We analyzed transcriptome data of 6918 BC samples from five independent cohorts, 81 xenograft triple-negative BC tumors that respond differently to ICB treatment and 754 samples of different cancer types from patients treated with ICB agents. RESULTS: We found that among four Notch genes, the expression levels of NOTCH1 and NOTCH4 were positively associated with recurrence of ER-negative BC, and that combined expression of these two genes (named Notch14) further enhanced this association, which was comparable with that of the Notch pathway signature. Analysis of 1182 immune-related genes revealed that the expression levels of most HLA genes, particularly HLA-DMA and -DRA, were reversely associated with recurrence in ER-negative BC with low, but not high Notch14 expression. A combined expression signature of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA was more prognostic for ER-negative and triple-negative BCs than previously reported immune-related signatures. Furthermore, we found that the expression levels of these four genes were also synergistically associated with T cell exclusion score, infiltration of specific T cells and ICB efficacy in ER-negative BC, thereby providing a potential molecular mechanism for the synergistic effect of these genes on BC. CONCLUSIONS: Our data indicate that a gene signature composed of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA may serve as a potential promising biomarker for predicting ICB therapy efficacy and recurrence in ER-negative/triple-negative BCs.

Major Histocompatibility Complex Class II HLA-DRα Is Downregulated by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Lytic Transactivator RTA and MARCH8.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) maintains two modes of life cycle, the latent and lytic phases. To evade the attack of the cell host's immune system, KSHV switches from the lytic to the latent phase, a phase in which only a few of viral proteins are expressed. The mechanism by which KSHV evades the attack of the immune system and establishes latency has not been fully understood. Major histocompatibility complex class II (MHC-II) molecules are key components of the immune system defense mechanism against viral infections. Here we report that HLA-DRα, a member of the MHC-II molecules, was downregulated by the replication and transcription activator (RTA) protein encoded by KSHV ORF50, an important regulator of the viral life cycle. RTA not only downregulated HLA-DRα at the protein level through direct binding and degradation through the proteasome pathway but also indirectly downregulated the protein level of HLA-DRα by enhancing the expression of MARCH8, a member of the membrane-associated RING-CH (MARCH) proteins. Our findings indicate that KSHV RTA facilitates evasion of the virus from the immune system through manipulation of HLA-DRα. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) has a causal role in a number of human cancers, and its persistence in infected cells is controlled by the host's immune system. The mechanism by which KSHV evades an attack by the immune system has not been well understood. This work represents studies which identify a novel mechanism by which the virus can facilitate evasion of an immune system. We now show that RTA, the replication and transcription activator encoded by KSHV (ORF50), can function as an E3 ligase to degrade HLA-DRα. It can directly bind and induce degradation of HLA-DRα through the ubiquitin-proteasome degradation pathway. In addition to the direct regulation of HLA-DRα, RTA can also indirectly downregulate the level of HLA-DRα protein by upregulating transcription of MARCH8. Increased MARCH8 results in the downregulation of HLA-DRα. Furthermore, we also demonstrate that expression of HLA-DRα was impaired in KSHV de novo infection.

Regulation of interlocking gene regulatory network subcircuits by a small molecule inhibitor of retinoblastoma protein (RB) phosphorylation: cancer cell expression of HLA-DR.

The induction of the major histocompatibility (MHC), antigen-presenting class II molecules by interferon-gamma, in solid tumor cells, requires the retinoblastoma tumor suppressor protein (Rb). In the absence of Rb, a repressosome blocks the access of positive-acting, promoter binding proteins to the MHC class II promoter. However, a complete molecular linkage between Rb expression and the disassembly of the MHC class II repressosome has been lacking. By treating A549 lung carcinoma cells with a novel small molecule that prevents phosphorylation-mediated, Rb inactivation, we demonstrate that Rb represses the synthesis of an MHC class II repressosome component, YY1. The reduction in YY1 synthesis correlates with the advent of MHC class II inducibility; with loss of YY1 binding to the promoter of the HLA-DRA gene, the canonical human MHC class II gene; and with increased Rb binding to the YY1 promoter. These results support the concept that the Rb gene regulatory network (GRN) subcircuit that regulates cell proliferation is linked to a GRN subcircuit regulating a tumor cell immune function.

Correlation between lipid deposition, immune-inflammatory cell content and MHC class II expression in diffuse intimal thickening of the human aorta.

Inflammatory reactions driven by an accumulation in the intima of immune-inflammatory cells and focal lipid depositions are the hallmarks of atherogenesis. It is commonly accepted that immune-inflammatory cell accumulation and lipid deposition are associated with the very earlier stage of atherosclerosis but no study has yet focused on the determination of quantitative values of this association. The present study examined correlations between lipid deposition, immune-inflammatory cell content and major histocompatibility complex (MHC) class II molecule HLA-DR expression in diffuse intimal thickening (DIT), which is thought to represent the earliest macroscopic manifestation of atherosclerosis. In parallel consecutive tissue sections of DIT, lipids were examined by chromatographic analysis (including triglycerides, cholesteryl esters, free cholesterol and phospholipids), histochemically, using Oil Red O staining, and by electron microscopy. Immune-inflammatory cells and HLA-DR expression were examined immunohistochemically in consecutive sections of the same tissue specimens. The study revealed that lipids exhibited a non-uniform distribution throughout the intima. In the juxtaluminal sublayer, lipids were localized both intracellularly and extracellularly, whereas in the juxtamedial musculoelastic sublayer, lipids were present predominantly along elastic fibers. Lipid deposits were found to positively correlate with HLA-DR expression (r=0.79; p<0.001). The study also identified a positive correlation between lipid deposition and immune-inflammatory cell content but the correlation values varied between different sublayers of the tunica intima. The correlation between lipid deposition and immune-inflammatory cell content in the juxtaluminal sublayer of the intima was notably stronger (r=0.69; p<0.001) than in the juxtamedial musculoelastic layer (r=0.28; p<0.001). The findings of the present study support a view that lipid accumulation in the intima plays a role in the initiation of inflammatory reaction and that at the pre-lesional stage in the development of atherosclerosis, lipid-associated immune cell activation might occur primarily in the juxtaluminal portion of the intima.

Mild hyperthermia enhances human monocyte-derived dendritic cell functions and offers potential for applications in vaccination strategies.

Dendritic cell (DC)-based immunotherapy has been shown to be a promising strategy for anti-cancer therapy. Nevertheless, only a low overall clinical response rate has been observed in vaccinated patients with advanced cancer and therefore methods to improve DC immuno-stimulatory functions are currently under intense investigation. In this respect, we exposed human monocyte-derived DCs to a physiological temperature stress of 40°C for up to 24 h followed by analysis for (i) expression of different heat shock proteins, (ii) survival, (iii) cell surface maturation markers, (iv) cytokine secretion, and (v) migratory capacity. Furthermore, we examined the ability of heat-shocked DCs to prime naïve CD8(+) T cells after loading with MelanA peptide, by transfection with MelanA RNA, or by transduction with MelanA by an adenovirus vector. The results clearly indicate that in comparison to control DCs, which remained at 37°C, heat-treated cells revealed no differences concerning the survival rate or their migratory capacity. However, DCs exposed to thermal stress showed a time-dependent enhanced expression of the immune-chaperone heat shock protein 70A and both an up-regulation of co-stimulatory molecules such as CD80, CD83, and CD86 and of the inflammatory cytokine TNF-α. Moreover, these cells had a markedly improved capacity to prime autologous naïve CD8(+) T cells in vitro in an antigen-specific manner, independent of the method of antigen-loading. Thus, our strategy of heat treatment of DCs offers a promising means to improve DC functions during immune activation which, as a physical method, facilitates straight-forward applications in clinical DC vaccination protocols.