T cell lessons from the rheumatoid arthritis synovium SCID mouse model: CD3-rich synovium lacks response to CTLA-4Ig but is successfully treated by interleukin-17 neutralization.

OBJECTIVE: To provide an intermediate step between classic arthritis models and clinical trials, the rheumatoid arthritis (RA) synovium SCID mouse model is a valuable tool for use during preclinical research. We undertook this study to investigate the validity of this humanized mouse model using anti-tumor necrosis factor (anti-TNF) and anti-interleukin-1 (anti-IL-1) treatment and to investigate the direct effect of T cells- and B cell-related therapies on the transplanted RA synovial tissue. METHODS: CB17/SCID mice were engrafted with human RA synovial tissue and systemically treated with anti-TNF, anti-IL-1, anti-IL-17, CTLA-4Ig, anti-CD20, or isotype control antibodies. RESULTS: Validation of the model with anti-TNF treatment significantly reduced serum cytokine levels and decreased histologic inflammation, whereas anti-IL-1 therapy did not show any effect on the RA synovial grafts. In mice engrafted with B cell-rich synovial tissue, anti-CD20 treatment showed clear therapeutic effects. Surprisingly, CTLA-4Ig treatment did not show any effects in this transplantation model, despite prescreening of the synovial tissue for the presence of CD3+ T cells and the costimulatory molecules CD80 and CD86. In contrast, great therapeutic potential was observed for anti-IL-17 treatment, but only when CD3+ T cells were abundantly present in the RA synovial tissue. CONCLUSION: This human RA synovium SCID mouse model enabled us to show that CTLA-4Ig lacks direct effects on T cell activation processes in the synovial tissue. Further evidence was obtained that IL-17 might indeed be an interesting therapeutic target in RA patients with CD3-rich synovial tissue. Further characterization of the RA patients' individual synovial profiles is of great importance for achieving tailored therapy.

Discovery of selective and orally available spiro-3-piperidyl ATP-competitive MK2 inhibitors.

The identification of a potent, selective, and orally available MK2 inhibitor series is described. The initial absence of oral bioavailability was successfully tackled by moving the basic nitrogen of the spiro-4-piperidyl moiety towards the electron-deficient pyrrolepyridinedione core, thereby reducing the pK(a) and improving Caco-2 permeability. The resulting racemic spiro-3-piperidyl analogues were separated by chiral preparative HPLC, and the activity towards MK2 inhibition was shown to reside mostly in the first eluting stereoisomer. This led to the identification of new MK2 inhibitors, such as (S)-23, with low nanomolar biochemical inhibition (EC(50) 7.4 nM) and submicromolar cellular target engagement activity (EC(50) 0.5 µM).

Therapeutic antibody targeting of CD97 in experimental arthritis: the role of antigen expression, shedding, and internalization on the pharmacokinetics of anti-CD97 monoclonal antibody 1B2.

CD97 is a member of the EGF-TM7 family of adhesion class receptors, with a proposed role in inflammatory cell recruitment. Neutralization of murine CD97 with the anti-mCD97 mAb 1B2 was efficacious in prevention of murine collagen-induced arthritis, a model with features resembling rheumatoid arthritis. Here, the therapeutic potential of neutralizing CD97 in arthritis was studied with emphasis on the 1B2 pharmacokinetics. Mice with established arthritis were treated with anti-mCD97 or anti-TNF-alpha serum. Ab pharmacokinetics and biodistribution were studied in diseased and nondiseased mice using labeled 1B2. The impact of CD97 expression on Ab pharmacokinetics was studied using CD97 knockout mice. Treatment with 1B2 showed an efficacy comparable to anti-TNF-alpha treatment. Pharmacokinetic analysis of 1B2 in wild-type and CD97 knockout mice indicated a dose-dependent Ab clearance, due to specific interaction with CD97. Biodistribution studies showed accumulation of 1B2 in spleen and lung. In vitro studies using murine splenocytes revealed that CD97 when bound to Ab was internalized. Moreover, soluble CD97 was detected in the supernatant, suggesting Ag shedding. Finally, in arthritic mice, higher levels of soluble CD97 were found and 1B2 treatment led to specific targeting of inflamed paws, resulting in a higher clearance rate of 1B2 in arthritic mice than in wild-type mice. In conclusion, our data support a therapeutic value of CD97 neutralization in experimental arthritis. The pharmacokinetic profile of the 1B2 Ab illustrates the complexity of Ab elimination from an organism and stresses the importance of understanding Ag-Ab interactions when developing therapeutic mAbs.

Biophysical and Immunological Characterization and In Vivo Pharmacokinetics and Toxicology in Nonhuman Primates of the Anti-PD-1 Antibody Pembrolizumab.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.

APRIL signaling via TACI mediates immunosuppression by T regulatory cells in multiple myeloma: therapeutic implications.

We investigate here how APRIL impacts immune regulatory T cells and directly contributes to the immunosuppressive multiple myeloma (MM) bone marrow (BM) microenvironment. First, APRIL receptor TACI expression is significantly higher in regulatory T cells (Tregs) than conventional T cells (Tcons) from the same patient, confirmed by upregulated Treg markers, i.e., Foxp3, CTLA-4. APRIL significantly stimulates proliferation and survival of Tregs, whereas neutralizing anti-APRIL monoclonal antibodies (mAbs) inhibit these effects. Besides TACI-dependent induction of cell cycle progression and anti-apoptosis genes, APRIL specifically augments Foxp3, IL-10, TGFß1, and PD-L1 in Tregs to further enhance Treg-inhibited Tcon proliferation. APRIL further increases MM cell-driven Treg (iTreg) via TACI-dependent proliferation associated with upregulated IL-10, TGFß1, and CD15s in iTreg, which further inhibits Tcons. Osteoclasts producing APRIL and PD-L1 significantly block Tcon expansion by iTreg generation, which is overcome by combined treatment with anti-APRIL and anti-PD1/PD-L1 mAbs. Finally, APRIL increases IL-10-producing B regulatory cells (Bregs) via TACI on BM Bregs of MM patients. Taken together, these results define novel APRIL actions via TACI on Tregs and Bregs to promote MM cell survival, providing the rationale for targeting APRIL/TACI system to alleviate the immunosuppressive BM milieu and improve patient outcome in MM.

Bacillus Calmette-Guérin-Induced Trained Immunity Is Not Protective for Experimental Influenza A/Anhui/1/2013 (H7N9) Infection in Mice.

Avian influenza A of the subtype H7N9 has been responsible for almost 1,600 confirmed human infections and more than 600 deaths since its first outbreak in 2013. Although sustained human-to-human transmission has not been reported yet, further adaptations to humans in the viral genome could potentially lead to an influenza pandemic, which may have severe consequences due to the absence of pre-existent immunity to this strain at population level. Currently there is no influenza A (H7N9) vaccine available. Therefore, in case of a pandemic outbreak, alternative preventive approaches are needed, ideally even independent of the type of influenza virus outbreak. Bacillus Calmette-Guérin (BCG) is known to induce strong heterologous immunological effects, and it has been shown that BCG protects against non-related infection challenges in several mouse models. BCG immunization of mice as well as human induces trained innate immune responses, resulting in increased cytokine responses upon subsequent ex vivo peripheral blood mononuclear cell restimulation. We investigated whether BCG (Statens Serum Institut-Denmark)-induced trained immunity may protect against a lethal avian influenza A/Anhui/1/2013 (H7N9) challenge. Here, we show that isolated splenocytes as well as peritoneal macrophages of BCG-immunized BALB/c mice displayed a trained immunity phenotype resulting in increased innate cytokine responses upon ex vivo restimulation. However, after H7N9 infection, no significant differences were found between the BCG immunized and the vehicle control group at the level of survival, weight loss, pulmonary influenza A nucleoprotein staining, or histopathology. In conclusion, BCG-induced trained immunity did not result in protection in an oseltamivir-sensitive influenza A/Anhui/1/2013 (H7N9) challenge mouse model.

Corrigendum: Bacillus Calmette-Guérin-Induced Trained Immunity Is Not Protective for Experimental Influenza A/Anhui/1/2013 (H7N9) Infection in Mice.

[This corrects the article DOI: 10.3389/fimmu.2018.00869.].

DHEA metabolism in arthritis: a role for the p450 enzyme Cyp7b at the immune-endocrine crossroad.

For dehydroepiandrosterone (DHEA) both immunosuppressive and immuno-stimulating properties have been described. The immunosuppressive effects may be explained by the conversion of DHEA into androgens and/or estrogens. The described immuno-stimulating effects of DHEA may be due to the conversion of DHEA into 7alpha-hydroxy-DHEA (7alpha-OH-DHEA) by the activity of the p450 enzyme, Cyp7b. 7alpha-OH-DHEA is thought to have anti-glucocoticoid activity preventing the anti-inflammatory action of endogenous glucocorticoids. To investigate a putative role of Cyp7b in the arthritic process, tissues from both the murine collagen-induce arthritis (CIA) model and from patients with rheumatoid arthritis (RA) were studied. We determined the Cyp7b expression levels in synovial tissue and the level of 7alpha-OH-DHEA in both serum and arthritic joints of mice with CIA. Our studies showed that the severity of arthritis correlates with increased Cyp7b activity. Next, we investigated Cyp7b expression and activity in RA patients where the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) are known to control the disease process. Fibroblast-like synoviocytes (FLS), isolated from RA synovial biopsies were found to express Cyp7b mRNA. In addition, Cyp7b enzymatic activity was detected in these cells. We also investigated whether Cyp7b activity is regulated by cytokines. Proinflammatory (e.g., TNF-alpha and IL-1beta) cytokines were found to stimulate Cyp7b activity and the anti-inflammatory cytokine transforming growth factor-beta (TGF-beta) was found to suppress Cyp7b activity in FLS. Next, we studied which signal transduction pathway is involved in the TNF-alpha-mediated induction of Cyp7b activity in human FLS. The results show a role for nuclear factor kappa B (NFkappaB) and activator protein-1 (AP-1) in the regulation of Cyp7b expression. Finally, we established that the effects of DHEA or 7alpha-OH-DHEA on the immune system can not be explained by glucocorticoid receptor (GR) engagement. The role of the p450 enzyme Cyp7b in DHEA metabolism and its relevance in the arthritic process will be discussed.

Exposure to Candida albicans polarizes a T-cell driven arthritis model towards Th17 responses, resulting in a more destructive arthritis.

BACKGROUND: Fungal components have been shown very effective in generating Th17 responses. We investigated whether exposure to a minute amount of C. albicans in the arthritic joint altered the local cytokine environment, leading to enhanced Th17 expansion and resulting in a more destructive arthritis. METHODOLOGY: Chronic SCW arthritis was induced by repeated injection with Streptococcus pyogenes (SCW) cell wall fragments into the knee joint of C57Bl/6 mice, alone or in combination with the yeast of C. albicans or Zymosan A. During the chronic phase of the arthritis, the cytokine levels, mRNA expression and histopathological analysis of the joints were performed. To investigate the phenotype of the IL-17 producing T-cells, synovial cells were isolated and analyzed by flowcytometry. PRINCIPAL FINDINGS: Intra-articular injection of either Zymosan A or C. albicans on top of the SCW injection both resulted in enhanced joint swelling and inflammation compared to the normal SCW group. However, only the addition of C. albicans during SCW arthritis resulted in severe chondrocyte death and enhanced destruction of cartilage and bone. Additionally, exposure to C. albicans led to increased IL-17 in the arthritic joint, which was accompanied by an increased synovial mRNA expression of T-bet and RORγT. Moreover, the C. albicans-injected mice had significantly more Th17 cells in the synovium, of which a large population also produced IFN-γ. CONCLUSION: This study clearly shows that minute amounts of fungal components, like C. albicans, are very potent in interfering with the local cytokine environment in an arthritic joint, thereby polarizing arthritis towards a more destructive phenotype.

PD-1 blockade augments Th1 and Th17 and suppresses Th2 responses in peripheral blood from patients with prostate and advanced melanoma cancer.

Negative costimulation on T cells is exploited by both prostate cancer and melanoma to evade antitumor immunity. Blocking such mechanisms restores antitumor immunity as was demonstrated by the improved survival of patients with metastatic melanoma after treatment with an antibody blocking the CTLA-4 inhibitory receptor (ipilimumab). Enhanced expression of another inhibitory immunoreceptor, programmed death-1 (PD-1), and its ligand, PD-L1, was found to correlate with a poor prognosis in prostate cancer and melanoma. PD-1-blocking antibodies are being developed to modulate antitumor immune responses. To support preclinical and clinical development of anti-PD-1 therapy, we sought to develop biomarker assays that can detect the effect of PD-1-blocking agents in whole blood and peripheral blood mononuclear cells. In this study, we assessed the effect of PD-1 blockade in modulating super antigen (staphylococcus enterotoxin B)-induced and recall antigen (tetanus toxoid)-induced T-cell reactivity in vitro using whole blood and peripheral blood mononuclear cells from patients with advanced melanoma, prostate cancer, and healthy controls. PD-1 blockade was found to shift antigen-induced cellular reactivity toward a proinflammatory Th1/Th17 response, as evidenced by enhanced production of interferon γ, interleukin (IL)-2, tumor necrosis factor α, IL-6, and IL-17 and reduced production of the Th2 cytokines IL-5 and IL-13. It is interesting to note that suppression of Th2 responsivity was seen with whole blood cells only from patients with cancer. Taken together, we identified novel biomarker assays that might be used to determine the functional consequences of PD-1 blockade in peripheral blood cells from patients with cancer. How these assays translate to the local antitumor response remains to be established in a clinical setting.