Human Primary and Memory Cytotoxic T Lymphocyte Responses Are Efficiently Induced by Means of CD40-activated B Cells As Antigen-presenting Cells: Potential for Clinical Application

Blood. May, 2002  |  Pubmed ID: 11964299

CD40 engagement is the major signal that induces B cells to efficiently present antigen to T cells. We previously demonstrated that human peripheral blood-derived CD40-activated B cells (CD40-B cells) function as antigen-presenting cells (APCs). Here, we have established a culture system to generate these APCs under clinically applicable conditions using guanylic acid-grade soluble trimeric CD40 ligand. To monitor APC function and antigen loading for these cells, simple and efficient quality control assays have been developed. Using this approach, we demonstrate that CD40-B cells from healthy donors and cancer patients are fully functional and equally expanded in long-term cultures. These B cells boost robust memory T-cell responses, but more importantly, they also prime naive T-cell responses against neoantigens ex vivo. CD40-B cells overcome current obstacles, such as the difficulty of isolation, generation, and long-term expansion observed with other APCs. Therefore, they are an excellent source of professional APCs for immune assessment, antigen discovery, and antigen-specific immunotherapy.

Viral Antigen-specific CD8+ T-cell Responses Are Impaired in Multiple Myeloma

British Journal of Haematology. Jun, 2003  |  Pubmed ID: 12786794

Multiple myeloma (MM) is associated with defects of humoral and cellular immunity, however, little is known about the frequency and function of antigen-specific CD8+ T cells. Such information might be critical for the development of immunotherapy for MM patients. As a model, we assessed the frequency and proliferation of CD8+ T cells specific for HLA-A*0201-restricted immunodominant epitopes from influenza A (Inf A) and Epstein-Barr virus (EBV). Experiments in identical twins demonstrated reduced numbers of antigen-specific T cells after ex-vivo antigenic challenge in the MM twin when compared with the healthy twin. Similarly, the proliferation and frequency of EBV- and Inf A-specific T cells was also significantly reduced in a cohort of 24 previously untreated or conventionally treated MM patients when compared with 19 healthy individuals. In contrast, MM patients studied after receiving an autologous stem cell transplantation showed strikingly higher frequencies of EBV-specific T cells with potential to proliferate ex vivo, suggesting that EBV-specific T cells are readily expandable under these circumstances. These data identify an impaired response of CD8+ T cells in MM patients, which might in part explain the relatively limited success of anti-MM immunisations. Prospective studies will determine whether such immune assessment strategies may identify patients more likely to benefit from cancer immunotherapy.

The Shared Tumor-associated Antigen Cytochrome P450 1B1 is Recognized by Specific Cytotoxic T Cells

Blood. Nov, 2003  |  Pubmed ID: 12869499

Cytochrome P450 1B1 (CYP1B1), a drug-metabolizing extrahepatic enzyme, was recently shown to be overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for anticancer therapy, particularly cancer immunotherapeutics. We identified HLA-A*0201-binding peptides and a naturally processed and presented T-cell epitope capable of inducing CYP1B1-specific cytotoxic T lymphocytes (CTLs) in HLA-A2 transgenic mice. Furthermore, the induction of CYP1B1-specific T cells was demonstrated in healthy donors and cancer patients. These T cells efficiently lysed target cells pulsed with the cognate peptide. More important, HLA-A2-matched tumor cell lines and primary malignant cells were also recognized by CYP1B1-specific CTLs. These findings form the basis of a phase 1 clinical trial exploring a DNA-based vector encoding CYP1B1 for widely applicable cancer immunotherapy conducted at the Dana-Farber Cancer Institute.

Autoantibodies Frequently Detected in Patients with Aplastic Anemia

Blood. Dec, 2003  |  Pubmed ID: 12947009

Although accumulating evidence strongly suggests that aplastic anemia (AA) is a T cell-mediated autoimmune disease, no target antigens have yet been described for AA. In autoimmune diseases, target autoantigens frequently induce not only cellular T-cell responses but also humoral B-cell responses. We hypothesized that the presence of antigen-specific autoantibodies could be used as a "surrogate marker" for the identification of target T-cell autoantigens in AA patients. We screened a human fetal liver library for serologic reactivity against hematopoietic stem/progenitor cell antigens and isolated 32 genes. In 7 of 18 AA patients, an immunoglobulin G (IgG) antibody response was detected to one of the genes, kinectin, which is expressed in all hematopoietic cell lineages tested including CD34+ cells. No response to kinectin was detected in healthy volunteers, multiply transfused non-AA patients, or patients with other autoimmune diseases. Epitope mapping of IgG autoantibodies against kinectin revealed that the responses to several of the epitopes were shared by different AA patients. Moreover, CD8+ cytotoxic T cells raised against kinectin-derived peptides suppressed the colony formation of granulocyte macrophage colony-forming units (CFU-GMs) in an HLA class I-restricted fashion. These results suggest that kinectin may be a candidate autoantigen that is involved in the pathophysiology of AA.

O-Linked Glycans Control Glycoprotein Processing by Antigen-presenting Cells: a Biochemical Approach to the Molecular Aspects of MUC1 Processing by Dendritic Cells

European Journal of Immunology. Dec, 2003  |  Pubmed ID: 14635032

MUC1 is a glycoprotein overexpressed in breast cancer and other adenocarcinomas, and is known to elicit cellular and humoral immunity directed against unglycosylated peptide epitopes in the repeat domain. Based on immunological evidence that O-linked glycans on repeat peptides remain intact during processing by dendritic cells (DC), we used MUC1 as a model to address the question which role O-linked glycans play in this process. We were able to identify the sites of proteolysis in MUC1 repeats and the enzyme(s) involved, and elucidated the site-specific effects of O-glycosylation on MUC1 processing by human and mouse DC. Peptides generated by the cellular processing machinery from native mucin or (glyco)peptides suggest specific cleavage at Gly13-Ser14, His20-Gly1 and Thr3-Ser4 peptide bonds in the tandem repeat GVTSAPDTRPAPGSTAPPAH resulting in the initial formation of STA27 or GVT20 and SAP17 as the final product with intact O-glycosylation. Human cathepsin L and the corresponding mouse enzyme in low-density endosomes were identified in vitro to catalyze this site-specific MUC1 proteolysis. O-Glycosylation controls the processing by preventing proteolysis of the Thr3-Ser4 peptide bond if either amino acid is glycosylated, and is responsible for the inertness of tumor-associated MUC1 glycoforms to effective DC processing by masking this cleavage site.

PH-triggered Microparticles for Peptide Vaccination

Journal of Immunology (Baltimore, Md. : 1950). Aug, 2004  |  Pubmed ID: 15294974

Improving vaccine delivery to human APCs is a way to increase the CTL response to vaccines. We report the use of a novel pH-triggered microparticle that exploits the ability of APCs to cross-present MHC I-restricted Ags that have been engulfed in the low pH environment of the phagosome. A model MHC class I-restricted peptide Ag from the influenza A matrix protein was encapsulated in spray-dried microparticles composed of dipalmitoylphosphatidylcholine and the pH-sensitive polymethacrylate Eudragit E100. Release of the peptide from the particle was triggered by a drop in pH to the acidity normally found in the phagosome. The particles were efficiently phagocytosed by human monocytes and dendritic cells with minimal cellular toxicity and no functional impairment. Encapsulation of the peptide in the microparticles resulted in efficient presentation of the peptide to CD8(+) T cells by human dendritic cells in vitro, and was superior to unencapsulated peptide or peptide encapsulated in an analogous pH-insensitive particle. Vaccination of human HLA-A*0201 transgenic mice with peptide encapsulated in pH-triggering microparticles resulted in priming of CTL responses. These microparticles can be modified to coencapsulate a range of adjuvants along with the Ag of interest. Encapsulation of MHC I epitopes in pH-triggered microparticles increases Ag presentation and may improve CD8(+) T cell priming to peptide vaccines against viruses and cancer.

DCs and CD40-activated B Cells: Current and Future Avenues to Cellular Cancer Immunotherapy

Trends in Immunology. Dec, 2004  |  Pubmed ID: 15530836

Despite the still poorly understood complexity of tumor-host immune interactions, the use of cellular vaccines (particularly dendritic cells) has made it possible to reliably generate tumor antigen-specific T cells, both in animal models and in humans. These encouraging pre-clinical results have led to a translation of these immunotherapeutic strategies into clinical trials. With numerous trials still underway, their general outcome has so far been disappointing, and the discrepancy between pre-clinical data and clinical response rates is striking. Thus, either the pre-clinical models have not been representative of the human situation or the translation into human clinical trials is still sub-optimal. Here we suggest new avenues of clinical research to further improve cellular cancer immunotherapy.

Identification of a New HLA-A*0201-restricted Cryptic Epitope from CYP1B1

International Journal of Cancer. Journal International Du Cancer. Jun, 2005  |  Pubmed ID: 15688394

Cytochrome P450 1B1 (CYP1B1) was recently shown to be a candidate tumor antigen broadly expressed in solid and hematologic malignancies. Nevertheless, use of such self-antigens as targets for immune intervention can be limited because of loss of high-avidity T cells during negative selection in the thymus. Recent data suggest that targeting of cryptic epitopes may represent a way to circumvent such self-tolerance and induce efficient antitumor CTL responses. Here, we present the identification and characterization of a novel, cryptic HLA-A*0201-binding peptide from CYP1B1. The nanomer CYP246 was identified by epitope deduction using algorithms to predict HLA-A*0201-binding peptides. CYP246 is characterized by strong initial HLA-A*0201 binding but a short MHC/peptide binding half-life. Expansion of high-avidity CTL was readily possible using autologous CD40-activated B cells from normal donors and cancer patients as antigen-presenting cells, suggesting that an intact T-cell repertoire can be expanded for this epitope. Lysis of CYP1B1-expressing, HLA-A*0201+ tumor cell lines and primary tumor cells confirmed that sufficient levels of CYP246 are presented by tumor cells for effector CTL killing. These findings indicate that CYP246 is a candidate cryptic epitope for immune interventions in which tumor CYP1B1 is targeted.

Engagement of CD83 Ligand Induces Prolonged Expansion of CD8+ T Cells and Preferential Enrichment for Antigen Specificity

Blood. Feb, 2006  |  Pubmed ID: 16239433

Following T-cell receptor and CD28 signaling, CD8+ T cells express a receptor for CD83, a molecule up-regulated on functionally mature dendritic cells. Although this expression pattern suggests that CD83 is involved in adaptive immunity, little is known about its function in the periphery, and the existence of its ligand on T cells is controversial. We demonstrate that the engagement of the CD83 ligand (CD83L) preferentially enriches and significantly amplifies the number of antigen-specific CD8+ T cells. Coengagement of the T-cell receptor, CD28, and CD83L supports priming of naive CD8+ T cells that retain antigen specificity and cytotoxic function for more than 6 months. Therefore, engagement of the CD83L provides a unique signal to activated CD8+ T cells that could be exploited to generate long-lived antigen-specific cytotoxic T cells for the treatment of cancer and infection.

CD25 and Indoleamine 2,3-dioxygenase Are Up-regulated by Prostaglandin E2 and Expressed by Tumor-associated Dendritic Cells in Vivo: Additional Mechanisms of T-cell Inhibition

Blood. Jul, 2006  |  Pubmed ID: 16522817

Immune tolerance is a central mechanism counteracting tumor-specific immunity and preventing effective anticancer immunotherapy. Induction of tolerance requires a specific environment in which tolerogenic dendritic cells (DCs) play an essential role deviating the immune response away from effective immunity. It was recently shown that maturation of DCs in the presence of PGE2 results in upregulation of indoleamine 2,3-dioxygenase (IDO) providing a potential mechanism for the development of DC-mediated Tcell tolerance. Here, we extend these findings, demonstrating a concomitant induction of IDO and secretion of soluble CD25 after DC maturation in the presence of PGE2. While maturation of DCs induced IDO expression on transcriptional level, only integration of PGE2 signaling led to up-regulation of functional IDO protein as well as significant expression of cell-surface and soluble CD25 protein. As a consequence, T-cell proliferation and cytokine production were significantly inhibited, which was mediated mainly by IDO-induced tryptophan depletion. Of importance, we demonstrate that different carcinoma entities associated with elevated levels of PGE2 coexpress CD25 and IDO in peritumoral dendritic cells, suggesting that PGE2 might influence IDO expression in human DCs in the tumor environment. We therefore suggest PGE2 to be a mediator of early events during induction of immune tolerance in cancer.

Dendritic Cells Are Significantly Reduced in Non-Hodgkin's Lymphoma and Express Less CCR7 and CD62L

Leukemia & Lymphoma. Apr, 2006  |  Pubmed ID: 16690519

Despite the lack of tumor control, infiltration of immune cells has been demonstrated for several malignancies including non-Hodgkin's lymphoma. Since dendritic cells play a pivotal role in the initiation and control of the immune response, the frequency and phenotype of recently described sub-types of dendritic cells in non-Hodgkin's lymphoma were characterized. Myeloid and plasmacytoid dendritic cells were analysed in 55 non-Hodgkin's lymphoma and 33 reactive lymph nodes by flow cytometry and immunohistochemistry. Overall frequency of dendritic cells in reactive lymph nodes was higher than in non-Hodgkin's lymphoma while the pDC/mDCs ratio was comparable. The low frequency of dendritic cells in infiltrated lymph nodes was confirmed by immunohistochemistry; however, no significant difference in the distribution within lymphoid and tumor tissue was detected. For further characterization of the dendritic cells in non-Hodgkin's lymphoma, the expressions of adhesion molecules, costimulatory molecules, chemokine receptors and activation markers were assessed. Interestingly, a significantly decreased expression of CD62L and CCR7, receptors necessary for homing to lymph nodes, was identified in dendritic cells in non-Hodgkin's lymphoma, potentially explaining the lack of these cells. Taken together, dendritic cells are phenotypically altered and reduced in number in NHL, potentially contributing to the loss of tumor control in these patients.

Cyclin D1-specific Cytotoxic T Lymphocytes Are Present in the Repertoire of Cancer Patients: Implications for Cancer Immunotherapy

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Oct, 2008  |  Pubmed ID: 18927298

Cyclin D1, a key cell cycle regulator, is overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for cancer immunotherapy. Nevertheless, it had previously been suggested that efficient T cells recognizing cyclin D1-derived epitopes are absent from the repertoire because of thymic deletion. We attempted to induce autologous CTL from healthy donors and patients with cyclin D1-overexpressing tumors using a highly efficient T-cell expansion system based on CD40-activated B cells as antigen-presenting cells.

Using CD40-activated B Cells to Efficiently Identify Epitopes of Tumor Antigens

Journal of Immunotherapy (Hagerstown, Md. : 1997). Feb-Mar, 2009  |  Pubmed ID: 19238014

The rapid development of genomics and proteomics has accelerated the discovery of antigens that play a role in host-tumor interaction and can be potentially targeted in tumor immunotherapy. Several independent approaches to characterize such antigens and identify the relevant epitopes have been developed. However, the detection, expansion, and characterization of antigen-specific T cells are essential steps common to all strategies. Efficient identification of epitopes, in particular in a preclinical setting, is often hampered by lack of significant numbers of antigen presenting cells at sufficient purity that readily expand low-frequency T-cell precursors. Using the cylins as a model family of self-tumor antigens, we show that CD40-activated primary human B cells can be used very efficiently to identify novel epitopes and characterize such tumor antigen-specific T cells.

Nail Dystrophy Due to Dose-intensive Chemotherapy

International Journal of Hematology. Jul, 2009  |  Pubmed ID: 19543953

The Shared Tumor Associated Antigen Cyclin-A2 is Recognized by High-avidity T-cells

International Journal of Cancer. Journal International Du Cancer. Nov, 2009  |  Pubmed ID: 19681121

Cyclin-A2, a key cell cycle regulator, has been shown to be overexpressed in various types of malignancies with little expression in normal tissue. Such tumor-associated genes potentially are useful targets for cancer immunotherapy. However, high-avidity cyclin-specific T cells are considered to be thymically deleted. We identified at least one nonameric HLA-A*0201 binding cyclin-A2 epitope by a reverse immunology approach. Using a highly efficient T-cell expansion system that is based on CD40-activated B (CD40-B) cells as sole antigen-presenting cells we successfully generated cyclin-A2 specific CTL from HLA-A*0201(+) donors. Interestingly, high-avidity cyclin-A2 specific CTL lines, which recognized peptide-pulsed and antigen expressing target cells, were indeed generated by stimulation with CD40-B cells when pulsed with low concentrations of peptide, whereas CD40-B cells pulsed at saturating concentrations could only induce low-avidity CTL, which recognized peptide-pulsed target cells only. One high-avidity CTL line was subcloned and CTL clones, whose peptide concentration required for half-maximal lysis were less than 1 nM, could lyse cyclin-A2 expressing tumor cells. Taken together, cyclin A2 is an attractive candidate for immune intervention in a significant number of cancer patients and high-avidity T cells can be readily generated using CD40-B cells as antigen-presenting cells.

T-cell Responses to Cyclin B1 Are Not Restricted to P53-overexpressing Tumors

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Nov, 2009  |  Pubmed ID: 19903794

The Role of B Cells in the Pathogenesis of Graft-versus-host Disease

Blood. Dec, 2009  |  Pubmed ID: 19749094

Allogeneic hematopoietic stem cell transplantation is an established treatment modality for malignant and nonmalignant hematologic diseases. Acute and chronic graft-versus-host diseases (GVHDs) are a major cause of morbidity and mortality after allogeneic stem cell transplantation. T cells have been identified as key players in the graft-versus-host reaction and, therefore, most established drugs used against GVHD target T cells. Despite our knowledge on the pathogenesis of the GVH reaction, success of established therapies for prevention and treatment of GHVD is unsatisfactory. Recently, animal and human studies demonstrated that B cells are involved in the immunopathophysiology of acute and chronic GVHD. Early phase clinical trials of B-cell depletion with rituximab have shown beneficial effects on both acute and chronic GVHD. This review summarizes the current experimental and clinical evidence for the involvement of B cells in the pathogenesis of acute and chronic GVHD and discusses the clinical implications for the management of patients undergoing allogeneic stem cell transplantation.

Cyclins Against Cancer: a Novel Family of Tumor Antigens?

Immunotherapy. Sep, 2010  |  Pubmed ID: 20874640

Toll-like Receptor 9 Agonists As Cancer Therapeutics

Expert Opinion on Investigational Drugs. Mar, 2011  |  Pubmed ID: 21254877

INTRODUCTION: Toll-like receptor 9 (TLR9) agonists, commonly referred to as CpG oligodeoxynucleotides (ODN), have been added to the arsenal of anti-cancer drugs as monotherapy or in combination with chemotherapy, radiotherapy and other immunotherapeutic approaches as they increase antigen presentation and boost anti-tumor T- and B-cell responses. Several synthetic TLR9 agonists have been developed for clinical grade use and displayed substantial efficacy in the preclinical and clinical models. AREAS COVERED: This review summarizes TLR9 signaling and the impact of TLR9 agonists on the immune response. The most recent experimental and clinical data are analyzed as well as the development of new TLR9 agonists in current clinical trials. EXPERT OPINION: Application of TLR9 agonists, in particular, combination strategies with chemo- or radiotherapy seem a promising and efficient immunotherapeutic approach in cancer patients even with refractory disease. Simultaneous application of TLR9 agonists aims at supporting the patient's immune response and overcoming specific immunosuppressant strategies developed by tumors. Combinatory approaches of the future might also seek for synergism of TLR9 agonists with other immunomodulatory strategies such as B-cell activation using the CD40-CD40L system.

Potential of Toll-like Receptor 9 Agonists in Combined Anticancer Immunotherapy Strategies: Synergy of PAMPs and DAMPs?

Immunotherapy. Mar, 2011  |  Pubmed ID: 21395370

Targeting Malignant B Cells As Antigen-presenting Cells: TLR-9 Agonist Induces Systemic Regression of Lymphoma

Expert Review of Vaccines. Mar, 2011  |  Pubmed ID: 21434797

Evaluation of: Brody JD, Ai WZ, Czerwinski DK et al. In situ vaccination with a TLR9 agonist induces systemic lymphoma regression: a Phase I/II study. J. Clin. Oncol. 28(28), 4324-4332 (2010). Despite high response rates of the follicular B-cell lymphoma to monoclonal antibodies, the clinical course of lymphoma is still characterized by a continuous pattern of relapse. Brody and colleagues treated 15 patients with relapsed, low-grade lymphoma using low-dose radiotherapy applied to one of the tumor sites with combined injection of a TLR-9 agonist at the same site. This strategy induced specific immunity and tumor regression in several patients with an objective response rate of 27%. The results indicate an involvement of the tumor TLR-9-expressing B cells acting as antigen-presenting cells. TLR-9 in situ vaccination combined with local radiotherapy clearly warrants further in-depth analysis and investigation in a Phase III randomized trial, and may provide a new opportunity for the treatment of B-cell malignancies.

The Cyclins: a Family of Widely Expressed Tumor Antigens?

Expert Review of Vaccines. Mar, 2011  |  Pubmed ID: 21434806

Continuous cell division is a hallmark of cancer and cell-cycle regulators therefore represent relevant target molecules for tumor therapy. Among these targets the cyclins are of particular interest as they are overexpressed in various tumor entities with little expression in normal tissue. Here we review evidence that these molecules are recognized by the immune system, summarize why cyclins A, B and D in particular appear to be interesting targets for active and passive immunotherapy, and discuss whether the entire family could be an interesting novel class of tumor antigens for cancer treatment and prevention.