Nucleotide sequence encoding coTCRcys.

Differentiation status of T cells from an NPC patient transduced with the coTCRcys receptor.

Minimal mispairing with the exogenous TCR b chain shown using Mock- and WT TCR-transduced T-cells from an NPC patient.

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts.

Abstract Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)–associated posttransplant lymphomas, and more limited success has been reported using this approach to treat a far more common EBV-associated malignancy, nasopharyngeal carcinoma (NPC). However, current approaches using EBV-transformed lymphoblastoid cell lines to reactivate EBV-specific T cells for infusion take 2 to 3 months of in vitro culture and favor outgrowth of T cells targeting viral antigens expressed within EBV+ lymphomas, but not in NPC. Here, we explore T-cell receptor (TCR) gene transfer to rapidly and reliably generate T cells specific for the NPC-associated viral protein LMP2. We cloned a human leukocyte antigen (HLA) A*1101-restricted TCR, which would be widely applicable because 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms, we have optimized expression of the TCR and demonstrated high-avidity antigen-specific function (proliferation, cytotoxicity, and cytokine release) in both CD8+ and CD4+ T cells. The engineered T cells also inhibited LMP2+ epithelial tumor growth in a mouse model. Furthermore, transduced T cells from patients with advanced NPC lysed LMP2-expressing NPC cell lines. Using this approach, within a few days large numbers of high-avidity LMP2-specific T cells can be generated reliably to treat NPC, thus providing an ideal clinical setting to test TCR gene transfer without the risk of autoimmunity through targeting self-antigens. Cancer Immunol Res; 3(10); 1138–47. ©2015 AACR.

The substantial T lymphocyte infiltrate found in cases of nasopharyngeal carcinoma (NPC) has been implicated in the promotion of both tumor growth and immune escape. Conversely, because malignant NPC cells harbor the Epstein-Barr virus, this tumor is a candidate for virus-specific T cell-based therapies. Preventing the accumulation of tumor-promoting T cells or enhancing the recruitment of tumor-specific cytotoxic T cells offers therapeutic potential. However, the mechanisms involved in T cell recruitment to this tumor are poorly understood. Comparing memory T cell subsets that have naturally infiltrated NPC tissue with their counterparts from matched blood revealed enrichment of CD8+, CD4+, and regulatory T cells expressing the chemokine receptor CXCR6 in tumor tissue. CD8+ and (nonregulatory) CD4+ T cells also were more frequently CCR5+ in tumor than in blood. Ex vivo studies demonstrated that both receptors were functional. CXCL16 and CCL4, unique chemokine ligands for CXCR6 and CCR5, respectively, were expressed by the malignant cells in tumor tissue from the majority of NPC cases, as was another CCR5 ligand, CCL5. The strongest expression of CXCL16 was found on tumor-infiltrating cells. CCL4 was detected on the tumor vasculature in a majority of cases. These findings suggest that CXCR6 and CCR5 play important roles in T cell recruitment and/or retention in NPC and have implications for the pathogenesis and treatment of this tumor.

Background: Evidence suggests that some patients with renal cell carcinoma (RCC) respond to immunomodulatory therapies that activate T lymphocytes. A prerequisite for effective T cell therapy is efficient targeting of effector T cells to the tumour site, yet the molecular basis of T cell recruitment to RCC is unknown. Furthermore, some T cells that naturally infiltrate this cancer are regulatory T cells (Tregs) that may suppress antitumour immune responses.Objective: Determine the mechanisms of effector and regulatory T cell recruitment to RCC to allow targeted therapy that promotes local anti-tumour immunity.Design, setting, and participants: Tumour-infiltrating and peripheral blood T cells were collected from 70 patients undergoing nephrectomy for RCC.Measurements: T cells were analysed by multicolour flow cytometry for expression of 19 chemokine receptors and 7 adhesion molecules. Receptors that were expressed at higher levels on tumour-infiltrating lymphocytes (TILs) compared with matched peripheral blood lymphocytes (PBLs) were analysed further for their ability to mediate migration responses in TILs and for expression of corresponding ligands in tumour tissue.Results and limitations: Three chemokine receptors-CCR5, CXCR3, and CXCR6-were significantly overexpressed on TILs compared with matched PBLs (n = 16 cases) and were capable of promoting migration in vitro. Their corresponding ligands CCL4-5, CXCL9-11, and CXCL16 were all detected in RCC tissue. However, since they were present in all cases studied, it was not possible to correlate ligand expression with levels of T cell infiltration. Foxp3+ Tregs were enriched within TILs compared with matched PBLs and expressed high levels of CCR5, CXCR3, and CXCR6, as well as CCR6, the ligand for which (CCL20) was detectable in RCC tissue.Conclusions: Our data support a role for CCR5, CXCR3, and CXCR6 in the selective recruitment of T cells into RCC tissue and, together with CCR6, in the recruitment of Tregs. (C) 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.

The substantial T lymphocyte infiltrate found in cases of nasopharyngeal carcinoma (NPC) has been implicated in the promotion of both tumor growth and immune escape. Conversely, because malignant NPC cells harbor the Epstein-Barr virus, this tumor is a candidate for virus-specific T cell-based therapies. Preventing the accumulation of tumor-promoting T cells or enhancing the recruitment of tumor-specific cytotoxic T cells offers therapeutic potential. However, the mechanisms involved in T cell recruitment to this tumor are poorly understood. Comparing memory T cell subsets that have naturally infiltrated NPC tissue with their counterparts from matched blood revealed enrichment of CD8 , CD4 , and regulatory T cells expressing the chemokine receptor CXCR6 in tumor tissue. CD8 and (nonregulatory) CD4 T cells also were more frequently CCR5 in tumor than in blood. Ex vivo studies demonstrated that both receptors were functional. CXCL16 and CCL4, unique chemokine ligands for CXCR6 and CCR5, respectively, were expressed by the malignant cells in tumor tissue from the majority of NPC cases, as was another CCR5 ligand, CCL5. The strongest expression of CXCL16 was found on tumor-infiltrating cells. CCL4 was detected on the tumor vasculature in a majority of cases. These findings suggest that CXCR6 and CCR5 play important roles in T cell recruitment and/or retention in NPC and have implications for the pathogenesis and treatment of this tumor. (Am J Pathol 2012, 180:1215–1222; DOI: 10.1016/j.ajpath.2011.11.032) Undifferentiated nasopharyngeal carcinoma (NPC) is one of the most common cancers in Southeast Asia, with an incidence of up to 28 cases per 100,000 men in regions of southern China. Radiotherapy alone or with chemotherapy cures the majority of NPC cases, although the proximity of the tumor to radiosensitive organs frequently results in complications that reduce quality of life. Furthermore, half of all patients present with advanced disease (stage III–IV) where 5-year disease-specific survival rates are only 72%. The malignant cells of NPC are associated with a sizeable lymphoid stroma mainly consisting of / TCR T cells, with both CD4 and CD8 subsets present in variable proportions. It has been suggested that these tumor infiltrating lymphocytes (TIL) may be required for tumor growth, possibly through intercellular signaling via CD40 ligand, CD27, and/or CD28 on TIL engaging corresponding receptors/ligands (CD40, CD70, CD80/86) on the malignant cells. Alternatively, TIL may represent an ongoing immune response to the tumor, possibly targeting Epstein-Barr virus (EBV) proteins that are expressed in the malignant cells. However, at least 10% of NPC TIL are of a regulatory T cell (Treg) phenotype (CD4 CD25highFoxp3 ). Tregs suppress immune responses through cytokine release and/or cell–cell contact and have been detected in many human cancers where they may promote immune escape by tumor cells. Thus, TIL within NPC may play a role in tumor growth and/or immune escape. Preventing the accumulation of these cells within the tumor could therefore have therapeutic potential. Alternatively, ensuring efficient delivery of tumor-specific cytotoxic T cells from the circulation to the tumor tissue could overcome immune evasion mechanisms and destroy malignant cells. Indeed, NPC is considered a good candidate for T cell–based therapies and

Undifferentiated nasopharyngeal carcinoma (NPC) is a very good candidate disease for treatment with adoptive T cell transfer. Uniformly Epstein-Barr virus (EBV)+, these tumours reportedly have functional antigen processing and presentation machinery and express viral proteins that contain known cytolytic T cell target epitopes. This raises the possibility that boosting relevant EBV-specific T cell immunity in NPC patients might defeat the tumour. The persistent nature of EBV infection may also encourage the long-term survival of therapeutically administered virus-specific cells. However, efficient delivery of tumour-specific T cells from the circulation to solid tumour tissue is a clear requirement for effective cellular therapy, yet the mechanisms by which T cells gain entry to NPC tumours have not yet been determined. The malignant cells of NPC are usually associated with a substantial lymphoid infiltrate mainly consisting of T cells. Using unmanipulated diagnostic biopsy samples, we have characterised chemokine receptor expression (CR) on tumour-infiltrating T cells, and established whether specific chemokine ligands are detectable at the NPC tumour site. We found that functional CXCR6 and CCR5 were expressed on tumour infiltrating T cells, consistent with the presence of specific ligands for these receptors at the tumour site. Furthermore, our data suggests that effector and regulatory cells may use shared homing mechanisms to gain entry to NPC tumours.

OBJECTIVE High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts. METHODS Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor alpha (TNFalpha), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-kappaB activation assay. RESULTS Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3-induced secretion of TNFalpha, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFalpha blockade ruled out autocrine TNFalpha-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-kappaB activation was required for production of both IL-6 and CCL5. CONCLUSION Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell-recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.

Epstein-Barr Virus (EBV)-positive post-transplant lymphoproliferative disease (PTLD) can be successfully treated by infusing T cells reactivated in vitro with EBV-transformed B lymphoblastoid cell lines (LCLs). T cell lines generated in this way are dominated by reactivities to the EBNA 3 viral proteins. However, such an approach may not be appropriate to treat other more common EBV-associated malignancies since, unlike PTLD, these tumours do not express the immunodominant EBNA 3 proteins; instead viral protein expression is restricted to relatively weak antigens. Furthermore, generating such T cell lines can take many weeks of in vitro culture. T cell receptor (TCR) gene transfer offers an alternative approach whereby T cells of defined specificity can be generated rapidly and reliably from all patients. We are exploring this method to treat undifferentiated Nasopharyngeal carcinoma (NPC), a tumour that occurs at high frequency in Southeast Asia (incidence >20/100,000 population) and where >50% of cases express the EBV protein LMP2. To ensure TCR gene transfer could be widely applicable to treat this cancer we have focussed on the T cell response to an LMP2 epitope that is restricted through HLA A*1101, an allele carried by >50% of the Chinese population. Furthermore the target epitope sequence is conserved in EBV strains present in NPC tumours. Having generated a high avidity T cell clone specific for this epitope the genes encoding the TCR a and b chain were cloned into the same MP71 retroviral vector, separated by a self-cleaving 2A peptide to ensure equal expression. Retrovirus was generated using Phoenix A packaging cells and following stimulation with anti-CD3 antibody, T cells from five A*1101+ donors (including an NPC patient) were transduced with this virus. Three days later, HLA:peptide pentamer staining detected the transferred TCR in 12-17% of CD8+ T cells. Staining for the b chain indicated that mispairing with endogenous a chains can occur in 0-8% of T cells. The TCR-transduced T cells expanded rapidly in vitro in response to antigen, showed high avidity for the target peptide (10-10M), and lysed autologous fibroblasts expressing LMP2. Significantly, they also recognised EBV-transformed LCLs which express physiological levels of LMP2 in an HLA A*1101-restricted manner. Preliminary data indicate that some CD4+ T cells transduced with this TCR also recognised A11.01+ LCLs suggesting they might provide a helper function in vivo to aid persistence of LMP2-specific CD8+ effectors.

Fibroblast-like synoviocytes (FLS) are potentially directly involved in the propagation of inflammation. We have previously shown evidence of an expanded activated population of natural killer (NK) cells in spondylarthritis (SpA) patients. In the present study, we sought to determine whether the interaction between NK cells and FLS from SpA patients results in a proinflammatory response. Autologous NK cells and FLS were obtained from 6 patients with SpA, 4 patients with rheumatoid arthritis (RA), and 8 patients with osteoarthritis (OA). Physical interactions between NK cells and FLS were studied by time-lapse phase-contrast microscopy. Fluorescence-activated cell sorting was used to study the activation, proliferation, and survival of NK cells in contact with FLS. Cytokine and stromal factor production were measured by a multiple cytokine bead assay. NK cells both adhered to and migrated beneath the FLS monolayer (pseudoemperipolesis). FLS from SpA and RA patients supported increased pseudoemperipolesis, activation, cytokine production, and survival of NK cells. The production of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, IL-1β, and IL-15, was increased in cocultures of NK cells and FLS, particularly in those from RA and SpA patients. Production of interferon-γ, RANTES, and matrix metalloproteinase 3 (MMP-3) by NK cell and FLS coculture was greatest in SpA patients. Surface expression of IL-15 on FLS was significantly increased in SpA and RA patients, but not OA patients. Blockade with an IL-15 monoclonal antibody resulted in increased apoptosis of NK cells. FLS promote the migration, activation, and survival of NK cells. The interaction of NK cells with FLS results in increased IL-15 expression by FLS and the production of proinflammatory chemokines, cytokines, and MMPs, which may contribute to joint inflammation. This response was much more marked in SpA and RA patients as compared with OA patients.

Objective. Fibroblast-like synoviocytes (FLS) are potentially directly involved in the propagation of inflammation. We have previously shown evidence of an expanded activated population of natural killer (NK) cells in spondylarthritis (SpA) patients. In the present study, we sought to determine whether the interaction between NK cells and FLS from SpA patients results in a proinflammatory response.Methods. Autologous NK cells and FLS were obtained from 6 patients with SpA, 4 patients with rheumatoid arthritis (RA), and 8 patients with osteoarthritis (OA). Physical interactions between NK cells and FLS were studied by time-lapse phase-contrast microscopy. Fluorescence-activated cell sorting was used to study the activation, proliferation, and survival of NK cells in contact with FLS. Cytokine and stromal factor production were measured by a multiple cytokine bead assay.Results. NK cells both adhered to and migrated beneath the FLS monolayer (pseudoemperipolesis). FLS from SpA and RA patients supported increased pseudoemperipolesis, activation, cytokine production, and survival of NK cells. The production of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, IL-1 beta, and IL-15, was increased in cocultures of NK cells and FLS, particularly in those from RA and SpA patients. Production of interferon-gamma, RANTES, and matrix metalloproteinase 3 (MMP-3) by NK cell and FLS coculture was greatest in SpA patients. Surface expression of IL-15 on FLS was significantly increased in SpA and RA patients, but not OA patients. Blockade with an IL-15 monoclonal antibody resulted in increased apoptosis of NK cells.Conclusion. FLS promote the migration, activation, and survival of NK cells. The interaction of NK cells with FLS results in increased IL-15 expression by FLS and the production of proinflammatory chemokines, cytokines, and MMPs, which may contribute to joint inflammation. This response was much more marked in SpA and RA patients as compared with OA patients.