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.

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, TGFβ1, 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, TGFβ1, 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.

Background and purpose: The p38 kinase regulates the release of proinflammatory cytokines including tumour-necrosis factor-a (TNFa) and is regarded as a potential therapeutic target in rheumatoid arthritis (RA). Using the novel p38 inhibitor Org 48762-0, we investigated the therapeutic potential of p38 inhibition and compared this to anti-mouse (m)TNFa antibody treatment in murine collagen-induced arthritis (CIA). Experimental approach: Pharmacological profiles of Org 48762-0 were characterized in kinase assays, cellular assays and in lipopolysaccharide (LPS)-induced inflammation in mice. The effects of Org 48762-0 and of mTNFa-neutralization on established arthritis were examined in murine CIA. Key results: Org 48762-0 potently inhibited p38a kinase with a high degree of kinase selectivity. In cellular assays, Org 48762-0 reduced LPS-induced TNFa release. Oral administration of Org 48762-0 in mice showed drug-like pharmacokinetic properties and inhibited LPS-induced cytokine production. These pharmacological characteristics of Org 48762-0 prompted a comparison of therapeutic efficacy with mTNFa-neutralization in CIA. Org 48762-0 and anti-mTNFa antibody treatment equally inhibited development of arthritis when evaluated macroscopically. Radiological analyses revealed protection against bone damage for both treatments, although statistical difference was reached with Org 48762-0 treatment only. Further, micro-computed tomographical and histopathological analyses confirmed the protective effects of Org 48762-0 on joint damage. Conclusions and implications: Pharmacological targeting of p38 kinase provided good protection against joint tissue damage in CIA. In our experiments, neutralization of mTNFa produced less prominent suppression of bone damage. Our data suggest a therapeutic potential for selective and potent p38 inhibitors in RA.

Objective. The cytochrome P450 enzyme CYP7B catalyzes the conversion of dehydroepiandrosterone (DHEA) into 7␣-hydroxy-DHEA (7␣-OH-DHEA). This metabolite can stimulate the immune response. We previously reported that the severity of murine collageninduced arthritis is correlated with CYP7B messenger RNA (mRNA) expression and activity in the arthritic joint. The purpose of this study was to investigate the presence of 7␣-OH-DHEA in synovial samples and the cytokine regulation of CYP7B activity in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).Methods. The presence of 7␣-OH-DHEA was examined in synovial biopsy tissues, synovial fluid, and serum by radioimmunoassay. The effect of cytokines on CYP7B mRNA expression and CYP7B activity in FLS was examined by determining the formation of the CYP7B enzyme product 7␣-OH-DHEA with the use of high-performance liquid chromatography.Results. The CYP7B enzyme product 7␣-OH-DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor ␣ (TNF␣), interleukin-1␣ (IL-1␣), IL-1␤, and IL-17 up-regulated CYP7B activity in an FLS cell line 2-10-fold. Enhanced CYP7B activity was correlated with an increase in CYP7B mRNA. The cytokine transforming growth factor ␤ inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNF␣ increased CYP7B activity in all cell lines tested.Conclusion. Exposure to the proinflammatory cytokines TNF␣, IL-1␣, IL-1␤, and IL-17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7␣-OH-DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.

Objective. The endogenous steroid dehydroepiandrosterone (DHEA) has been reported to play a role in rheumatoid arthritis (RA). DHEA is metabolized by the P450 enzyme CYP7B into 7␣-OH-DHEA, which has immunostimulating properties. This study was undertaken to investigate the putative role of CYP7B in arthritis using murine collagen-induced arthritis (CIA), an interleukin-1␤ (IL-1␤)-dependent model.Methods. DBA/1J mice were immunized and administered a booster with type II collagen. The presence of 7␣-OH-DHEA was determined in both arthritic and nonarthritic joints and the serum of CIA mice by radioimmunoassay. CYP7B messenger RNA (mRNA) expression was analyzed in synovial biopsy samples, and in fibroblast-like synoviocytes (FLS) isolated from these synovial biopsy samples, by reverse transcriptasepolymerase chain reaction (RT-PCR). In addition, the regulatory role of IL-1␤ on CYP7B activity in FLS was determined using RT-PCR, Western blotting, and highperformance liquid chromatography.Results. In knee joint synovial biopsy samples from arthritic mice, 7␣-OH-DHEA levels were 5-fold higher than in nonarthritic mice. Elevated levels of 7␣-OH-DHEA were accompanied by an increase in CYP7B mRNA expression and were positively correlated with disease severity. In serum, no differences in 7␣-OH-DHEA levels were observed between arthritic and nonarthritic mice. Incubation of FLS with IL-1␤ resulted in a dose-dependent increase in 7␣-OH-DHEA formation. In addition, IL-1␤ enhanced CYP7B mRNA and CYP7B protein levels in FLS.Conclusion. Disease progression in CIA is correlated with enhanced CYP7B activity, which leads to locally enhanced 7␣-OH-DHEA levels. Elevated IL-1␤ levels within the arthritic joint may regulate this increase in CYP7B activity.