Reduced human leukocyte antigen expression in advanced-stage Ewing sarcoma: implications for immune recognition.

Ewing sarcoma (EWS) is a tumour most commonly arising in bone, although on occasion in soft tissue, with a poor prognosis in patients with refractory or relapsed disease, despite multimodal therapy. Immunotherapeutic strategies based on tumour-reactive T and/or natural killer cells may improve the treatment of advanced-stage EWS. Since cellular immune recognition critically depends on human leukocyte antigen (HLA) expression, knowledge about HLA expression in EWS is crucial in the design of cellular immunotherapeutic strategies. Constitutive and IFNgamma-induced HLA class I expression was analysed in EWS cell lines (n = 6) by flow cytometry, using antibodies against both monomorphic and allele-specific antigens. Expression of antigen processing pathway components and beta-2 microglobulin (beta2m) was assessed by western blot. Expression of class II transactivator (CIITA), and its contribution to HLA class II expression, was evaluated by qRT-PCR, transduction assays, and flow cytometry. beta2m/HLA class I and class II expression was validated in EWS tumours (n = 67) by immunofluorescence. Complete or partial absence of HLA class I expression was observed in 79% of EWS tumours. Lung metastases consistently lacked HLA class I and sequential tumours demonstrated a tendency towards decreased expression upon disease progression. Together with absent or low constitutive expression levels of specific HLA class I loci and alleles, and differential induction of identical alleles by IFNgamma in different cell lines, these results may reflect the existence of an immune escape mechanism. Inducible expression of TAP-1/-2, tapasin, LMP-2/-7, and the beta2m/HLA class I complex by IFNgamma suggests that regulatory mechanisms are mainly responsible for heterogeneity in constitutive class I expression. EWSs lack IFNgamma-inducible HLA class II, due to lack of functional CIITA. The majority of EWS tumours, particularly if advanced-stage, exhibit complete or partial absence of both classes of HLA. This knowledge will be instrumental in the design of cellular immunotherapeutic strategies for advanced-stage EWS.

NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways.

INTRODUCTION: Ewing sarcoma (EWS) is a malignant bone-associated sarcoma, with poor prognosis in case of metastasis or relapse. To explore the feasibility of natural killer (NK) cell mediated immunotherapy and to identify molecular mechanisms involved, the susceptibility of EWS to NK cells was investigated. METHODS AND RESULTS: All EWS cell lines tested (n=7) were lysed by purified allogeneic NK cells from healthy donors, and the efficacy of lysis was increased by activating NK cells with interleukin-15 (IL-15). FACS analysis and immunohistochemistry revealed that EWS cell lines as well as primary tumor cells expressed ligands for the activating NK cell receptors NKG2D and DNAM-1. NK cell cytotoxicity to EWS cells critically depended on the combination of NKG2D and DNAM-1 signaling, since blocking either of these receptors abrogated lysis by resting NK cells. Cytokine-activated NK cells more efficiently recognized EWS cells, since only combined, but not single blockade of NKG2D and DNAM-1 by antibodies inhibited lysis of EWS cells. Induction or blockade of HLA class I on EWS cells did not significantly influence lysis. This suggests that predominantly activating, rather than inhibitory signals on EWS cells determined susceptibility to NK cell cytotoxicity. NK cell cytotoxicity to EWS cells and K562 was reduced in EWS patients at diagnosis (n=11) compared to age matched controls, despite normal NK cell numbers and increased expression of NKG2D. The impaired function of these NK cells was restored after activation with IL-15 in vitro. CONCLUSION: These results demonstrate that EWS cells are potentially susceptible to NK cell cytotoxicity due to the expression of activating NK cell receptor ligands. The use of cytokine-activated NK cells rather than resting NK cells in immunotherapy may be instrumental to optimize NK cell reactivity to EWS.

Expression of cellular FLICE inhibitory protein, caspase-8, and protease inhibitor-9 in Ewing sarcoma and implications for susceptibility to cytotoxic pathways.

PURPOSE: Ewing sarcoma is a common pediatric bone tumor with an unfavorable prognosis for metastatic or recurrent disease. Cellular immunotherapy may provide new treatment options and depends on the cytolytic death receptor and perforin/granzyme pathways. Expression of death receptor pathway inhibitor cellular FLICE inhibitory protein (cFLIP), initiator caspase-8, and granzyme B inhibitor protease inhibitor-9 (PI-9) have been reported to determine susceptibility to cell- and chemotherapy-mediated killing in several tumor types. Here, we have studied their in vitro and in vivo expression in Ewing sarcoma and the implications for susceptibility to cytotoxicity. EXPERIMENTAL DESIGN: Ewing sarcoma cell lines (n = 8) were tested for cFLIP, PI-9, and caspase-8 expression. Functional significance was tested by anti-Fas antibody (death receptor pathway) or natural killer cell (perforin/granzyme pathway) treatment. Immunohistochemistry was done on 28 sections from 18 patients. In half of the cases, sequential material, including metastases, was available. RESULTS: Although all tested Ewing sarcoma cell lines expressed cFLIP, resistance to CD95/Fas-mediated apoptosis was only observed in two cell lines lacking caspase-8 expression. PI-9 was expressed at low levels in four of eight Ewing sarcoma cell lines, but positive cell lines remained susceptible to perforin/granzyme-mediated killing. In primary Ewing sarcoma, including metastases, cFLIP was abundantly expressed in 18 of 18 patients. Caspase-8 was expressed in all patients but showed more intertumoral and intratumoral variation in both intensity and heterogeneity of staining. PI-9, in contrast, was undetectable. CONCLUSIONS: The expression patterns of cFLIP, caspase-8, and the absence of PI-9 provide a rationale to preferentially exploit the perforin/granzyme pathway in cytotoxic therapies against Ewing sarcoma.

Splenic suppressor of cytokine signaling 3 transgene expression affects T cell responses and prevents development of collagen-induced arthritis.

OBJECTIVE: Members of the suppressor of cytokine signaling (SOCS) family are key negative intracellular regulators of cytokine and growth factor responses, including those that regulate immune responses in autoimmune disorders, such as rheumatoid arthritis (RA). The aim of this study was to investigate modulation of T cell immunity for the treatment of experimental arthritis, via enhanced expression of SOCS-3 in splenic antigen-presenting cells (APCs) obtained after intravenous injection of adenovirus encoding SOCS-3. METHODS: DBA/1 mice were immunized with type II collagen, and adenovirus vectors were administered by intravenous injection before the clinical onset of collagen-induced arthritis (CIA). Splenic cellular responses were analyzed by measuring cytokine production, using Luminex multi-analyte technology. Th cell populations were analyzed by flow cytometry. RESULTS: Systemic delivery of adenovirus encoding SOCS-3 resulted in enhanced transgene expression in splenic APCs, which led to decreased production of interleukin-23 (IL-23), IL-6, and tumor necrosis factor alpha, but significantly higher production of antiinflammatory IL-10, by these cells. Fluorescence-activated cell sorting analysis showed increased numbers of splenic CD4+ T cells after SOCS-3 treatment. In the presence of SOCS-3-transduced APCs, however, purified splenic CD3+ T cells showed reduced antigen-specific proliferation and a significant reduction in the production of interferon-gamma (-43%), IL-4 (-41%), and IL-17 (-70%). Interestingly, the altered splenic cellular responses were accompanied by a protective effect on CIA development, and histologic analysis of knee joints showed reduced joint inflammation and connective tissue destruction. CONCLUSION: This study demonstrates effective prevention of CIA after intravenously induced overexpression of SOCS-3; this is probably caused by the generation of tolerogenic APCs, which have an inhibitory effect on Th1, Th2, and especially, Th17 cell activity.

Male IL-6 gene knock out mice developed more advanced osteoarthritis upon aging.

OBJECTIVE: Interleukin-6 (IL-6) is expressed in osteoarthritic joints but its function in osteoarthritis (OA) is unknown. To study this, spontaneous and experimental OA were evaluated in IL-6 deficient (IL-6(-/-)) mice. DESIGN: Histology of knees of 18-23-month-old wild type (wt) and IL-6(-/-) mice was compared for signs of OA. Cartilage proteoglycan (PG) density was measured by image analysis on safranin-O stained whole knee sections. Chondrocyte PG synthesis was measured ex vivo by (35)S-sulfate incorporation. Knee bone mineral density (BMD) was measured by dual energy x-ray absorptiometry. In young mice (3 months), OA was induced by intra-articular injection of collagenase. RESULTS: The incidence of extensive cartilage loss at both lateral and medial sides was markedly higher in old IL-6(-/-) males, but not in females, as compared to their wt controls. Compared to age-matched wt mice, reduced ex vivo PG synthesis was found during aging in IL-6(-/-) males, without affecting their cartilage PG density. IL-6(-/-) males showed more extensive extracellular matrix deposition in the collateral ligaments and subchondral bone sclerosis, predominantly at the medial side. Total knee BMD decreased more in IL-6(-/-) (-23%) than in wt (-10%) males during aging. Collagenase-induced OA showed a similar degree of joint pathology in both strains, implying that OA susceptibility was not different at younger age. CONCLUSIONS: Upon aging, IL-6(-/-) male mice developed more severe spontaneous OA. Reduced PG synthesis and BMD values might be indicative for an impaired repair response in IL-6(-/-) mice. This suggests a protective role for IL-6 in age-related OA in male mice.

Adenoviral transfer of murine oncostatin M elicits periosteal bone apposition in knee joints of mice, despite synovial inflammation and up-regulated expression of interleukin-6 and receptor activator of nuclear factor-kappa B ligand.

Oncostatin M (OSM) has been described as a bone-remodeling factor either stimulating osteoblast activity or osteoclast formation in vitro. To elucidate the in vivo effect of OSM on bone remodeling, we injected an adenoviral vector encoding murine OSM in knee joints of mice. OSM strongly induced interleukin (IL)-6 gene expression, a known mediator of osteoclast development. We investigated the OSM effect in wild-type and IL-6-deficient mice and found a similar degree of OSM-induced joint inflammation. Within the first week of inflammation, the periosteum along the femur and tibia increased in cell number and stained positive for the osteoblast marker alkaline phosphatase. At these sites bone apposition occurred in both strains as demonstrated by Goldner and Von Kossa staining. In vitro OSM enhanced the effect of bone morphogenetic protein-2 on osteoblast differentiation. Immunohistochemistry demonstrated expression of receptor activator of nuclear factor-kappa B ligand (RANKL) and its receptor, receptor activator of nuclear factor-kappa B (RANK), in the periosteum but osteoclasts were not detected at sites of bone apposition. Induced mRNA expression for the receptor activator of nuclear factor-kappa B ligand inhibitor osteoprotegerin probably controlled osteoclast development during OSM overexpression. Our results show that OSM favors bone apposition at periosteal sites instead of resorption in vivo. This effect was not dependent on or inhibited by IL-6.

Local activation of STAT-1 and STAT-3 in the inflamed synovium during zymosan-induced arthritis: exacerbation of joint inflammation in STAT-1 gene-knockout mice.

OBJECTIVE: STAT proteins play an important role in cytokine signaling. Some investigators have reported preferential activation of STAT-1, and others have reported preferential activation of STAT-3, in response to endogenous interleukin-6 (IL-6), in patients with rheumatoid arthritis. The present study was undertaken to investigate synovial STAT-1 and STAT-3 activation in an experimental animal model of arthritis. METHODS: Zymosan was injected intraarticularly into naive wild-type (WT), IL-6(-/-), and STAT-1(-/-) mice to induce arthritis. Western blots of synovial lysates were probed with phosphospecific antibodies to detect STAT-1/STAT-3 activation. Inflammation was assessed histologically. Synovial gene expression of the STAT-induced feedback inhibitors suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 in WT and STAT-1(-/-) mice was investigated by reverse transcriptase-polymerase chain reaction. RESULTS: STAT-3 was activated in inflamed synovium of WT mice throughout the course of disease, whereas activated STAT-1 was observed only during the chronic phase. In IL-6(-/-) mice, STAT activation was limited to STAT-3 on day 1. Although macrophage influx was not inhibited, disease went into remission after day 7 in IL-6(-/-) mice. STAT-1 deficiency resulted in exacerbation of chronic joint inflammation and granuloma formation. In STAT-1(-/-) mice, STAT-3 activation in the inflamed joints was unaltered as compared with WT mice. However, synovial SOCS-1, but not SOCS-3, gene expression was markedly reduced in STAT-1(-/-) mice. CONCLUSION: The results in the IL-6(-/-) mice suggest that STAT-3 is involved in the chronicity of ZIA. Exacerbation of arthritis in STAT-1(-/-) mice suggests an opposing effect of STAT-1, i.e., suppression of joint inflammation. The expression of SOCS-1 could be the underlying mechanism by which STAT-1 controls joint inflammation.

Growth plate damage, a feature of juvenile idiopathic arthritis, can be induced by adenoviral gene transfer of oncostatin M: a comparative study in gene-deficient mice.

OBJECTIVE: To investigate the involvement of proinflammatory and destructive mediators in oncostatin M (OSM)-induced joint pathology, using gene-deficient mice. METHODS: An adenoviral vector expressing murine OSM was injected into the joints of naive wild-type mice and mice deficient for interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha (TNFalpha), or inducible nitric oxide synthase (iNOS). Reverse transcription-polymerase chain reaction was used to study gene expression. Inflammation and cartilage proteoglycan (PG) depletion were assessed by histology. OSM and IL-1 levels in synovial fluid from patients with juvenile idiopathic arthritis (JIA) were measured by enzyme-linked immunosorbent assay. RESULTS: Adenoviral expression of murine OSM led to joint inflammation, bone apposition, chondrophyte formation, articular cartilage PG depletion, and VDIPEN neoepitope expression in wild-type mice. A unique and consistent observation was the focal PG depletion and disorganization of the growth plate cartilage during the first week of inflammation. Synovial IL-1beta, IL-6, TNFalpha, and iNOS gene expression was strongly induced. Of these factors, only deficiency in IL-1 markedly reduced inflammation and PG depletion and completely prevented growth plate damage. In addition, this is the first study in which OSM was detected in JIA synovial fluid. Most samples were also IL-1beta positive. CONCLUSION: IL-1, but not IL-6, TNFalpha, or iNOS, plays an important role in joint disease induced by intraarticular gene transfer of OSM in mice. The effect of OSM on murine connective tissue and the presence of OSM in human synovial fluid make involvement of OSM in human arthropathies very likely.