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Background & Aims Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice. Methods We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2−negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells. Results Peptide GPC3 367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3 367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3 367 multimer and secreted interferon-γ when cultured with GPC3 367 , but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8 + T cells that expressed the transgenic T-cell receptor specifically bound GPC3 367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice. Conclusions We identified a GPC3 367 -specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC. [ABSTRACT FROM AUTHOR]

Abstract: This review summarizes our studies of the past several years on the development of third generation dendritic cell (DC) vaccines. These developments have implemented two major innovations in DC preparation: first, young DCs are prepared within 3days and, second, the DCs are matured with the help of Toll-like receptor agonists, imbuing them with the capacity to produce bioactive IL-12 (p70). Based on phenotype, chemokine-directed migration, facility to process and present antigens, and stimulatory capacity to polarize Th1 responses in CD4+ T cells, induce antigen-specific CD8+ CTL and activate natural killer cells, these young mDCs display all the important properties needed for initiating good antitumor responses in a vaccine setting. [Copyright &y& Elsevier]

Background: Altered intakes of n-3 and n-6 polyunsaturated fatty acids were suggested to modulate allergic disease, but intervention trials yielded inconclusive results. Because allergies are primed in early infancy and in utero, the fetus might be more accessible to nutritional intervention strategies. Objective: We sought to investigate how supplementation of pregnant women with a fish oil (FO) preparation modulates allergy-related immune parameters in mothers and offspring. Methods: We performed a multicenter, randomized, double-blind, placebo-controlled trial. Three hundred eleven pregnant women received daily either FO with 0.5 g of docosahexaenoic acid and 0.15 g of eicosapentaenoic acid, 400 μg of methyl-tetra-hydrofolic acid, both, or placebo from the 22nd gestational week. TH1/TH2-related molecules were quantified in 197 maternal and 195 cord blood samples by using real-time RT-PCR. Data are given as geometric means [95% CIs]. Results: FO supplementation was associated with increased TGF-β mRNA in maternal (0.85 [0.8-0.89]; placebo: 0.68 [0.64-0.72]) and cord blood (0.85 [0.81-0.9]; placebo: 0.75 [0.71-0.79]). IL-1 (0.69 [0.66-0.73]; placebo: 0.83 [0.79-0.88]) and IFN-γ (0.54 [0.51-0.57]; placebo: 0.65 [0.61-0.69]) were decreased in mothers only (P < .001). Cord blood mRNA levels of IL-4 (0.54 [0.52-0.57]; placebo: 0.64 [0.61-0.68]), IL-13 (0.61 [0.58-0.65]; placebo: 0.85 [0.80-0.89]), CCR4 (0.70 [0.67-0.73]; placebo: 0.88 [0.84-0.92]; all P < .001), and natural killer (P < .001) and CCR3+CD8+ T cells (P < .04) were decreased in the FO group. Conclusion: Supplementation with FO during pregnancy is associated with decreased mRNA levels of TH2-related molecules in the fetus and decreased maternal inflammatory cytokines. We speculate that both effects are mediated by TGF-β. [Copyright &y& Elsevier]

Background: Asthma is characterized by a TH2 immune response. CD4+CD25hi regulatory T cells (Tregs) have been proposed to prevent allergic diseases through suppression of TH2 responses. Objective: We sought to investigate the role of CD4+CD25hi T cells in children with asthma. Methods: CD4+CD25hi Tregs and forkhead/winged-helix transcription factor FOXP3 mRNA levels were quantified in peripheral blood and bronchoalveolar lavage fluid (BALF) of 18 children with asthma, 10 children with chronic cough, and 13 control subjects without lung diseases. CD4+CD25hi T cells were isolated from peripheral blood and BALF of asthmatic patients and control subjects, and their capacity to suppress proliferation and cytokine/chemokine production of autologous responder T cells was analyzed. Results: CD4+CD25hi T cells were decreased in BALF of asthmatic children compared with values in children with cough or control subjects. In children with asthma, inhaled corticosteroid treatment was associated with increased percentages of CD4+CD25hi T cells in peripheral blood and BALF. Isolated BALF and peripheral blood CD4+CD25hi T cells from nonasthmatic subjects suppressed proliferation and cytokine/chemokine production by CD4+CD25− responder T cells. BALF CD4+CD25hi T cells from asthmatic subjects failed to suppress proliferation and production of TH2-associated cytokines and chemokines by CD4+CD25− responder T cells, which was restored after use of inhaled corticosteroids. Conclusion: These findings provide the first evidence that pulmonary CD4+CD25hi Tregs are impaired in pediatric asthma. Clinical implications: Pulmonary Tregs might represent a therapeutic target in pediatric asthma. [Copyright &y& Elsevier]

Background: Pseudomonas aeruginosa infection determines the course of cystic fibrosis (CF) lung disease. Studies in human peripheral blood indicate that P aeruginosa infection is associated with a predominant TH2 immune response, whereas TH1 responses are accompanied by a better pulmonary outcome. Objective: Analyses of peripheral blood may not correspond directly with the local pulmonary immune response. Therefore, we asked whether the TH1/TH2 response is altered in bronchoalveolar lavage fluid from P aeruginosa–infected patients with CF. Methods: Bronchoalveolar lavage fluid was obtained from 12 patients with CF chronically infected with P aeruginosa, 11 noninfected patients with CF, and 8 healthy controls. Pulmonary CXCR3+ (TH1) and CCR4+ (TH2) expressing CD4+ and CD8+ lymphocytes were quantified by flow cytometry. Levels of TH1-associated (IL-2, IFN-γ, IFN-γ inducible T cell-α chemoattractant, Monokine induced by IFN-γ, IFN-γ inducible protein of 10 kd) and TH2-associated (IL-4, IL-5, IL-10, thymus and activation-regulated chemokine [TARC], macrophage-derived chemokine) cytokines and chemokines and a panel of proinflammatory molecules were quantified at the protein level. Chemokines mRNA levels were assessed by real time RT-PCR. Results: P aeruginosa–infected patients with CF had significantly higher levels of pulmonary CCR4+CD4+ (TH2) cells, IL-4, IL-13, and TARC and lower levels of IFN-γ compared with noninfected patients with CF and healthy controls. Bronchoalveolar lavage fluid levels of IL-4, IL-13, and TARC correlated inversely with FEV1 in P aeruginosa–infected patients with CF. Conclusion: These results reveal the prevalence of a pulmonary TH2 immune response in P aeruginosa–infected patients with CF. The modulation of the pulmonary TH2 response in P aeruginosa infection may be an option for the treatment of P aeruginosa lung disease in patients with CF. [Copyright &y& Elsevier]

Background: Cough is a frequent symptom in children, but the differentiation of asthmatic cough from cough of other origins can be difficult. Chemokines recruit T lymphocytes to inflamed tissues, and the corresponding chemokine receptors are differentially expressed on TH1 and TH2 cells. Objective: We sought to determine whether levels of TH1/TH2-related chemokines and their receptors differ in bronchoalveolar lavage fluid (BALF) from 12 children with allergic asthma, 15 nonatopic children with chronic cough, and 10 children without airway disease. Methods: The TH1-related (IFN-γ–inducible protein of 10 kd [IP-10], IFN-γ–inducible T cell α chemoattractant [ITAC], monokine induced by IFN-γ [Mig], and IFN-γ) and TH2-related (thymus- and activation-regulated chemokine [TARC], macrophage-derived chemokine [MDC], IL-5, and IL-4) chemokines and cytokines were quantified in BALF by ELISA and a particle-based multiplex array. Percentages of pulmonary lymphocytes expressing CXCR3+ and CCR5+ (TH1) and CCR4+ and CCR3+ (TH2) chemokine receptors were determined in BALF by flow cytometry. Results: Pulmonary CCR4+CD4+ cells and levels of TARC and MDC were significantly increased in asthmatic children versus children with chronic cough or without airway disease. In asthmatic children CCR4+CD4+ cells correlated positively with levels of TARC, MDC, and serum IgE levels and negatively with FEV1. In contrast, CXCR3+CD8+ cells and levels of ITAC were significantly increased in children with non-atopic chronic cough compared with the other groups. In children with chronic cough, CXCR3+CD8+ cells correlated with levels of ITAC and IFN-γ. Conclusion: Pulmonary CCR4+CD4+ and CXCR3+CD8+ cells and their ligands TARC, MDC, and ITAC clearly differentiate asthmatic children from nonatopic children with chronic cough. The analysis of these markers could facilitate the diagnostic discrimination of asthma versus other reasons for chronic cough in children. [Copyright &y& Elsevier]

Summary: Proteolytic activity of the mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) paracaspase is required for survival of the activated B cell subtype of diffuse large B cell lymphoma (ABC-DLBCL). We have identified distinct derivatives of medicinal active phenothiazines, namely mepazine, thioridazine, and promazine, as small molecule inhibitors of the MALT1 protease. These phenothiazines selectively inhibit cleavage activity of recombinant and cellular MALT1 by a noncompetitive mechanism. Consequently, the compounds inhibit anti-apoptotic NF-κB signaling and elicit toxic effects selectively on MALT1-dependent ABC-DLBCL cells in vitro and in vivo. Our data provide a conceptual proof for a clinical application of distinct phenothiazines in the treatment of ABC-DLBCL. [ABSTRACT FROM AUTHOR]

Abstract: The immune response against prostate cancer seems to be inefficient although tumour cells show an over-expression of tumour-associated antigens suggesting that regulatory networks inhibit immune cell function locally. To address this proposition, lymphocytes within prostate cancer-inflicted tissue were analysed for the expression of markers associated with negative regulatory function and exhaustion. Prostate cancer, benign prostatic hyperplasia and healthy prostate tissues were investigated by immunohistology for CD25, FOXP3, PD-1 and B7-H1. We had previously documented that prostate cancer islets are surrounded by clustered accumulations of CD3+ lymphocytes, which lack perforin and interferon-gamma (IFNγ) expression, thus are apparently quiescent. Here, we report that these clusters contain numerous CD25+ and FOXP3+ cells. These markers are associated with regulatory T cells, and their presence in lymphocyte clusters near prostate cancer regions indicates an environment with negative impact on immune response against cancer cells. Consistent with this hypothesis, cells expressing PD-1 and its ligand B7-H1, which are markers associated with exhaustion of lymphocyte function, were also detected in the lymphocyte clusters. Expression of molecules associated with inhibition and exhaustion of lymphocytes may reflect events contributing to ineffective immune responses against cancer cells. [Copyright &y& Elsevier]

Abstract: Previous studies have analyzed transfer of RNA-encoded tumor-associated antigens (TAAs) into immature dendritic cells (DCs) because of their exceptional ability to internalize antigens. Concerns have been raised regarding the use of immature DCs in clinical studies because of their capacity to tolerize T cells. Therefore, we focused on optimizing RNA transfer into mature DCs using the method of electroporation and obtained high protein expression in 90% of mature DCs. Particular emphasis was placed on quantifying RNA transfer. Reconstitution of peptide–MHC (pMHC) ligands on RNA-pulsed DCs was measured with the help of effector-memory cytotoxic T lymphocytes (CTLs) specific for the melanoma-associated antigens tyrosinase and mutated cyclin-dependent kinase 4. In contrast to single-species RNA, transfer of native tumor-derived RNA or amplified tumor RNA into DCs resulted in very low or no capacity to reactivate these CTLs. A correlation was found between TAA message levels in tumor-derived RNA and pMHC ligand reconstitution on DCs. These results demonstrate that even TAAs with highly immunogenic mutated epitopes are not necessarily transferred when tumor-derived RNA is transfected into the DCs, thereby resulting in a lack of DC capacity to reactivate some preexisting effector-memory CTLs. [Copyright &y& Elsevier]

Summary: Th2 cells play a central role in type I allergies. However, the source of interleukin-4 which may lead to a Th1/Th2 imbalance is unknown. Vα24+CD161+ Natural killer T (NKT) cells secrete high amounts of interleukin-4 and/or interferon-γ and are assumed to participate in the initiation of Th1/Th2 immune responses. Their contribution to the development of Th2-dependent type I allergies is controversial.Our objective in this paper was to determine whether Vα24+CD161+ NKT cells differ in atopic and non-atopic adults.Venous blood was obtained from thirteen atopic and sixteen healthy adult probands. Vα24+CD161+ NKT cells were determined in CD4+, CD8bright/dim and CD4−CD8− lymphocytes by flow cytometry. At the molecular level, the amounts of T cell receptor (TCR) AV24-AJ18 transcripts were quantified with respect to TCRAV24 chain transcripts alone or to all TCR alpha chain transcripts. To detect potential inserted nucleotides in the N-region, a novel real-time PCR-based technology was applied.Both CD4+ and CD4−CD8− NKT cells were present at higher frequencies than CD8+ NKT cells in all probands. CD8dim NKT cell levels were lower in healthy individuals, although not statistically significantly different to the patients. Amounts of AV24-AJ18 transcripts in relation to total TCR alpha-chains and to TCRAV24 alone were equal in both proband groups. N-region diversity was detected in four clones from four different individuals, but altered the amino acid sequence in only one clone of an atopic donor.Analysis of Vα24+CD161+ NKT cell frequencies at both the cellular and molecular levels failed to reveal significant differences in peripheral blood of atopic and non-atopic probands. If NKT cells contribute to development of type I allergies they must do so at earlier times or in other locations. [Copyright &y& Elsevier]