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An abscopal effect is a clinical observation whereby a local treatment is associated with regression of metastatic cancer at a site distant from the primary location of treatment. Here, we describe the clinical systemic effect induced by regional hyperthermia combined with low-dose chemotherapy and provide immunologic correlates. A 15-year-old patient had been diagnosed with alveolar rhabdomyosarcoma (ARMS). All previous treatment options failed in the patient including haploidentical stem cell transplantation and donor lymphocyte infusion. The patient presented with local and metastatic disease, and upon admission, underwent regional hyperthermia combined with low-dose chemotherapy. Immediately following therapy severe skin reactions were observed. Skin biopsies revealed an intraepithelial lymphocytic infiltration dominated by CD3+/CD8+ T cells with a regular network of dendritic cells. Clinical images compared before and during sequential treatment cycles showed complete metabolic response of the local tumor for more than 10 months of therapy. In addition, metastases completely regressed although they were not direct targets of regional hyperthermia. The systemic effect was associated with enhanced frequency of NK cells and T cells expressing the lectin-like natural-killer group 2 D activating receptor (NKG2D), an increase of the CD56bright subset of NK cells, as well as an increase of effector/memory and effector CD8+ and CD4+ T cells in the blood while the percentage of CD25+FOXP3+ regulatory T cells declined. Regional hyperthermia combined with low-dose chemotherapy had the potential to create a systemic effect which was associated with activation of NK cells and T cells. [ABSTRACT FROM AUTHOR]

Abstract: Background: Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo‐organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen‐specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non‐human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno‐specific Treg would benefit research on immune tolerance in xenotransplantation using this model system. Method: Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti‐inflammatory cytokines. After loading with porcine‐specific (PS) in vitro‐transcribed RNA (ivtRNA), tolDC were used to induce CD4+ T cells to become porcine‐specific Treg (PSTreg) in cocultures supplemented with IL‐2 and rapamycin for 10 days. Anti‐inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine‐specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity. Results: TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL‐10 and TGF‐β1, whereas IL‐12p40 and IFN‐γ were not expressed. PSTreg were successfully generated in cocultures of CD4+ T cells and PS ivtRNA‐loaded tolDC. They exhibited a CD3+ CD4+ CD25+ CD127low/− CD45RAlow Foxp3+ phenotype and were characterized by high expression of IL‐10 and TGF‐β1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL‐10 and TGF‐β1 cytokine upon interaction with PSTeff and suppressing IFN‐γ expression on PSTeff. Conclusion: In this study, a fast 3‐day method to generate baboon‐derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine‐antigen specificity and expression of IL‐10 and TGF‐β1. Porcine‐specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons. [ABSTRACT FROM AUTHOR]

Background: Adoptive immunotherapy offers great potential for treating many types of cancer but its clinical application is hampered by cross-reactive T cell responses in healthy human tissues, representing serious safety risks for patients. We previously developed a computational tool called Expitope for assessing cross-reactivity (CR) of antigens based on tissue-specific gene expression. However, transcript abundance only indirectly indicates protein expression. The recent availability of proteome-wide human protein abundance information now facilitates a more direct approach for CR prediction. Here we present a new version 2.0 of Expitope, which computes all naturally possible epitopes of a peptide sequence and the corresponding CR indices using both protein and transcript abundance levels weighted by a proposed hierarchy of importance of various human tissues.Results: We tested the tool in two case studies: The first study quantitatively assessed the potential CR of the epitopes used for cancer immunotherapy. The second study evaluated HLA-A*02:01-restricted epitopes obtained from the Immune Epitope Database for different disease groups and demonstrated for the first time that there is a high variation in the background CR depending on the disease state of the host: compared to a healthy individual the CR index is on average two-fold higher for the autoimmune state, and five-fold higher for the cancer state.Conclusions: The ability to predict potential side effects in normal tissues helps in the development and selection of safer antigens, enabling more successful immunotherapy of cancer and other diseases. [ABSTRACT FROM AUTHOR]

T-cell receptor (TCR) immunotherapy uses T cells engineered with new TCRs to enable detection and killing of cancer cells. Efficacy of TCR immunotherapy depends on targeting antigenic peptides that are efficiently presented by the best-suited major histocompatibility complex (MHC) molecules of cancer cells. However, efficient strategies are lacking to easily identify TCRs recognizing immunodominant peptide-MHC (pMHC) combinations utilizing any of the six possible MHC class I alleles of a cancer cell. We generated an MHC cell library and developed a platform approach to detect, isolate, and re-express TCRs specific for immunodominant pMHCs. The platform approach was applied to identify a human papillomavirus (HPV16) oncogene E5-specific TCR, recognizing a novel, naturally processed pMHC (HLA-B*15:01) and a cytomegalovirus-specific TCR targeting an immunodominant pMHC (HLA-B*07:02). The platform provides a useful tool to isolate in an unbiased manner TCRs specific for novel and immunodominant pMHC targets for use in TCR immunotherapy. [ABSTRACT FROM AUTHOR]

As cancer strikes, individuals vary not only in terms of factors that contribute to its occurrence and development, but as importantly, in their capacity to respond to treatment. While exciting new therapeutic options that mobilize the immune system against cancer have led to breakthroughs for a variety of malignancies, success is limited to a subset of patients. Pre-existing immunological features of both the host and the tumor may contribute to how patients will eventually fare with immunotherapy. A broad understanding of baseline immunity, both in the periphery and in the tumor microenvironment, is needed in order to fully realize the potential of cancer immunotherapy. Such interrogation of the tumor, blood, and host immune parameters prior to treatment is expected to identify biomarkers predictive of clinical outcome as well as to elucidate why some patients fail to respond to immunotherapy. To approach these opportunities for progress, the Society for Immunotherapy of Cancer (SITC) reconvened the Immune Biomarkers Task Force. Comprised of an international multidisciplinary panel of experts, Working Group 4 sought to make recommendations that focus on the complexity of the tumor microenvironment, with its diversity of immune genes, proteins, cells, and pathways naturally present at baseline and in circulation, and novel tools to aid in such broad analyses. [ABSTRACT FROM AUTHOR]

Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. [ABSTRACT FROM AUTHOR]

Identifying T-cell receptors (TCRs) that bind tumor-associated antigens (TAAs) with optimal affinity is a key bottleneck in the development of adoptive T-cell therapy of cancer. TAAs are unmutated self proteins, and T cells bearing high-affinity TCRs specific for such antigens are commonly deleted in the thymus. To identify optimal-affinity TCRs, we generated antigen-negative humanized mice with a diverse human TCR repertoire restricted to the human leukocyte antigen (HLA) A*02:01 (ref. 3). These mice were immunized with human TAAs, for which they are not tolerant, allowing induction of CD8+ T cells with optimal-affinity TCRs. We isolate TCRs specific for the cancer/testis (CT) antigen MAGE-A1 (ref. 4) and show that two of them have an anti-tumor effect in vivo. By comparison, human-derived TCRs have lower affinity and do not mediate substantial therapeutic effects. We also identify optimal-affinity TCRs specific for the CT antigen NY-ESO. Our humanized mouse model provides a useful tool for the generation of optimal-affinity TCRs for T-cell therapy. [ABSTRACT FROM AUTHOR]

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]

Our previously reported phase I clinical trial with the allogeneic gene–modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (TH1/TH2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies. [ABSTRACT FROM AUTHOR]

Adoptive transfer of Τ cells expressing transgenic TCR with antitumor specificity provides a hopeful new therapy for patients with advanced cancer. To fulfill a large need for TCR with high affinity and specificity for various tumor entities, we sought to identify parameters for rapid selection of CTL clones with suitable characteristics. Twelve CTL clones displaying different Ag sensitivities for the same peptide-MHC epitope of the melanoma-associated Ag tyrosinase were analyzed in detail. Better MHC-muItimer binding and slower multimer release are thought to reflect stronger TCR-peptide-MHC interactions; thus, these parameters would seem well suited to identify higher avidity CTL. However, large disparities were found comparing CTL multimer binding with peptide sensitivity. In contrast, CD8+ CTL with superior Ag sensitivity mediated good tumor cytotoxicity and also secreted the triple combination of IFN-γ, IL-2, and TNF-&agr;, representing a Thl pattern often missing in lower avidity CTL. Furthermore, recipient lymphocytes were imbued with high Ag sensitivity, superior tumor recognition, as well as capacity for Thl polycytokine secretion after transduction with the TCR of a high-avidity CTL. Thus, Th1 polycytokine secretion served as a suitable parameter to rapidly demark cytotoxic CD8+ Τ cell clones for further TCR evaluation. [ABSTRACT FROM AUTHOR]

The hyaluronan-mediated motility receptor (HMMR/Rhamm) is overexpressed in numerous tumor types, including acute lymphoid leukemia and acute myeloid leukemia (AML). Several studies have reported the existence of T-cell responses directed against HMMR in AML patients that are linked to better clinical outcome. Therefore, we explored the use of HMMR-specific TCRs for transgenic expression in lymphocytes and their in vivo impact on HMMR+ solid tumors and disseminated leukemia. We obtained TCRs via an in vitro priming approach in combination with CD137-mediated enrichment. Recipient lymphocytes expressing transgenic TCR revealed the specific tumor recognition pattern seen with the original T cells. Adoptive transfer experiments using a humanized xenograft mouse model resulted in significantly retarded solid tumor outgrowth, which was enhanced using IL-15-conditioned, TCR-transgenic effector memory cells. These cells also showed an increased potency to retard the outgrowth of disseminated AML, and this was further improved using CD8-enriched effector memory cells. To define a safe clinical setting for HMMR-TCR gene therapy, we analyzed transgenic T-cell recognition of hematopoietic stem cells (HSCs) and found on-target killing of HLA-A2+ HSCs. Our findings clearly limit the use of HMMR-TCR therapy to MHC-mismatched HSC transplantation, in which HLA-A2 differences can be used to restrict recognition to patient HSCs and leukemia. [ABSTRACT FROM AUTHOR]

The apoptosis inhibitor protein survivin is overexpressed in many tumors, making it a candidate target molecule for various forms of immunotherapy. To explore survivin as a target antigen for adoptive T cell therapy using lymphocytes expressing survivin-specific transgenic T cell receptors (Tg-TCRs), we isolated HLA-A2-allorestricted survivin-specific T cells with high functional avidity. Lymphocytes expressing Tg-TCRs were derived from these T cells and specifically recognized HLA-A2+ survivin+ tumor cells. Surprisingly, HLA-A2+ but not HLA-A2- lymphocytes expressing Tg-TCRs underwent extensive apoptosis over time. This demise was caused by HLA-A2-restricted fratricide that occurred due to survivin expression in lymphocytes, which created ligands for Tg-TCR recognition. Therefore, survivin-specific TCR gene therapy would be limited to application in HLA-A2-mismatched stem cell transplantation. We also noted that lymphocytes that expressed survivin-specific Tg-TCRs killed T cell clones of various specificities derived from HLA-A2+ but not HLA-A2- donors. These results raise a general question regarding the development of cancer vaccines that target proteins that are also expressed in activated lymphocytes, since induction of high-avidity T cells that expand in lymph nodes following vaccination or later accumulate at tumor sites might limit themselves by self-MHC-restricted fratricide while at the same time inadvertently eliminating neighboring T cells of other specificities. [ABSTRACT FROM AUTHOR]

The development of immunotherapies for renal cell carcinoma (RCC) has been the subject of research for several decades. In addition to cytokine therapy, the benefit of various adoptive cell therapies has again come into focus in the past several years. Nevertheless, success in fighting this immunogenic tumor is still disappointing. RCC can attract a multitude of different effector cells of both the innate and adaptive immune system, including natural killer (NK) cells, γδ T cells, NK-like T cells, peptide-specific T cells, dendritic cells (DC), and regulatory T cells (Tregs). Based on intensive research on the biology and function of different immune cells, we now understand that individual cell types do not act in isolation but function within a complex network of intercellular interactions. These interactions play a pivotal role in the efficient activation and function of effector cells, which is a prerequisite for successful tumor elimination. This review provides a current overview of the diversity of effector cells having the capacity to recognize RCC. Aspects of the functions and anti-tumor properties that make them attractive candidates for adoptive cell therapies, as well as experience in clinical application are discussed. Improved knowledge of the biology of this immune network may help us to effectively harness various effector cells, placing us in a better position to develop new therapeutic strategies to successfully fight RCC. [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: The microbial environment in early infancy or even in utero may modulate the risk to develop allergic disease. Since Toll-like receptors (TLR) recognize microbial products, we hypothesized that maternal allergies may be associated with decreased levels of TLR2, TLR4 and CD14 mRNA in mothers and their offspring. 185 healthy pregnant women from Germany (n = 48), Hungary (n = 50) and Spain (n = 87) were enrolled in a European multicenter study. Levels of TLR2, TLR4 and CD14 mRNA were quantified in maternal peripheral blood samples taken at delivery and placental cord blood samples. Numbers of TLR2+, TLR4+ and CD14+ monocytes were quantified by flow cytometry in 42 cord blood samples obtained from the German participants. Bivariate and multivariate regression analyses were performed. Maternal allergies were associated with significantly lower levels of TLR2/4/CD14 mRNA in maternal blood and cord blood samples. Maternal and fetal TLR2/4/CD14 mRNA levels were significantly correlated with each other (TLR2 r = 0.42; TLR4 r = 0.58; CD14 r = 0.54). The results suggest that maternal allergy status may affect allergic risk in offspring through a decreased expression of fetal TLR2/4/CD14. [Copyright &y& Elsevier]

T-cell-mediated loss of pancreatic β-cells is the crucial event in the development of type 1 diabetes. The phenotypic characteristics of disease-associated T-cells in type 1 diabetes have not yet been defined. The negative results from two intervention trials (the Diabetes Prevention Trial-Type 1 Diabetes and the European Nicotinamide Diabetes Intervention Trial) illustrate the need for technologies to specifcally monitor ongoing autoimmune reactions. We used fluorescence-activated cell sorter analysis to study surface marker expression on T-cell lines specific for two major type 1 diabetes autoantigens, GAD65 and proinsulin. We then applied this knowledge in a cross-sectional approach to delineate the phenotype of circulating memory T-cells. The autoreactive T-cells of patients could be distinguished from those of control subjects by their coexpression of CD25 and CD134. Autoantigen-specific T-cells that recognized multiple GAD65- and preproinsulin-derived peptides and coexpressed CD25+CD134- were confined to patients (n = 32) and pre-diabetic probands (n = 5). Autoantigen-reactive T-cells in control subjects (n = 21) were CD25+CD134- and recognized fewer autoantigen-derived peptides. Insulin therapy did not induce CD25+CD134+ T-cells in type 2 diabetic patients. The coexpression of CD25 and the costimulatory molecule CD134 on memory T-cells provides a novel marker for type 1 diabetes-associated T-cell immunity. The CD134 costimulatory molecule may also provide a novel therapeutic target in type 1 diabetes. [ABSTRACT FROM AUTHOR]

Initial vaccine developments for renal cell carcinoma (RCC) have concentrated on cell-based approaches in which tumor cells themselves provide mixtures of unknown tumor-associated antigens as immunizing agents. Antigens derived from autologous tumors can direct responses to molecular composites characteristic of individual tumors, whereas antigens derived from allogeneic tumor cells must be commonly shared by RCC. Three types of cell-based vaccine for RCC have been investigated: isolated tumor cell suspensions, gene modified tumor cells and dendritic cells (DCs) expressing RCC-associated antigens. Approaches using genetic modification of autologous RCC have included ex vivo modification of tumor cells or modification of tumors in vivo. We have used gene-modification of allogeneic tumor cell lines to create generic RCC vaccines. More recently, emphasis has shifted to the use of DCs as cell-based vaccines for RCC. DCs have moved to a position of central interest because of their excellent stimulatory capacity, combined with their ability to process and present antigens to both naive CD4 and CD8 cells. The long impasse in identifying molecular targets for specific immunotherapy of RCC is now rapidly being overcome through the use of tools and information emerging from human genome research. Identification of candidate molecules expressed by RCC using cDNA arrays, combined with protein arrays and identification of peptides presented by MHC molecules, allow specific vaccines to be tailored to the antigenic profile of individual tumors, providing the basis for development of patient-specific vaccines. [ABSTRACT FROM AUTHOR]

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]

Background: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. Methods: SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40(+) and EpCAM(+)). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met(+) human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. Results: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase(+) melanoma cells by TCR-, as well as of CEA(+) colon cancer cells by chimeric antigen receptor (CAR)-modified T cells. Conclusions: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT. [ABSTRACT FROM AUTHOR]

Background: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. Methods: SV40 T antigen–specific T cells from T cell receptor (TCR)-I–transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR–anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40+ and EpCAM+). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met+ human tumor cell lines. Differences between experimental conditions were analyzed using the Student’s t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. Results: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR–transduced T cells to immobilized c-Met and recognition of tyrosinase+ melanoma cells by TCR-, as well as of CEA+ colon cancer cells by chimeric antigen receptor (CAR)–modified T cells. Conclusions: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT. [ABSTRACT FROM PUBLISHER]

Adoptive cell therapies continually evolve through science-based innovation. Specialized innovations for TCR-T therapies are described here that are embedded in an End-to-End Platform for TCR-T Therapy Development which aims to provide solutions for key unmet patient needs by addressing challenges of TCR-T therapy, including selection of target antigens and suitable T cell receptors, generation of TCR-T therapies that provide long term, durable efficacy and safety and development of efficient and scalable production of patient-specific (personalized) TCR-T therapy for solid tumors. Multiple, combinable, innovative technologies are used in a systematic and sequential manner in the development of TCR-T therapies. One group of technologies encompasses product enhancements that enable TCR-T therapies to be safer, more specific and more effective. The second group of technologies addresses development optimization that supports discovery and development processes for TCR-T therapies to be performed more quickly, with higher quality and greater efficiency. Each module incorporates innovations layered onto basic technologies common to the field of immunology. An active approach of "evolution by innovation" supports the overall goal to develop best-in-class TCR-T therapies for treatment of patients with solid cancer., Competing Interests: The contributing author is an employee and a shareholder of Medigene AG., (Copyright © 2023 Schendel.)

Intensive induction chemotherapy achieves complete remissions (CR) in >60% of patients with acute myeloid leukemia (AML) but overall survival (OS) is poor for relapsing patients not eligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Oral azacytidine may be used as maintenance treatment in AML in first remission, but can be associated with substantial side effects, and less toxic strategies should be explored. Twenty AML patients in first CR (CR1) ineligible for allo-HSCT were treated with FDC101, an autologous RNA-loaded mature dendritic cell (mDC) vaccine expressing two leukemia-associated antigens (LAAs). Each dose consisted of 2.5-5 × 10 6 mDCs per antigen, given weekly until week 4, at week 6, and then monthly, during the 2-year study period. Patients were followed for safety and long-term survival. Treatment was well tolerated, with mild and transient injection site reactions. Eleven of 20 patients (55%) remained in CR, while 4 of 6 relapsing patients achieved CR2 after salvage therapy and underwent allo-HSCT. OS at five years was 75% (95% CI: 50-89), with 70% of patients ≥60 years of age being long-term survivors. Maintenance therapy with this DC vaccine was well tolerated in AML patients in CR1 and was accompanied by encouraging 5-year long-term survival., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Patients with high-risk prostate cancer (PC) can experience biochemical relapse (BCR), despite surgery, and develop noncurative disease. The present study aimed to reduce the risk of BCR with a personalized dendritic cell (DC) vaccine, given as adjuvant therapy, after robot-assisted laparoscopic prostatectomy (RALP)., Methods: Twelve weeks after RALP, 20 patients with high-risk PC and undetectable PSA received DC vaccinations for 3 years or until BCR. The primary endpoint was the time to BCR. The immune response was assessed 7 weeks after surgery (baseline) and at one-time point during the vaccination period., Results: Among 20 patients, 11 were BCR-free over a median of 96 months (range: 84-99). The median time from the end of vaccinations to the last follow-up was 57 months (range: 45-60). Nine patients developed BCR, either during (n = 4) or after (n = 5) the vaccination period. Among five patients diagnosed with intraductal carcinoma, three experienced early BCR during the vaccination period. All patients that developed BCR remained in stable disease within a median of 99 months (range: 74-99). The baseline immune response was significantly associated with the immune response during the vaccination period (p = 0.015). For patients diagnosed with extraprostatic extension (EPE), time to BCR was longer in vaccine responders than in non-responders (p = 0.09). Among 12 patients with the International Society of Urological Pathology (ISUP) grade 5 PC, five achieved remission after 84 months, and all mounted immune responses., Conclusion: Patients diagnosed with EPE and ISUP grade 5 PC were at particularly high risk of developing postsurgical BCR. In this subgroup, the vaccine response was related to a reduced BCR incidence. The vaccine was safe, without side effects. This adjuvant first-in-man Phase I/II DC vaccine study showed promising results. DC vaccines after curative surgery should be investigated further in a larger cohort of patients with high-risk PC., (© 2021 The Authors. The Prostate published by Wiley Periodicals LLC.)

Background: The cancer-testis antigen MAGE-A4 is an attractive target for T-cell-based immunotherapy, especially for indications with unmet clinical need like non-small cell lung or triple-negative breast cancer., Methods: An unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived epitope (GVYDGREHTV) that was properly processed and presented on human leukocyte antigen (HLA)-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high-avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies., Results: A MAGE-A4-reactive, HLA-A2-restricted T-cell receptor (TCR) was isolated from primed T cells of an HLA-A2-negative donor. The respective TCR-T-cell (TCR-T) product bbT485 was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared with TCR-Ts expressing a TCR derived from a tolerized T-cell repertoire to self-antigens. This natural high-avidity TCR was found to be CD8 co-receptor independent, allowing effector functions to be elicited in transgenic CD4 + T helper cells. These CD4 + TCR-Ts supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells and upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines on tumor-specific stimulation., Conclusion: The extensive pre-clinical assessment of safety and in vivo potency of bbT485 provide the basis for its use in TCR-T immunotherapy studies. The ability of this non-mutated high-avidity, co-receptor-independent TCR to activate CD8 + and CD4 + T cells could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T-cell subsets that goes beyond what is currently tested in the clinic., Competing Interests: Competing interests: KD, TH, LP, GL, DJS, DS, and CE are employees and DJS is a Managing Director of Medigene Immunotherapies GmbH, a subsidiary of Medigene AG, Planegg, Germany. CE and DS are designated as inventors on two patent applications (PCT/EP2020/058779 and PCT/US20/31796) related to this work that have been filed by Medigene Immunotherapies GmbH and bluebird bio Inc. KPG, LJP, PRN, and MSM are current employees at bluebird bio Inc., Cambridge, USA. ID was an employee of bluebird bio., Cambridge, USA during her contributions to this publication. ID is a current employee of ElevateBio, Waltham, USA. KPG, LJP, ID, PRN, and MSM are current equity holders at bluebird bio., Cambridge, USA., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In the last years, immunotherapies have shown tremendous success as treatments for multiple types of cancer. However, there are still many obstacles to overcome in order to increase response rates and identify effective therapies for every individual patient. Since there are many possibilities to boost a patient's immune response against a tumor and not all can be covered, this review is focused on T cell receptor-mediated therapies. CD8 + T cells can detect and destroy malignant cells by binding to peptides presented on cell surfaces by MHC (major histocompatibility complex) class I molecules. CD4 + T cells can also mediate powerful immune responses but their peptide recognition by MHC class II molecules is more complex, which is why the attention has been focused on CD8 + T cells. Therapies based on the power of T cells can, on the one hand, enhance T cell recognition by introducing TCRs that preferentially direct T cells to tumor sites (so called TCR-T therapy) or through vaccination to induce T cells in vivo . On the other hand, T cell activity can be improved by immune checkpoint inhibition or other means that help create a microenvironment favorable for cytotoxic T cell activity. The manifold ways in which the immune system and cancer interact with each other require not only the use of large omics datasets from gene, to transcript, to protein, and to peptide but also make the application of machine learning methods inevitable. Currently, discovering and selecting suitable TCRs is a very costly and work intensive in vitro process. To facilitate this process and to additionally allow for highly personalized therapies that can simultaneously target multiple patient-specific antigens, especially neoepitopes, breakthrough computational methods for predicting antigen presentation and TCR binding are urgently required. Particularly, potential cross-reactivity is a major consideration since off-target toxicity can pose a major threat to patient safety. The current speed at which not only datasets grow and are made available to the public, but also at which new machine learning methods evolve, is assuring that computational approaches will be able to help to solve problems that immunotherapies are still facing., (Copyright © 2019 Mösch, Raffegerst, Weis, Schendel and Frishman.)

Organ transplantation remains the most effective treatment for patients with late stage organ failure. Transgenic pigs provide an alternative organ donor source to the limited availability of human organs. However, cellular rejection still remains to be the obstacle for xenotransplantation. Superior to other methods, antigen-specific regulatory T cells (Treg) alleviate cellular rejection with fewer side effects. Here we demonstrate the use of a fast method to provide tolerogenic dendritic cells (tolDC) that can be used to generate effective porcine-specific Treg cells (PSTreg). TolDC were produced within three days from human monocytes in medium supplemented with anti-inflammatory cytokines. Treg were generated from naïve CD4 + T cells and induced to become PSTreg by cocultivation with porcine-antigen-loaded tolDC. Results showed that PSTreg exhibited the expected phenotype, CD4 + CD25 + CD127 low/- Foxp3 + , and a more activated phenotype. The specificity of PSTreg was demonstrated by suppression of effector T cell (Teff) activation markers of different stages and inhibition of Teff cell proliferation. TolDC and PSTreg exhibited high expression of IL-10 and TGF-β1 at both protein and RNA levels, and PSTreg also highly expressed IL-35 at RNA levels. Upon restimulation, PSTreg retained the activated phenotype and specificity. Taken together, the newly developed procedure allows efficient generation of highly suppressive PSTreg.

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 GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 multimer and secreted interferon-γ when cultured with GPC3367, 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 GPC3367 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 GPC3367-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., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine.

Dendritic cell (DC)-based immunotherapy is a promising strategy for the elimination of minimal residual disease in patients with acute myeloid leukemia (AML). Particularly, patients with a high risk of relapse who are not eligible for hematopoietic stem cell transplantation could benefit from such a therapeutic approach. Here, we review our extensive studies on the development of a protocol for the generation of DCs with improved immunogenicity and optimized for the use in cell-based immunotherapy. This new generation DC vaccine combines the production of DCs in only 3 days with Toll-like receptor-signaling-induced cell maturation. These mature DCs are then loaded with RNA encoding the leukemia-associated antigens Wilm's tumor protein 1 and preferentially expressed antigen in melanoma in order to stimulate an AML-specific T-cell-based immune response. In vitro as well as in vivo studies demonstrated the enhanced capacity of these improved DCs for the induction of tumor-specific immune responses. Finally, a proof-of-concept Phase I/II clinical trial is discussed for post-remission AML patients with high risk for disease relapse.

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.

Posttransplantation lymphoproliferative disease (PTLD) associated with Epstein-Barr virus (EBV) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation. PTLD is efficiently prevented by adoptive transfer of EBV-specific T cells from the donor. To make EBV-specific T cells available in urgent clinical situations, we developed a rapid protocol for their isolation by overnight stimulation of donor blood cells with peptides derived from 11 EBV antigens, interferon-gamma surface capture, and immunomagnetic separation. Six patients with PTLD received 1 transfusion of EBV-specific T cells. No response was seen in 3 patients who had late-stage disease with multiorgan dysfunction at the time of T-cell transfer. In 3 patients who received T cells at an earlier stage of disease, we observed complete and stable remission of PTLD. Two patients have remained free from EBV-associated disease for more than 2 years. CD8(+) T cells specific for EBV early antigens rapidly expanded after T-cell transfer, temporarily constituted greater than 20% of all peripheral blood lymphocytes, and were maintained throughout the observation period. Thus, a rapid and sustained reconstitution of a protective EBV-specific T-cell memory occurred after the infusion of small numbers of directly isolated EBV-specific T cells.

Immunotherapy with whole cell cancer vaccines has been tested in various tumor types. This study investigated the safety profile and antitumor activity of an allogeneic prostate carcinoma cell line, LNCaP, expressing recombinant human interleukin-2 and human interferon-gamma. Thirty HLA-A*0201-matched patients with progressive, castration-resistant prostate cancer received four intradermal injections on days 1, 15, 29, and 92, and then every 90 days, as long as no tumor progression occurred. Three patients received a dose level of 7.5 million cells, and 27 patients received 15 million cells per injection. The primary study criteria were safety and the difference in prostate-specific antigen doubling time (PSA-DT), determined in the pretreatment phase (before the start of vaccination) and in the trial treatment phase (during vaccination). No dose-limiting or autoimmune toxicity was seen. During vaccination there was a significant prolongation of the PSA-DT compared with the prevaccination period (prolongation from 63 to 114 days; p < 0.01; intention to treat). In addition, results showed a period of PSA stabilization of at least 12 weeks, together with stable bone scans in 12 of 30 patients, and 3 patients sustained a >50% decrease in PSA versus baseline. The median overall survival time from first vaccination was 32 months (mean value, 34 months). Immune monitoring revealed T cell stimulation in the majority of patients. This vaccine strategy was found to be safe and well tolerated and was accompanied by prolongation of PSA-DT. The results of this trial warrant clinical development of this vaccine.

Adoptive transfer of T cells expressing transgenic T-cell receptors (TCRs) with antitumor function is a hopeful new therapy for patients with advanced tumors; however, there is a critical bottleneck in identifying high-affinity TCR specificities needed to treat different malignancies. We have developed a strategy using autologous dendritic cells cotransfected with RNA encoding an allogeneic major histocompatibility complex molecule and a tumor-associated antigen to obtain allo-restricted peptide-specific T cells having superior capacity to recognize tumor cells and higher functional avidity. This approach provides maximum flexibility because any major histocompatibility complex molecule and any tumor-associated antigen can be combined in the dendritic cells used for priming of autologous T cells. TCRs of allo-restricted T cells, when expressed as transgenes in activated peripheral blood lymphocytes, transferred superior function compared with self-restricted TCR. This approach allows high-avidity T cells and TCR specific for tumor-associated self-peptides to be easily obtained for direct adoptive T-cell therapy or for isolation of therapeutic transgenic TCR sequences.

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 mug of methyl-tetra-hydrofolic acid, both, or placebo from the 22nd gestational week. T(H)1/T(H)2-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-beta 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-gamma (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 T(H)2-related molecules in the fetus and decreased maternal inflammatory cytokines. We speculate that both effects are mediated by TGF-beta.

Background: Prostate cancer is the most common cancer of men in the Western world. Despite the over-expression of tumor-associated antigens, like PSA or PSMA, immune activation is inefficient. The goal of this investigation was to assess in situ characteristics of prostate cancer-infiltrating lymphocytes and to determine their activation status and effector function., Methods: We compared 17 carcinoma containing tissues, four benign prostatic hyperplasia tissues and eight healthy prostate tissues regarding lymphocyte subset composition, locoregional distribution, and functional status using immunohistological staining of cryopreserved tissues. For determination of lymphocyte subsets, serial sections were stained with CD3, CD4, and CD8 antibodies. Activation status and effector function were studied using CD69, interferon-gamma (IFN gamma), perforin, and CD3 zeta chain antibodies. T-cell-receptor repertoire (TCR) analysis was made to determine the complexity of infiltrating lymphocytes., Results: CD3+, CD4+, and CD69+ T lymphocytes were prominent in tissues derived from patients with prostate carcinoma. CD8+ lymphocytes were significantly less than CD4+ lymphocytes. IFN gamma and perforin were downregulated on infiltrating lymphocytes compared to cells of healthy prostate tissue. Very few lymphocytes were detected within cancerous lesions whereas surrounding tissues showed extensive lymphocyte cluster formation. The TCR repertoire of infiltrating lymphocytes was broad and similar to that of healthy prostate tissue, giving no evidence for specific lymphocyte recruitment., Conclusions: In the prostate cancer microenvironment, CD4+ T lymphocytes dominated while CD8+ T cells were sparse. The lymphocytes exhibited signs of disturbed effector function. Consequently, the immune response against autologous tumor cells is likely to be inefficient in controlling tumor growth., ((c) 2007 Wiley-Liss, Inc.)

Background: Galactooligosaccharides (GOS) and long-chain fructooligosaccharides (lcFOS) proliferate bifidobacteria in infant gut microbiota. However, it is not known how GOS and FOS influence the microbiota of pregnant women and whether a potential prebiotic effect is transferred to the offspring., Objectives: We aimed to test how supplementation with GOS and lcFOS (GOS/lcFOS) in the last trimester of pregnancy affects maternal and neonatal gut microbiota. Variables of fetal immunity were assessed as a secondary outcome., Design: In a randomized, double-blind, placebo-controlled pilot study, 48 pregnant women were supplemented 3 times/d with 3 g GOS/lcFOS (at a ratio of 9:1) or maltodextrin (placebo) from week 25 of gestation until delivery. Percentages of bifidobacteria and lactobacilli within total bacterial counts were detected by fluorescent in situ hybridization and quantitative polymerase chain reaction in maternal and neonatal (days 5, 20, and approximately 182) stool samples. Variables of fetal immunity were assessed in cord blood by using flow cytometry and cytokine multiplex-array analysis., Results: The proportions of bifidobacteria in the maternal gut were significantly higher in the supplemented group than in the placebo group (21.0% and 12.4%, respectively; P = 0.026); the proportion of lactobacilli did not differ between the groups. In neonates, bifidobacteria and lactobacilli percentages, diversity and similarity indexes, and fetal immune parameters did not differ significantly between the 2 groups. Mother-neonate similarity indexes of bifidobacteria decreased over time., Conclusions: GOS/lcFOS supplementation has a bifidogenic effect on maternal gut microbiota that is not transferred to neonates. The increased maternal bifidobacteria did not affect fetal immunity as measured by a comprehensive examination of cord blood immunity variables.

Background: Asthma is characterized by a T(H)2 immune response. CD4(+)CD25(hi) regulatory T cells (Tregs) have been proposed to prevent allergic diseases through suppression of T(H)2 responses., Objective: We sought to investigate the role of CD4(+)CD25(hi) T cells in children with asthma., Methods: CD4(+)CD25(hi) 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(+)CD25(hi) 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(+)CD25(hi) 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(+)CD25(hi) T cells in peripheral blood and BALF. Isolated BALF and peripheral blood CD4(+)CD25(hi) T cells from nonasthmatic subjects suppressed proliferation and cytokine/chemokine production by CD4(+)CD25(-) responder T cells. BALF CD4(+)CD25(hi) T cells from asthmatic subjects failed to suppress proliferation and production of T(H)2-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(+)CD25(hi) Tregs are impaired in pediatric asthma., Clinical Implications: Pulmonary Tregs might represent a therapeutic target in pediatric asthma.

In Germany lung cancer is the leading cause of cancer-associated death in men. Surgery, chemotherapy and radiation may enhance survival of patients suffering from lung cancer but the enhancement is typically transient and mostly absent with advanced disease; eventually more than 90% of lung cancer patients will die of disease. New approaches to the treatment of lung cancer are urgently needed. Immunotherapy may represent one new approach with low toxicity and high specificity but implementation has been a challenge because of the poor antigenic characterization of these tumors and their ability to escape immune responses. Several different immunotherapeutic treatment strategies have been developed. This review examines the current state of development and recent advances with respect to non-specific immune stimulation, cellular immunotherapy (specific and non-specific), therapeutic cancer vaccines and gene therapy for lung cancer. The focus is primarily placed on immunotherapeutic cancer treatments that are already in clinical trial or well progressed in preclinical studies. Although there seems to be a promising future for immunotherapy in lung cancer, presently there is not standard immunotherapy available for clinical routine.

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 T(H)2 immune response, whereas T(H)1 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 T(H)1/T(H)2 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(+) (T(H)1) and CCR4(+) (T(H)2) expressing CD4(+) and CD8(+) lymphocytes were quantified by flow cytometry. Levels of T(H)1-associated (IL-2, IFN-gamma, IFN-gamma inducible T cell-alpha chemoattractant, Monokine induced by IFN-gamma, IFN-gamma inducible protein of 10 kd) and T(H)2-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(+) (T(H)2) cells, IL-4, IL-13, and TARC and lower levels of IFN-gamma compared with noninfected patients with CF and healthy controls. Bronchoalveolar lavage fluid levels of IL-4, IL-13, and TARC correlated inversely with FEV(1) in P aeruginosa-infected patients with CF., Conclusion: These results reveal the prevalence of a pulmonary T(H)2 immune response in P aeruginosa-infected patients with CF. The modulation of the pulmonary T(H)2 response in P aeruginosa infection may be an option for the treatment of P aeruginosa lung disease in patients with CF.

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 T H 1 and T H 2 cells., Objective: We sought to determine whether levels of T H 1/T H 2-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 T H 1-related (IFN-gamma-inducible protein of 10 kd [IP-10], IFN-gamma-inducible T cell alpha chemoattractant [ITAC], monokine induced by IFN-gamma [Mig], and IFN-gamma) and T H 2-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 + (T H 1) and CCR4 + and CCR3 + (T H 2) 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 FEV 1 . 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-gamma., 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.

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. Valpha24+CD161+ Natural killer T (NKT) cells secrete high amounts of interleukin-4 and/or interferon-gamma 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 Valpha24+CD161+ NKT cells differ in atopic and non-atopic adults. Venous blood was obtained from thirteen atopic and sixteen healthy adult probands. Valpha24+CD161+ NKT cells were determined in CD4+, CD8(bright/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. CD8(dim) 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 Valpha24+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.

Background: Early exposure to bacterial endotoxin has been proposed to protect against allergy development in children. Whether endotoxin is able to direct T-cell differentiation into a predominance of type 1 immunity is still unresolved., Objective: We sought to compare the effects of endotoxin and mite and cat allergens on T-cell differentiation in infants., Methods: In a random population sample of 135 2-year-old children of an ongoing birth-cohort study, peripheral blood CD4+ and CD8+ T-cell subsets were defined by the expression of the chemokine receptors CCR5 and CCR3 as surrogate markers for type 1 and type 2 T cells, respectively. Endotoxin and mite and cat allergens were measured in house dust collected from the mother's mattress at the child's age of 3 months to assess early exposure., Results: In the CD4+ T-cell subset, endotoxin levels were positively associated with high proportions of type 1 CCR5+ cells (odds ratio for fourth exposure quartile [OR(Q4)], 7.68; 95% CI, 1.35-43.75), whereas cat allergen levels were associated with increased proportions of type 2 CCR3+ cells (OR(Q4), 4.07; 95% CI, 1.05-15.85). In contrast to endotoxin, allergen levels were associated with CD8+ T cells, showing an inverse relationship between mite allergen concentrations and high proportions of CCR5+ or CCR3+ cells (CCR5+ cells: OR(Q4), 0.14; 95% CI, 0.03-0.74; CCR3+ cells: OR(Q4), 0.16; 95% CI, 0.03-0.89) and a positive association of cat allergen levels with increased proportions of CCR5+ cells (OR(Q4), 9.24, 95% CI, 1.61-53.10), as well as CCR3+ cells (OR(Q3), 6.64; 95% CI, 1.21-36.51)., Conclusion: Our results indicate that endotoxin has the potential to promote the development of type 1 CD4+ T cells, whereas mite and cat allergens primarily modify the proportion of CD8+ cells of both types.

Recent reports have demonstrated that EBV can be used as a target of specific CTL-based treatments in severe chronic EBV, immunoblastic B cell lymphoma and Hodgkin's disease (HD). Based upon the promising results form these in vivo studies, it has been suggested that an antigen-specific CTL-based immunotherapy may be of benefit in treating EBV-associated tumors such as HD and nasopharyngeal carcinoma (NPC) which express the potentially immunogenic antigens, LMP1 and LMP2a. Recent work form our group has demonstrated that LMP2a-specific CTLs may be generated in vitro using autologous antigen presenting cells which have been transfected with polyadenylated LMP2a RNA in the presence of a cationic lipid. In this study, we demonstrate that the presence of the lipid enhances dendritic cell (DC) transfection efficiency and appears to protect the intracellular LMP2a RNA from degradation by cellular RNAses. Significantly, these improvements resulted in the transfected DCs having a superior ability to stimulate autologous T cell proliferation. These LMP2a + DCs were used to stimulate LMP2a-specific effector cells which were predominantly a mixture of cytotoxic and helper CD4+ T cells. The molecular mechanisms whereby these CD4+ T cells lyzed their LMP2a-expressing targets was investigated and we show that, although expressing Fas ligand on their surface, LMP2a-specific CD4+ effector cells kill their targets using the Ca2+-dependent perforin/granzyme pathway which is the same mechanism used by CD8+ CTLs.

Lymphokine activated killer (LAK) cells represent mixtures of natural killer (NK) and non-MHC-restricted CTLs that have the capacity to lyse a variety of tumor cells and MHC class I-negative target cells. Although it is clear that NK cells are negatively regulated by interactions with MHC class Ia or class Ib molecules, the regulation of LAK-derived T cells has not been clarified to date. In the studies presented here, we demonstrate that IFN-gamma treatment of tumor cells can induce their resistance to LAK-derived T cells in a manner similar to that seen for NK cells. The IFN-gamma-mediated suppression of LAK activity correlates with increased MHC class I expression by the tumor cells, and the inhibition of LAK-mediated cytotoxicity can be reversed in the presence of class I-specific antibody. Furthermore, the expression of MHC class Ia or class Ib molecules in class I-negative cell lines can reduce their susceptibility to LAK-mediated cytotoxicity. This principle of negative regulation by MHC class I molecules applies to LAK-derived T cells generated from peripheral blood lymphocytes of renal cell carcinoma patients and healthy, control donors. Although LAK-derived T cells can be inhibited in their lytic activity through interactions with MHC class Ia and class Ib molecules, they do not express the known inhibitory receptors specific for these ligands that are found on NK cells. Apparently, LAK-derived T cells are negatively regulated by as yet undefined inhibitory receptors.

The allogeneic mixed lymphocyte culture (MLC) has served as an important experimental system for elucidating the cellular and molecular basis of human lymphocyte responses. Complex mixtures of lymphocytes are stimulated by disparate alloantigens, inducing cellular activation and generating a cytokine milieu that is an excellent breeding ground for the proliferation and differentiation of many distinct lymphocyte subsets. Cloning of individual lymphocytes following alloactivation has allowed various cytotoxic lymphocytes to be isolated and characterized with respect to phenotype and specificity. These analyses have revealed that all types of cytotoxic effector cells are regulated by interactions with MHC-peptide ligands, however, the consequences of these interactions can result in opposite functional outcomes. In this review we summarize how allogeneic MHC class I-peptide ligands positively or negatively regulate the activities of four distinct groups of cytotoxic lymphocytes and how this information might be transferred into clinical use.