Spatial analysis reveals targetable macrophage-mediated mechanisms of immune evasion in hepatocellular carcinoma minimal residual disease.

Hepatocellular carcinoma (HCC) frequently recurs from minimal residual disease (MRD), which persists after therapy. Here, we identified mechanisms of persistence of residual tumor cells using post-chemoembolization human HCC (n = 108 patients, 1.07 million cells) and a transgenic mouse model of MRD. Through single-cell high-plex cytometric imaging, we identified a spatial neighborhood within which PD-L1 + M2-like macrophages interact with stem-like tumor cells, correlating with CD8+ T cell exhaustion and poor survival. Further, through spatial transcriptomics of residual HCC, we showed that macrophage-derived TGFß1 mediates the persistence of stem-like tumor cells. Last, we demonstrate that combined blockade of Pdl1 and Tgfß excluded immunosuppressive macrophages, recruited activated CD8+ T cells and eliminated residual stem-like tumor cells in two mouse models: a transgenic model of MRD and a syngeneic orthotopic model of doxorubicin-resistant HCC. Thus, our spatial analyses reveal that PD-L1+ macrophages sustain MRD by activating the TGFß pathway in stem-like cancer cells and targeting this interaction may prevent HCC recurrence from MRD.

Cholesterol-binding motifs in STING that control endoplasmic reticulum retention mediate anti-tumoral activity of cholesterol-lowering compounds.

The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.

T-cell derived extracellular vesicles prime macrophages for improved STING based cancer immunotherapy.

A key phenomenon in cancer is the establishment of a highly immunosuppressive tumour microenvironment (TME). Despite advances in immunotherapy, where the purpose is to induce tumour recognition and hence hereof tumour eradication, the majority of patients applicable for such treatment still fail to respond. It has been suggested that high immunological activity in the tumour is essential for achieving effective response to immunotherapy, which therefore have led to exploration of strategies that triggers inflammatory pathways. Here activation of the stimulator of interferon genes (STING) signalling pathway has been considered an attractive target, as it is a potent trigger of pro-inflammatory cytokines and types I and III interferons. However, immunotherapy combined with targeted STING agonists has not yielded sustained clinical remission in humans. This suggests a need for exploring novel adjuvants to improve the innate immunological efficacy. Here, we demonstrate that extracellular vesicles (EVs), derived from activated CD4+ T cells (T-EVs), sensitizes macrophages to elevate STING activation, mediated by IFNγ carried on the T-EVs. Our work support that T-EVs can disrupt the immune suppressive environment in the tumour by reprogramming macrophages to a pro-inflammatory phenotype, and priming them for a robust immune response towards STING activation.

Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis.

BACKGROUND: Lymphocyte activation gene-3 (LAG-3) inhibits T cell activation and interferes with the immune response by binding to MHC-II. As antigen presentation is central in rheumatoid arthritis (RA) pathogenesis, we studied aspects of LAG-3 as a serological marker and mediator in the pathogenesis of RA. Since Galectin-3 (Gal-3) is described as an additional binding partner for LAG-3, we also aimed to study the functional importance of this interaction. METHODS: Plasma levels of soluble (s) LAG-3 were measured in early RA patients (eRA, n = 99) at baseline and after 12 months on a treat-to-target protocol, in self-reportedly healthy controls (HC, n = 32), and in paired plasma and synovial fluid (SF) from chronic RA patients (cRA, n = 38). Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were examined for LAG-3 expression by flow cytometry. The binding and functional outcomes of LAG-3 and Gal-3 interaction were assessed with surface plasmon resonance (SPR) and in cell cultures using rh-LAG3, an antagonistic LAG-3 antibody and a Gal-3 inhibitor. RESULTS: Baseline sLAG-3 in the plasma was increased in eRA compared to HC and remained significantly elevated throughout 12 months of treatment. A high level of sLAG-3 at baseline was associated with the presence of IgM-RF and anti-CCP as well as radiographic progression. In cRA, sLAG-3 was significantly increased in SF compared with plasma, and LAG-3 was primarily expressed by activated T cells in SFMCs compared to PBMCs. Adding recombinant human LAG-3 to RA cell cultures resulted in decreased cytokine secretion, whereas blocking LAG-3 with an antagonistic antibody resulted in increased cytokine secretion. By SPR, we found a dose-dependent binding between LAG-3 and Gal-3. However, inhibiting Gal-3 in cultures did not further change cytokine production. CONCLUSIONS: sLAG-3 in the plasma and synovial fluid is increased in both early and chronic RA patients, particularly in the inflamed joint. High levels of sLAG-3 are associated with autoantibody seropositivity and radiographic progression in eRA, and LAG-3 plays a biologically active role in cRA by decreasing inflammatory cytokine production. This functional outcome is not affected by Gal-3 interference. Our results suggest that LAG-3 is a faceted regulator of inflammation in early and chronic RA.

MYC Overexpression Drives Immune Evasion in Hepatocellular Carcinoma That Is Reversible through Restoration of Proinflammatory Macrophages.

Cancers evade immune surveillance, which can be reversed through immune-checkpoint therapy in a small subset of cases. Here, we report that the MYC oncogene suppresses innate immune surveillance and drives resistance to immunotherapy. In 33 different human cancers, MYC genomic amplification and overexpression increased immune-checkpoint expression, predicted nonresponsiveness to immune-checkpoint blockade, and was associated with both Th2-like immune profile and reduced CD8 T-cell infiltration. MYC transcriptionally suppressed innate immunity and MHCI-mediated antigen presentation, which in turn impeded T-cell response. Combined, but not individual, blockade of PDL1 and CTLA4 could reverse MYC-driven immune suppression by leading to the recruitment of proinflammatory antigen-presenting macrophages with increased CD40 and MHCII expression. Depletion of macrophages abrogated the antineoplastic effects of PDL1 and CTLA4 blockade in MYC-driven hepatocellular carcinoma (HCC). Hence, MYC is a predictor of immune-checkpoint responsiveness and a key driver of immune evasion through the suppression of proinflammatory macrophages. The immune evasion induced by MYC in HCC can be overcome by combined PDL1 and CTLA4 blockade. SIGNIFICANCE: Macrophage-mediated immune evasion is a therapeutic vulnerability of MYC-driven cancers, which has implications for prioritizing MYC-driven hepatocellular carcinoma for combination immunotherapy.

T-cell immunoglobulin and mucin domain 3 is upregulated in rheumatoid arthritis, but insufficient in controlling inflammation.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease, that involves both pro- and anti-inflammatory mechanisms. The purpose of the present study is to investigate T-cell immunoglobulin and mucin domain 3 (Tim-3) in RA. METHODS: Plasma levels of soluble (s) Tim-3 in early RA (n=98), were followed, to evaluate association with treatment and disease activity, acquired from a prospective collected biobank (clinicaltrials.gov (NCT00660647)). We also investigate the influence of Tim-3 on spontaneous cytokine production in synovial fluid mononuclear cells (SFMC) from RA patients after addition of neutralizing anti-Tim-3's antibodies, either alone or in combination with neutralizing anti-Programmed Cell death protein 1 (PD-1) antibodies. RESULTS: Long-time stimulated CD4 T-cells expressed high levels of Tim-3, but tended to decrease their PD-1 expression. Tim-3 expression was exclusively seen co-expressed with PD-1 by CD3, CD4, CD45RO positive cells in the inflamed RA joint. Addition of neutralizing Tim-3 antibodies increased the secretion of IFNγ and MCP-1, in SFMC cultures from RA. Whereas neutralizing anti-PD-1 antibodies showed a broader impact on cytokine production. Finally, we observed that soluble Tim-3 is increased in plasma and is associated with disease activity in early RA. CONCLUSION: Taken together, our findings indicate disease-suppressive functions of Tim-3 in RA.

The MYC oncogene - the grand orchestrator of cancer growth and immune evasion.

The MYC proto-oncogenes encode a family of transcription factors that are among the most commonly activated oncoproteins in human neoplasias. Indeed, MYC aberrations or upregulation of MYC-related pathways by alternate mechanisms occur in the vast majority of cancers. MYC proteins are master regulators of cellular programmes. Thus, cancers with MYC activation elicit many of the hallmarks of cancer required for autonomous neoplastic growth. In preclinical models, MYC inactivation can result in sustained tumour regression, a phenomenon that has been attributed to oncogene addiction. Many therapeutic agents that directly target MYC are under development; however, to date, their clinical efficacy remains to be demonstrated. In the past few years, studies have demonstrated that MYC signalling can enable tumour cells to dysregulate their microenvironment and evade the host immune response. Herein, we discuss how MYC pathways not only dictate cancer cell pathophysiology but also suppress the host immune response against that cancer. We also propose that therapies targeting the MYC pathway will be key to reversing cancerous growth and restoring antitumour immune responses in patients with MYC-driven cancers.

Programmed death ligand 2 - A link between inflammation and bone loss in rheumatoid arthritis.

OBJECTIVE: Active rheumatoid arthritis (RA) is accompanied by increased appendicular and axial bone loss, closely associated to the degree of inflammation. The programmed death-1 (PD-1) pathway is important for maintaining peripheral tolerance, and its ligand PD-L2 has recently been associated with bone morphogenetic protein activity. Here, we report that PD-L2 plays a central role in RA osteoimmunology. METHODS: Femoral bone mineral density (BMD) and trabecular bone microstructure were evaluated by micro-CT in wild type (WT) and PD-L2-/- mice. Osteoclasts were generated from RA synovial fluid mononuclear cells and peripheral blood monocytes. The effects of recombinant PD-L2, was evaluated by tartrate-resistant acid phosphatase (TRAP) activity and the development of bone erosions in the presence of anti-citrullinated protein antibodies (ACPA). Plasma soluble (s)PD-L2 levels were measured in patients with early (e)RA (n â€‹= â€‹103) treated with methotrexate alone or in combination with the TNF inhibitor Adalimumab. RESULTS: PD-L2-/- mice had a decreased BMD and deteriorated trabecular bone microstructure that was not related to the RANKL/OPG pathway. PD-L2 decreased TRAP activity in osteoclasts and decreased ACPA-induced erosions. In the RA synovial membrane PD-L2 was highly expressed especially in the lining layer and plasma sPD-L2 levels were increased in eRA patients and decreased with treatment. One-year sPD-L2 correlated inversely with erosive progression two years after treatment initiation with methotrexate and placebo. CONCLUSION: PD-L2 regulates bone homeostasis in RA. Our findings provide new insight into the relationship between the immune system and bone homeostasis, and suggest a potential therapeutic target for limiting inflammatory bone loss in RA.

MYC functions as a switch for natural killer cell-mediated immune surveillance of lymphoid malignancies.

The MYC oncogene drives T- and B- lymphoid malignancies, including Burkitt's lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). Here, we demonstrate a systemic reduction in natural killer (NK) cell numbers in SRα-tTA/Tet-O-MYCON mice bearing MYC-driven T-lymphomas. Residual mNK cells in spleens of MYCON T-lymphoma-bearing mice exhibit perturbations in the terminal NK effector differentiation pathway. Lymphoma-intrinsic MYC arrests NK maturation by transcriptionally repressing STAT1/2 and secretion of Type I Interferons (IFNs). Treating T-lymphoma-bearing mice with Type I IFN improves survival by rescuing NK cell maturation. Adoptive transfer of mature NK cells is sufficient to delay both T-lymphoma growth and recurrence post MYC inactivation. In MYC-driven BL patients, low expression of both STAT1 and STAT2 correlates significantly with the absence of activated NK cells and predicts unfavorable clinical outcomes. Our studies thus provide a rationale for developing NK cell-based therapies to effectively treat MYC-driven lymphomas in the future.

CD46 activation induces distinct CXCL-10 response in monocytes and monocyte-derived dendritic cells.

CD46 is an important immune regulatory receptor with dual functions, however, the CD46 isoform distribution and the effect of CD46 activation on the cytokine production in monocytes and monocyte-derived dendritic cells (moDCs) is unclear. Here, we show that CD46 activation of moDCs downregulates LPS-induced CXCL-10 expression, while the expression of CXCL-10 in monocytes is unaffected. Furthermore, the differentiation of moDCs induces a switch towards dominance of CYT-2 isoforms of CD46. These data indicate that CD46 activation exerts different functions in monocytes and moDCs and this correlates with a switch in CD46 isoform expression upon differentiation of moDCs.

CD46 is a potent co-stimulatory receptor for expansion of human IFN-γ-producing CD8+ T cells.

Similar to CD4+ T cells, precursor CD8+ T cells are thought to depend on a co-stimulatory signal through CD28 for proliferation and differentiation into effector cells. CD46 is another co-stimulatory receptor that promotes differentiation of CD4+ T-helper cells type 1 (Th1 cells) into a regulatory phenotype with a switch from IFN-γ towards IL-10-secretion over time. Whether CD46 exerts a similar function on CD8+ T cells remains to be fully elucidated. Here, we demonstrate that CD46 co-stimulation induced secretion of IFN-γ as well as expansion of IFN-γ-secreting CD8+ T cells. In contrast to CD46 co-stimulation of CD4+ T cells, CD8+ T cells did not differentiate into a regulatory IL-10-secreting phenotype. This demonstrates that CD46 is a co-stimulatory receptor on CD8+ T cells, and that it exerts separate functions during CD4+ and CD8+ T-cell differentiation.

Extracellular Vesicles Transfer the Receptor Programmed Death-1 in Rheumatoid Arthritis.

INTRODUCTION: Extracellular vesicles (EVs) have been recognized as route of communication in the microenvironment. They transfer proteins and microRNAs (miRNAs) between cells, and possess immunoregulatory properties. However, their role in immune-mediated diseases remains to be elucidated. We hypothesized a role for EVs in the rheumatoid arthritis (RA) joint, potentially involving the development of T cell exhaustion and transfer of the co-inhibitory receptor programmed death 1 (PD-1). METHODS: Synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from RA patients were investigated for PD-1 and other markers of T cell inhibition. EVs were isolated from RA plasma and synovial fluid. In addition, healthy control (HC) and RA PBMCs and SFMCs were cultured to produce EVs. These were isolated and investigated by immunogold electron microscopy (EM) and also co-cultured with lymphocytes and PD-1 negative cells to investigate their functions. Finally, the miRNA expression profiles were assessed in EVs isolated from RA and HC cell cultures. RESULTS: Cells from the RA joint expressed several T cell co-inhibitory receptors, including PD-1, TIM-3, and Tigit. ELISA demonstrated the presence of PD-1 in EVs from RA plasma and synovial fluid. Immunogold EM visualized PD-1 expression by EVs. Co-culturing lymphocytes and the PD-1 negative cell line, U937 with EVs resulted in an induction of PD-1 on these cells. Moreover, EVs from RA PBMCs increased proliferation in lymphocytes when co-cultured with these. All EVs contained miRNAs associated with PD-1 and other markers of T cell inhibition and the content was significantly lower in EVs from RA PBMCs than HC PBMCs. Stimulation of the cells increased the miRNA expression. However, EVs isolated from stimulated RA SFMCs did not change their miRNA expression profile to the same extend. CONCLUSION: EVs carrying both the PD-1 receptor and miRNAs associated with T cell inhibition were present in RA cell cultures. Upon stimulation, these miRNAs failed to be upregulated in EVs from RA SFMCs. This was in line with increased expression of T cell co-inhibitory markers on SFMCs. In conclusion, we suggest EVs to play a significant role in the RA microenvironment, potentially favoring the progression of T cell exhaustion.

Divergent tropism of HHV-6AGS and HHV-6BPL1 in T cells expressing different CD46 isoform patterns.

CD46 is a receptor for HHV-6A, but its role as a receptor for HHV-6B is controversial. The significance of CD46 isoforms for HHV-6A and HHV-6B tropism is unknown. HHV-6AGS was able to initiate transcription of the viral genes U7 and U23 in the CD46+CD134- T-cell lines Peer, Jurkat, Molt3, and SupT1, whereas HHV-6BPL1 was only able to do so in Molt3 and SupT1, which expressed a CD46 isoform pattern different from Peer and Jurkat. The HHV-6BPL1-susceptible T-cell lines were characterized by low expression of the CD46 isoform BC2 and domination of isoforms containing the cytoplasmic tail, CYT-1. A HHV-6BPL1 susceptible cell line, Be13, changed over time its CD46 isoform pattern to resemble Peer and Jurkat and concomitantly lost its susceptibility to HHV-6BPL1 but not HHV-6AGS infection. We propose that isoforms of CD46 impact on HHV-6B infection and thereby in part explain the distinct tropism of HHV-6AGS and HHV-6BPL1.

Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells.

CD46 is a glycoprotein with important functions in innate and adaptive immune responses. Functionally different isoforms are generated by alternative splicing at exons 7-9 (BC and C isoforms) and exon 13 (CYT-1 and CYT-2 isoforms) giving rise to BC1, BC2, C1 and C2. We developed a novel real-time PCR assay that allows quantitative comparisons between these isoforms. Their relative frequency in CD4+ T cells from 100 donors revealed a distribution with high interpersonally variability. Importantly, the distribution between the isoforms was not random and although splicing favoured inclusion of exon 8 (BC isoforms), exclusion of exon 8 (C isoforms) was significantly linked to exclusion of exon 13 (CYT-2 isoforms). Despite inter-individual differences, CD4+ and CD8+ T cells, B cells, NK cells and monocytes expressed similar isoform profiles intra-individually. However, memory/effector CD4+ T cells had a significantly higher frequency of CYT-2 when compared with naïve CD4+ T cells. Likewise, in vitro activation of naïve and total CD4+ T cells increased the expression of CYT-2. This indicates that although splicing factors determine a certain expression profile in an individual, the profile can be modulated by external stimuli. This suggests a mechanism by which alterations in CD46 isoforms may temporarily regulate the immune response.

The DR6 protein from human herpesvirus-6B induces p53-independent cell cycle arrest in G2/M.

HHV-6B infection inhibits cell proliferation in G2/M, but no protein has so far been recognized to exert this function. Here we identify the protein product of direct repeat 6, DR6, as an inhibitor of G2/M cell-cycle progression. Transfection of DR6 reduced the total number of cells compared with mock-transfected cells. Lentiviral transduction of DR6 inhibited host cell DNA synthesis in a p53-independent manner, and this inhibition was DR6 dose-dependent. A deletion of 66 amino acids from the N-terminal part of DR6 prevented efficient nuclear translocation and the ability to inhibit DNA synthesis. DR6-induced accumulation of cells in G2/M was accompanied by an enhanced expression of cyclin B1 that accumulated predominantly in the cytoplasm. Pull-down of cyclin B1 brought down pCdk1 with the inactivating phosphorylation at Tyr15. Together, DR6 delays cell cycle with an accumulation of cells in G2/M and thus might be involved in HHV-6B-induced cell-cycle arrest.

Bulk properties of the lipid bilayer are not essential for the thermal stability of Na,K-ATPase from shark rectal gland or pig kidney.

The thermal stability of Na,K-ATPase from pig kidney is markedly greater than that of Na,K-ATPase from shark salt glands. The role of the lipid bilayer is studied by solubilisation of the membrane-bound enzyme in the nonionic detergent octaethyleneglycoldodecylmonoether (C(12)E(8)), addition of excess dioleylphosphatidylcholine (DOPC) or palmitoyloleylphosphatidylcholine (POPC) and reconstitution of membranes by removal of detergent. At 54°C the reconstituted enzymatically active pig enzyme retains a high thermal stability, and reconstituted shark enzyme retains a low thermal stability, even with a 9-fold excess of DOPC. This result suggests that the origin of the difference in thermal stability is not related to bulk lipid properties of the native membranes.