A phase I study of IMC-001, a PD-L1 blocker, in patients with metastatic or locally advanced solid tumors.

Introduction IMC-001 is a fully human IgG1 monoclonal antibody that binds to human PD-L1 (programmed death-ligand 1). This study evaluated the safety, pharmacokinetics, and pharmacodynamics of IMC-001 in patients with advanced solid tumors. Materials and Methods This open-labeled phase I study used a standard 3 + 3 dose-escalation design, with doses ranging from 2 to 20 mg/kg. IMC-001 was administered intravenously every 2 weeks until disease progression or unacceptable toxicity. The dose-limiting toxicity window was defined as 21 days from the first dose. Results Fifteen subjects were included in 5 dose-escalation cohorts. No dose-limiting toxicity was observed, and the maximum tolerated dose was not reached. The most common adverse events (AEs) were general weakness, decreased appetite, fever, and cough. No grade 4 or 5 treatment emergent AEs were reported during the study. One subject in the 2 mg/kg cohort showed grade 2 immune-induced thyroiditis and diabetes mellitus suspected to be related to IMC-001. Over the dose range of 2-20 mg/kg IMC-001, the AUC0-14d, AUC0-∞, and Cmax generally increased in a dose-proportional manner for each step of dose escalation. Of the 15 enrolled patients, 1 subject with rectal cancer showed a partial response, and the disease control rate was 33.3%. Conclusions IMC-001 demonstrated a favorable safety profile up to 20 mg/kg administered intravenously every 2 weeks and showed preliminary efficacy in patients with advanced solid tumors. Based on pharmacokinetic and pharmacodynamic data, 20 mg/kg was selected as the recommended phase II dose. Clinical trial identification NCT03644056 (date of registration: August 23, 2018).

Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis.

Fibroblasts regulate tissue homeostasis, coordinate inflammatory responses, and mediate tissue damage. In rheumatoid arthritis (RA), synovial fibroblasts maintain chronic inflammation which leads to joint destruction. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to pathology. Here, we show functional and transcriptional differences between fibroblast subsets from human synovial tissues using bulk transcriptomics of targeted subpopulations and single-cell transcriptomics. We identify seven fibroblast subsets with distinct surface protein phenotypes, and collapse them into three subsets by integrating transcriptomic data. One fibroblast subset, characterized by the expression of proteins podoplanin, THY1 membrane glycoprotein and cadherin-11, but lacking CD34, is threefold expanded in patients with RA relative to patients with osteoarthritis. These fibroblasts localize to the perivascular zone in inflamed synovium, secrete proinflammatory cytokines, are proliferative, and have an in vitro phenotype characteristic of invasive cells. Our strategy may be used as a template to identify pathogenic stromal cellular subsets in other complex diseases.

Stromal cell cadherin-11 regulates adipose tissue inflammation and diabetes.

M2 macrophages, innate lymphoid type 2 cells (ILC2s), eosinophils, Tregs, and invariant NK T cells (iNKT cells) all help to control adipose tissue inflammation, while M1 macrophages, TNF, and other inflammatory cytokines drive inflammation and insulin resistance in obesity. Stromal cells regulate leukocyte responses in lymph nodes, but the role of stromal cells in adipose tissue inflammation is unknown. PDGFRα+ stromal cells are major producers of IL-33 in adipose tissue. Here, we show that mesenchymal cadherin-11 modulates stromal fibroblast function. Cadherin-11-deficient mice displayed increased stromal production of IL-33, with concomitant enhancements in ILC2s and M2 macrophages that helped control adipose tissue inflammation. Higher expression levels of IL-33 in cadherin-11-deficient mice mediated ILC2 activation, resulting in higher IL-13 expression levels and M2 macrophage expansion in adipose tissue. Consistent with reduced adipose tissue inflammation, cadherin-11-deficient mice were protected from obesity-induced glucose intolerance and adipose tissue fibrosis. Importantly, anti-cadherin-11 mAb blockade similarly improved inflammation and glycemic control in obese WT mice. These results suggest that stromal fibroblasts expressing cadherin-11 regulate adipose tissue inflammation and thus highlight cadherin-11 as a potential therapeutic target for the management of obesity.

Genetic polymorphism directs IL-6 expression in fibroblasts but not selected other cell types.

Interleukin (IL)-6 blockade is an effective treatment for rheumatoid arthritis (RA), and synovial fibroblasts are a major IL-6 producer in the inflamed joint. We found that human RA and osteoarthritis (OA) synovial fibroblasts derived from independent donors reproducibly segregated into low, medium, and high IL-6 producers, independent of stimulus, cell passage, or disease state. IL-6 expression pattern correlated strongly with total mRNA expression, not mRNA stability, suggesting transcriptional rather than posttranscriptional regulation. High-fibroblast IL-6 expression was significantly associated with the IL-6 proximal promoter single nucleotide polymorphism (SNP) rs1800795 minor allele (CC) genotype. In contrast, no association between this SNP and IL-6 production was detected in CD14(+) monocytes, another major producer of synovial IL-6. Luciferase expression assays confirmed that this SNP was associated with differential IL-6 expression in fibroblasts. To date, several association studies examining rs1800795 allele frequency and disease risk have reported seemingly conflicting results ranging from no association to association with either the major or minor allele across a spectrum of conditions, including cancer and autoimmune, cardiovascular, infectious, and metabolic diseases. This study points to a prominent contribution from promoter genetic variation in fibroblast IL-6 regulation, but not in other IL-6-producing cell types. We propose that some of the heterogeneity in these clinical studies likely reflects the cellular source of IL-6 in specific diseases, much of which may be produced by nonhematopoietic cells. These results highlight that functional analysis of disease-associated SNPs on gene expression and pathologic processes must consider variation in diverse cell types.

Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.

Lymph node stromal cells (LNSCs) closely regulate immunity and self-tolerance, yet key aspects of their biology remain poorly elucidated. Here, comparative transcriptomic analyses of mouse LNSC subsets demonstrated the expression of important immune mediators, growth factors and previously unknown structural components. Pairwise analyses of ligands and cognate receptors across hematopoietic and stromal subsets suggested a complex web of crosstalk. Fibroblastic reticular cells (FRCs) showed enrichment for higher expression of genes relevant to cytokine signaling, relative to their expression in skin and thymic fibroblasts. LNSCs from inflamed lymph nodes upregulated expression of genes encoding chemokines and molecules involved in the acute-phase response and the antigen-processing and antigen-presentation machinery. Poorly studied podoplanin (gp38)-negative CD31(-) LNSCs showed similarities to FRCs but lacked expression of interleukin 7 (IL-7) and were identified as myofibroblastic pericytes that expressed integrin α(7). Together our data comprehensively describe the transcriptional characteristics of LNSC subsets.

Modulation of matrix metalloproteinase production by rheumatoid arthritis synovial fibroblasts after cadherin 11 engagement.

OBJECTIVE: Cadherin 11 is a homophilic cell-to-cell adhesion molecule expressed on joint synovial fibroblasts. Absence of cadherin 11 in a mouse rheumatoid arthritis (RA) model led to striking reductions in cartilage erosion. Matrix metalloproteinases (MMPs) are enzymes expressed by synovial fibroblasts important for cartilage erosion. The objective of this study was to determine if synovial fibroblast MMP production is regulated by cadherin 11. METHODS: To mimic cadherin 11 engagement, human RA synovial fibroblasts were stimulated with a chimeric construct consisting of the cadherin 11 extracellular domain linked to the human IgG1 Fc domain (Cad-11-Fc). Effects on MMP production were measured by enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction analysis, and immunoblotting. RESULTS: Human Cad-11-Fc up-regulated MMP-1 and MMP-3 protein production by RA synovial fibroblasts, both alone and in synergy with tumor necrosis factor α. This up-regulation required cell cadherin 11 engagement, since a mutant Cad-11-Fc with reduced binding affinity stimulated significantly less MMP production. Also, short hairpin RNA (shRNA) cadherin 11 silencing almost completely inhibited Cad-11-Fc-induced MMP expression. Cad-11-Fc stimulation increased RA synovial fibroblast MMP messenger RNA levels. It also increased the phosphorylation of the MAPKs JNK, ERK, and p38 kinase, the phosphorylation of NF-κB p65, and the nuclear translocation of activator protein 1 transcription factor. MAPK and NF-κB inhibitors partially blocked RA synovial fibroblast MMP expression. CONCLUSION: Cadherin 11 engagement stimulates increased synthesis of several MMPs by RA synovial fibroblasts in a MAPK- and NF-κB-dependent manner. These results underscore the existence of a pathway by which cadherin 11 regulates MMP production and has important implications for joint destruction in RA.

Cadherin-11 regulates fibroblast inflammation.

Fibroblasts are important participants in inflammation. Although not leukocytes, their capacity to produce cytokines, chemokines, and other inflammatory factors locally in tissues suggests that they can contribute to inflammatory diseases. For example, fibroblasts in a rheumatoid arthritis (RA) joint are a dominant source of IL-6 and RANKL in the synovium, both of which are therapeutic targets for inflammation and bone erosion. Previously, we found that fibroblasts can be targeted by mAb directed against cadherin-11 (cad-11), a mesenchymal cadherin that fibroblasts selectively express. Targeting cad-11 significantly reduced inflammation as assessed by joint swelling and clinical inflammation scores. However, the mechanism by which anti-cad-11 reduced inflammation was not known. Here, we show that cad-11 engagement induces synovial fibroblasts to secret proinflammatory cytokines including IL-6. Cad-11 engagement strongly synergized with TNF-α and IL-1ß in the induction of IL-6. Importantly, cad-11 activated MAP kinases and NF-κB for IL-6 induction. IL-6 levels in ankles of inflamed joints were reduced in cad-11 mutant mice compared to wild-type mice with inflammatory arthritis. Thus, we suggest that cad-11 modulates synovial fibroblasts to evoke inflammatory factors that may contribute to the inflammatory process in RA.

Fibroblast-like synoviocytes in inflammatory arthritis pathology: the emerging role of cadherin-11.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease affecting the joint synovium. The normal synovium consists of a lining layer of fibroblast-like synoviocytes (FLS) and macrophages, one to three cells deep that overlies the loose connective tissue of the synovial sublining. During the course of RA, the synovium is the site of inflammation where immune cells are massively infiltrated, and the lining layer becomes hyperplastic and transforms into a pannus tissue that destroys articular cartilage and bone. FLS play an important role in this RA pathogenesis. In this review, we explain that cadherin-11, an adhesion molecule, is selectively expressed on FLS and required for synovial lining formation. In addition, cadherin-11 on FLS contributes to synovial inflammation and mediates cartilage degradation in a mouse model of inflammatory arthritis. Therefore, we suggest that FLS are critical regulators of synovial inflammation and arthritis pathology via mechanisms that are mediated by cadherin-11.

Alternative splicing regulates activation-induced cytidine deaminase (AID): implications for suppression of AID mutagenic activity in normal and malignant B cells.

The mutagenic enzyme activation-induced cytidine deaminase (AID) is required for immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in germinal center (GC) B cells. Deregulated expression of AID is associated with various B-cell malignancies and, currently, it remains unclear how AID activity is extinguished to avoid illegitimate mutations. AID has also been shown to be alternatively spliced in malignant B cells, and there is limited evidence that this also occurs in normal blood B cells. The functional significance of these splice variants remains unknown. Here we show that normal GC human B cells and blood memory B cells similarly express AID splice variants and show for the first time that AID splicing variants are singly expressed in individual normal B cells as well as malignant B cells from chronic lymphocytic leukemia patients. We further demonstrate that the alternative AID splice variants display different activities ranging from inactivation of CSR to inactivation or heightened SHM activity. Our data therefore suggest that CSR and SHM are differentially switched off by varying the expression of splicing products of AID at the individual cell level. Most importantly, our findings suggest a novel tumor suppression mechanism by which unnecessary AID mutagenic activities are promptly contained for GC B cells.

B lymphocyte stimulator regulates adaptive immune responses by directly promoting dendritic cell maturation.

B lymphocyte stimulator (BLyS) is a well-known direct costimulator of adaptive immune cells, particularly B lineage cells. However, we have reported recently that BLyS is also able to activate monocytes. Other innate immune cells, such as dendritic cells (DCs), play a key role in the initiation of adaptive immune responses and the purpose of the current study was to assess whether there is a direct role for BLyS in modulating human DC functions. In this study, we show that BLyS induces DC activation and maturation. Thus, BLyS strongly induced up-regulation of surface costimulatory molecule expression and secretion of specific cytokines and chemokines in DCs. BLyS-stimulated DCs (BLyS-DCs) were also able to augment allogeneic CD4 T cell proliferation to a greater extent than control DCs. BLyS-DCs secreted elevated levels of the major Th1-polarizing cytokine, IL-12p70, and they promoted naive CD4 T cell differentiation into Th1 T cells. Regarding BLyS receptor expression, DCs primarily express cytoplasmic transmembrane activator and CAML interactor; however, low levels of cell surface transmembrane activator and CAML interactor are expressed as well. Collectively, our data suggest that BLyS may modulate adaptive immune cells indirectly by inducing DC maturation.

Divergent effects of BAFF on human memory B cell differentiation into Ig-secreting cells.

B cell-activating factor belonging to the TNF family (BAFF) plays a critical role in B cell maturation, yet its precise role in B cell differentiation into Ig-secreting cells (ISCs) remains unclear. In this study, we find that upon isolation human naive and memory B (MB) cells have prebound BAFF on their surface, whereas germinal center (GC) B cells lack detectable levels of prebound BAFF. We attribute their lack of prebound BAFF to cell activation, because we demonstrate that stimulation of naive and MB cells results in the loss of prebound BAFF. Furthermore, the absence of prebound BAFF on GC B cells is not related to a lack of BAFF-binding receptors or an inability to bind exogenous BAFF. Instead, our data suggest that accessibility to soluble BAFF is limited within GCs, perhaps to prevent skewing of the conventional B cell differentiation program. In support of this concept, whereas BAFF significantly enhances ISC differentiation in response to T cell-dependent activation, we report for the first time the ability of BAFF to considerably attenuate ISC differentiation of MB cells in response to CpG stimulation, a form of T cell-independent activation. Our data suggest that BAFF may be providing regulatory signals during specific T cell-independent events, which protect the balance between MB cells and ISCs outside GCs. Taken together, these data define a complex role for BAFF in humoral immune responses and show for the first time that BAFF can also play an inhibitory role in B cell differentiation.

A role for BLyS in the activation of innate immune cells.

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) ligand superfamily. Although BLyS costimulates adaptive immune cells, the ability of BLyS to stimulate innate immune cells has not been described. Here, we show that BLyS strongly induces human monocyte survival, and activation as measured by proinflammatory cytokine secretion and up-regulation of costimulatory molecule expression. In addition, monocytes cultured with BLyS differentiated into macrophage-like cells. Regarding BLyS receptor(s) expression, freshly isolated monocytes bound low levels of exogenous BLyS and expressed primarily intracellular TACI, and cell surface TACI levels increased following monocyte activation. Of interest, bone marrow monocytes from some multiple myeloma patients expressed significant levels of cell surface TACI at isolation. Our findings indicate that BLyS plays a role in activating innate immune cells. Moreover, this study may explain more clearly why high BLyS production is often correlated with certain inflammatory autoimmune diseases and B-lymphocyte malignancies.

Proto-oncogene PML enhances antigen presentation by MHC class I molecules in human lung cancer cells.

Deficient expression of the mouse proto-oncogene PML is associated with tumor immune evasion, occurring through down-regulated expression of genes involved in antigen processing and presentation. We investigated whether the defective antigen presentation found in human lung cancer cells could be restored by the human homolog of PML. PML induced the expression of MHC class I heavy chain and beta2-microglobulin at the level of transcription, thereby restoring defective antigen presentation resolved in some, but not all, lung cancer cell lines. Furthermore, the capacity of PML to restore antigen presentation required its targeting into nuclear bodies. These findings might have important application in the development of antitumor immunotherapeutic strategies.