CD40-B Cells Research Articles

P2.03-37 The Efficiency of Octamer-4 Specific Cytotoxic T Cells Induce By CD40-B Cells in Killing Lung Cancer Stem-Like Cells

This study aimed to investigate the correlation of Octamer-4 (OCT4) expression with clinicopathological features and prognosis in lung adenocarcinoma patients, and to further explore the killing effect of OCT4 specific cytotoxic T cells (CTLs) on lung cancer stem cells (LCSCs). 257 lung adenocarcinoma patients underwent thoracic surgery were enrolled in this study and tissue samples were obtained during the operation. OCT4 expression was detected by immunofluorescence staining assay. CD154+ feeder cells were constructed to transfect CD40-B cells, and then mixed with OCT4 antigen peptides and CD8+ T lymphocytes extracted from peripheral blood of lung adenocarcinoma patients, subsequently the OCT4 specific CTLs were co-cultured with PC9 LCSCs to detect the killing efficacy. OCT4+ phenotype was illuminated to be associated with poor differentiation, worse disease free survival (DFS) and overall survival (OS). And Cox’s analysis revealed OCT4+ was an independent predictive factor for shorter DFS and OS. CD40-B-cells with antigen presenting capacity was successfully constructed indicated by elevated CD86+, human leukocyte antigens (HLA)-A+ and CD80+ cells percentage, and OCT4 specific CTLs was successfully activated suggested by increased CD3+, CD4+ and CD8+ cells percentage as well as elevated interleukin (IL-2) and interferon (IFN)-γ expressions. OCT4 specific CTLs presented an elevated cytotoxic activity on LCSCs at percentage 75.5% ± 8.2% compared with CMV pp65 CTLs (25.6% ± 5.1%) and blank control CTLs (20% ± 4.7%). In conclusion, OCT4 expression could be served as a convincing risk biomarker for prognosis in lung adenocarcinoma patients and potential target of CTLs as immuntherapy in killing LCSCs.

Isolation and expansion of OCT4/Sox2 specific cytotoxic T lymphocytes in vitro.

e14578 Background: The main reason for tumor MDR (multidrug resistance ,MDR) is the presence of CSC(cancer stem cells,CSC). In the CSC of lung cancer, OCT4 and Sox2 are closely related to the tumor MDR. Therefore, in the current study, we introduced a method for the generation of OCT4+/Sox2+ CSC specific CTL(cytotoxic T lymphocytes,CTL) through co-culture with CD154+ feeder layer cells. Methods: Lenti/CD154 was constructed. The NIH3T3 cells were incubated with Lenti/CD154 (103 vp per cell) for 24 h at 37 °C and 5% CO2. Then the NIH3T3 cells were subsequently maintained in fresh complete DMEM medium. Lenti/GFP was used as a control. On the 6th day post infection, the infection rate was determined by flow cytometry after incubation with CD154-PE antibody. The feeder layer cells were co-cultured with CD40B cells and the biomarkers of CD40B cells were determined by flow cytometry. The CTL were treated with OCT4 plus Sox2 antigen and then the biomarkers of CTL were examined by flow cytometry and IL-2 and IFNγ were determined by ELISA. Results: The rate of CD154+ NIH3T3 cells was 99.7%, which was higher than that of uninfected-cells (14.8%). After co-culture with CD154+ NIH3T3 cells, the rates of CD40B expressing CD80, CD86 and HLA-A were significantly increased. OCT4 plus Sox2 treatment further increased the CD3, CD4 and CD8 expression and elevated the secretion of IL-2 and IFNγ. After co-culture of CTL with MDR PC9-drived cancer stem cells (CSC) and parental PC9 cells at different ratio, CTL significantly killed the tumor cells compared with control. Conclusions: The generation and expansion of OCT/Sox2 specific CTL is a feasible method for the treatment of lung cancer.

CD40-activated B cells induce anti-tumor immunity in vivo.

The introduction of checkpoint inhibitors represents a major advance in cancer immunotherapy. Some studies on checkpoint inhibition demonstrate that combinatorial immunotherapies with secondary drivers of anti-tumor immunity provide beneficial effects for patients that do not show a strong endogenous immune response. CD40-activated B cells (CD40B cells) are potent antigen presenting cells by activating and expanding naïve and memory CD4+ and CD8+ and homing to the secondary lymphoid organs. In contrast to dendritic cells, the generation of highly pure CD40B cells is simple and time efficient and they can be expanded almost limitlessly from small blood samples of cancer patients. Here, we show that the vaccination with antigen-loaded CD40B cells induces a specific T-cell response in vivo comparable to that of dendritic cells. Moreover, we identify vaccination parameters, including injection route, cell dose and vaccination repetitions to optimize immunization and demonstrate that application of CD40B cells is safe in terms of toxicity in the recipient. We furthermore show that preventive immunization of tumor-bearing mice with tumor antigen-pulsed CD40B cells induces a protective anti-tumor immunity against B16.F10 melanomas and E.G7 lymphomas leading to reduced tumor growth. These results and our straightforward method of CD40B-cell generation underline the potential of CD40B cells for cancer immunotherapy.

Processing and MHC class II presentation of exogenous soluble antigen involving a proteasome-dependent cytosolic pathway in CD40-activated B cells.

Activated B cells have the capacity to present antigen and induce immune responses as potent antigen-presenting cells (APCs). As in other APCs, antigen presentation by B cells involves antigen internalization, antigen processing, and peptide loading onto MHC molecules. However, while the mechanism of antigen processing has been studied extensively in other APCs, this pathway remains elusive in B cells. The aim of this study was to investigate the MHC class II processing pathway in CD40-activated B cells (CD40Bs), as a model for activated, antigen-presenting B cells. Using CMV pp65 as a model antigen, we evaluated processing and presentation of the CD4+T-cell epitope 509-523 (K509) by human CD40Bs in ELISPOT assays. As expected, stimulation of specific CD4+T-cell clones was attenuated after pretreatment of CD40Bs with inhibitors of classic class II pathway components. However, proteasome inhibitors such as epoxomicin limited antigen presentation as well. This suggests that the antigen is processed in a non-classical, cytosolic MHC class II pathway. Further experiments with truncated protein variants revealed involvement of the proteasome in processing of the N and C extensions of the epitope. Access to the cytosol was shown to be size dependent. Epoxomicin sensitivity exclusively in CD40B cells, but not in dendritic cells, suggests a novel processing mechanism unique to this APC. Our data suggest that B cells process antigen using a distinct, non-classical class II pathway.

Tumorantigen-Specific CD40B Cells: Combining Enhanced Antigen-Presentation and Antibody-Secretion for Tumor Targeting

Efficient antigen presentation is a prerequisite for the development of a T-cell-mediated immune response. Dendritic cells (DCs) are the most prominent professional antigen-presenting cells (APCs). However, they have several disadvantages as cellular adjuvant in cancer immunotherapy. Therefore, an alternative approach was developed, in which polyclonal B cells can serve as potent APCs by treatment with the CD40 ligand. We demonstrated that CD40-activation dramatically improves antigen presentation by B cells, efficiently inducing naive and memory CD4+ and CD8+ T-cell responses. Moreover, these CD40-activated (CD40) B cells home to secondary lymphoid organs. However, antigen presentation by antigen-specific B cells is more effective compared to polyclonal B cells. Therefore, we use tumorantigen-specific B cells to improve the antigen-presenting function of CD40B cells. Purified human and murine tumorantigen-specific B cells highly upregulate activation markers upon CD40-stimulation, which results in an enhanced CD4+ and CD8+ T cell response in vitro and in vivo. This response is significantly lower in polyclonal CD40B cells and comparable to the stimulation induced by mature dendritic cells. Moreover, antigen-specific B cells could be stimulated in vitro to differentiate into antibody-secreting plasma cells. Treatment of E.G7 lymphoma-bearing mice with a combination of antigen-specific CD40B cells and plasma cells results in inhibition of tumor growth and prolonged survival. Moreover, antigen-specific B cells home to the tumor site and the spleen, where they encounter T cells. These results provide new insights into the role of activated antigen-specific B cells as APCs and their use for cancer immunotherapy. DisclosuresScheid:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

In Vitro and in Vivo Imaging of Initial T-B Cell Interactions in the Setting of B Cell-Based Cancer Immunotherapy

In recent years, there has been a growing interest in the use of B cells for cellular immunotherapy, since B cell-based cancer vaccines have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated (CD40) B cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo.The dynamic interactions of human antigen-presenting cells and antigen-specific T cells were observed by time-lapse videomicroscopy. The migratory and chemoattractant potential of CD40B cells was analyzed by flow cytometry and standard transwell migration assays. GFP+ CD40B cells or CD40B cells isolated from Luciferase+mice were used for subsequent in vivo studies.Murine CD40B cells show similar migratory and chemotactic characteristics compared to human CD40B cells. Upon CD40-activation, B cells upregulate the important molecules involved in lymh node homing (CD62L, CCR7/CDCR4), which are functional and induce chemotaxis of T cells in vitro. Striking differences were observed for interactions of human CD40B cells or DCs with T cells. Antigen-loaded CD40B cells differ from immature and mature DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived (7.5 min) and sequential interactions with cognate T cells. In vivo, CD40B cells migrate to the spleen and the lymph nodes, where they enrich in the B cell zone before traveling to B cell/ T cell boundary close to the T cell zone.CD40B cell interactions with T cells are dynamic and short-lived and thereby differ from DCs. Taken together, the migration behavior of CD40B cells and their interaction with T cells underline their potential as cellular adjuvant for cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

CD40-activated B cells as antigen-presenting cells: the final sprint toward clinical application

Efficient antigen presentation is a prerequisite for the development of a T-cell-mediated immune response in vitro and in vivo. CD40-activated B cells (CD40B cells) are a promising alternative to dendritic cells as professional APCs for immunotherapy. CD40 activation dramatically improves antigen presentation by normal and malignant B cells, efficiently inducing naive and memory CD4+ and CD8+ T-cell responses. Moreover, CD40B cells do not only attract T cells by release of chemokines, but also home to secondary lymphoid organs. Furthermore, CD40B cells can be expanded exponentially over several weeks at high purity without a loss of antigen-presenting function, providing an almost unlimited source of cellular adjuvant. Vaccination with CD40B cells was shown in mice and dogs to induce a specific immune response. This article summarizes the achievements of intense research on CD40B cells over the last decade, as well as novel developments critical for a rapid translation into clinical application.

IL-6 Production by Dendritic Cells Is Dispensable for CD8+Memory T-Cell Generation

Following activation, naïve CD8+ T cells will differentiate into effectors that differ in their ability to survive: some will persist as memory cells while the majority will die by apoptosis. Signals given by antigen-presenting cells (APCs) at the time of priming modulate this differential outcome. We have recently shown that, in opposition to dendritic cell (DC), CD40-activated B-(CD40-B) cell vaccination fails to efficiently produce CD8+ memory T cells. Understanding why CD40-B-cell vaccination does not lead to the generation of functional long-lived memory cells is essential to define the signals that should be provided to naïve T cells by APCs. Here we show that CD40-B cells produce very low amount of IL-6 when compared to DCs. However, supplementation with IL-6 during CD40-B-cell vaccination did not improve memory generation. Furthermore, IL-6-deficient DCs maintained the capacity to promote the formation of functional CD8+ effectors and memory cells. Our results suggest that in APC vaccination models, IL-6 provided by the APCs is dispensable for proper CD8+ T-cell memory generation.

Design of CD40 Agonists and Their Use in Growing B Cells for Cancer Immunotherapy

CD40 stimulation has produced impressive results in early-stage clinical trials of patients with cancer. Further progress will be facilitated by a better understanding of how the CD40 receptor becomes activated and the subsequent functions of CD40-stimulated immune cells. This review focuses on two aspects of this subject. The first is the recent recognition that signaling by CD40 is initiated when the receptors are induced to cluster within the membrane of responding cells. This requirement for CD40 clustering explains the stimulatory effects of certain anti-CD40 antibodies and the activity of many-trimer, but not one-trimer, forms of CD40 ligand (CD40L, CD154). The second topic is the use of these CD40 activators to expand B cells (“CD40-B cells”). As antigen-presenting cells (APCs), CD40-B cells are as effective as dendritic cells, with the important difference that CD40 B cells can be induced to proliferate in vitro, whereas DCs proliferate poorly if at all. As a result, the use of CD40-B cells as antigen-presenting cells (APCs) promises to streamline the generation of anti-tumor CD8+ T cells for the adoptive cell therapy (ACT) of cancer.

CD40-Activated B Cell Cancer Vaccine Improves Second Clinical Remission and Survival in Privately Owned Dogs with Non-Hodgkin's Lymphoma

Cell-based active immunotherapy for cancer is a promising novel strategy, with the first dendritic cell (DC) vaccine achieving regulatory approval for clinical use last year. Manufacturing remains arduous, especially for DC vaccines, and the prospect of using cell-based immunotherapy in the adjuvant setting or in combination with chemotherapy remains largely untested. Here, we used a comparative oncology approach to test the safety and potential efficacy of tumor RNA-loaded, CD40-activated B cells in privately owned dogs presenting with non-Hodgkin's lymphoma (NHL), a clinical scenario that represents not only a major problem in veterinary medicine but also a bona fide spontaneous animal model for the human condition. When administered to NHL dogs in remission after induction chemotherapy, CD40-B cells electroporated ex vivo with autologous tumor RNA safely stimulated immunity in vivo. Although chemotherapy plus CD40-B vaccination did not improve time-to-progression or lymphoma-specific survival compared to dogs treated with chemotherapy alone, vaccination potentiated the effects of salvage therapy and improved the rate of durable second remissions as well as subsequent lymphoma-specific survival following salvage therapy. Several of these relapsed dogs are now long-term survivors and free of disease for more than a year. Overall, these clinical and immunological results suggest that cell-based CD40 cancer vaccination is safe and synergizes with chemotherapy to improve clinical outcome in canine NHL. More broadly, our findings underscore the unique value of clinical investigations in tumor-bearing companion animals.

Detailed kinetics of the direct allo-response in human liver transplant recipients: new insights from an optimized assay.

Conventional assays for quantification of allo-reactive T-cell precursor frequencies (PF) are relatively insensitive. We present a robust assay for quantification of PF of T-cells with direct donor-specificity, and establish the kinetics of circulating donor-specific T cells after liver transplantation (LTx). B cells from donor splenocytes were differentiated into professional antigen-presenting cells by CD40-engagement (CD40-B cells). CFSE-labelled PBMC from LTx-recipients obtained before and at several time points after LTx, were stimulated with donor-derived or 3rd party CD40-B cells. PF of donor-specific T cells were calculated from CFSE-dilution patterns, and intracellular IFN-γ was determined after re-stimulation with CD40-B cells. Compared to splenocytes, stimulations with CD40-B cells resulted in 3 to 5-fold higher responding T-cell PF. Memory and naïve T-cell subsets responded equally to allogeneic CD40-B cell stimulation. Donor-specific CD4+ and CD8+ T-cell PF ranged from 0.5 to 19% (median: 5.2%). One week after LTx, PF of circulating donor-specific CD4+ and CD8+ T cells increased significantly, while only a minor increase in numbers of T cells reacting to 3rd party allo-antigens was observed. One year after LTx numbers of CD4+ and CD8+ T cells reacting to donor antigens, as well as those reacting to 3rd party allo-antigens, were slightly lower compared to pre-transplant values. Moreover, CD4+ and CD8+ T cells responding to donor-derived, as well as those reacting to 3rd party CD40-B cells, produced less IFN-γ. In conclusion, our alternative approach enables detection of allo-reactive human T cells at high frequencies, and after application we conclude that donor-specific T-cell PF increase immediately after LTx. However, no evidence for a specific loss of circulating T-cells recognizing donor allo-antigens via the direct pathway up to 1 year after LTx was obtained, underscoring the relative insensitiveness of previous assays.

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (>10(9) cells per patient) from healthy donors as well as cancer patients. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro. Furthermore they efficiently take up, process and present antigens to T cells. CD40-B cells were shown to not only prime naíve, but also expand memory T cells. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa). Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (>90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach.

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (>109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime naϊve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a). CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (>10(9) cells per patient) from healthy donors as well as cancer patients (Fig.1c,d). In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3). To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function. CD40-B cells efficiently take up, process and present antigens to T cells. They do not only prime naïve, but also expand memory T cells. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors.

Induction of myeloma-specific cytotoxic T lymphocytes ex vivo by CD40-activated B cells loaded with myeloma tumor antigens

We investigated to establish CD40-activated B cells (CD40-B cells) as alternative antigen-presenting cells (APCs) for the induction of myeloma-specific cytotoxic T lymphocytes (CTLs). To generate CD40-B cells, peripheral blood mononuclear cells were co-cultured with CD40L-transfected J558 cells in the presence of IL-4, insulin, transferrin, and cyclosporine for 14 days, and pulsed with myeloma lysates. The CD40-B cells consistently expressed high levels of CD80, CD86, CD54, CCR7, and HLA-DR. The CD40-B cells produced IL-12, IFN-gamma, and IL-6 during the culture period, but not IL-10. In addition, the CD40-B cells showed potent allogeneic T-cell stimulatory capacities that depended on the dose ratio and had the potential to polarize naïve T cells into Th1 subsets. The CD40-B cells loaded with tumor lysates induced strong target-specific CTLs, based on large numbers of IFN-gamma secreting cells and higher cytotoxic activity against target cells compared to the CD40-B cells without the tumor lysates. These results suggest that CD40-B cells loaded with myeloma lysates might provide alternative APCs for cellular immunotherapy in patients with myeloma.

CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential

Cellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC-vaccine trials, induction of tumour antigen-specific immunity is observed frequently and well-documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40-activated B cells (CD40-B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell-based vaccines we performed a single-patient analysis of 502 patients from DC-based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re-evaluated the CD40-B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always > 90% after 2 weeks and remains stable for several weeks. They have comparable antigen-presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40-B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen-presentation and migratory properties.

RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma

Cell-based vaccination strategies to induce functional tumor-specific T cells in cancer patients have focused on using autologous dendritic cells. An alternative approach is to use RNA-loaded CD40 activated B cells (CD40-B) that are highly efficient antigen-presenting cells capable of priming naive T cells, boosting memory T-cell responses and breaking tolerance to tumor antigens. The use of tumor RNA as the antigenic payload allows for gene transfer without viruses or vectors and permits major histocompatibility complex (MHC)-independent, multiple-antigen targeting. Here, we use CD40L transfected K562 cells to generate functional CD40-B cells from the peripheral blood of humans and dogs. Testing of RNA-loaded CD40-B cells in dogs allows not only for its development in veterinary medicine but also for determination of its safety and efficacy in a large animal model of spontaneous cancer prior to initiation of human clinical trials. We found that CD40-B cells from healthy humans, healthy dogs and tumor-bearing dogs express increased levels of immune molecules such as MHC and CCR7. Moreover, RNA-loaded CD40-B cells induce functional, antigen-specific T cells from healthy dogs and dogs with lymphoma. These findings pave the way for immunotherapy trials using tumor RNA-loaded CD40-B cells to stimulate antitumor immunity in a large animal model of spontaneous neoplasia.

Simultaneous Activation of Viral Antigen-specific Memory CD4+ and CD8+ T-cells Using mRNA-electroporated CD40-activated Autologous B-cells

Recently, it has become obvious that not only CD8 T-cells, but also CD4 T-helper cells are required for the induction of an effective, long-lasting cellular immune response. In view of the clinical importance of cytomegalovirus (CMV) and human immunodeficiency virus (HIV) infection, we developed 2 strategies to simultaneously reactivate viral antigen-specific memory CD4 and CD8 T-cells of CMV-seropositive and HIV-seropositive subjects using mRNA-electroporated autologous CD40-activated B cells. In the setting of HIV, we provide evidence that CD40-activated B cells can be cultured from HAART-naive HIV-1 seropositive patients. These cells not only express and secrete the HIV p24 antigen after electroporation with codon-optimized HIV-1 gag mRNA, but can also be used to in vitro reactivate Gag antigen-specific interferon-gamma-producing CD4 and CD8 autologous T-cells. For the CMV-specific approach, we applied mRNA coding for the pp65 protein coupled to the lysosomal-associated membrane protein-1 to transfect CD40-activated B cells to induce CMV antigen-specific CD4 and CD8 T-cells. More detailed analysis of the activated interferon-gamma-producing CMV pp65 tetramer positive CD8 T-cells revealed an effector memory phenotype with the capacity to produce interleukin-2. Our findings clearly show that the concomitant activation of both CD4 and CD8 (memory) T-cells using mRNA-electroporated CD40-B cells is feasible in CMV and HIV-1-seropositive persons, which indicates the potential value of this approach for application in cellular immunotherapy of infectious diseases.

CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants

AbstractCD40-activated B cells (CD40-B cells) have previously been introduced as an alternative source of antigen-presenting cells for immunotherapy. CD40-B cells can prime naive and expand memory T cells, and they can be generated in large numbers from very small amounts of peripheral blood derived from healthy individuals or cancer patients alike. Administration of CD40-B cells as a cellular adjuvant would require these cells to migrate toward secondary lymphoid organs and attract T cells in situ, processes guided by specific chemokines and chemokine receptors. Here, we demonstrate that primary, human CD40-B cells express a pattern of adhesion molecules and chemokine receptors necessary for homing to secondary lymphoid organs and have the capacity to migrate to cognate ligands. Furthermore, we show that CD40-B cells express important T-cell attractants and induce strong T-cell chemotaxis. These findings further support the use of CD40-B cells as cellular adjuvant for cancer immunotherapy.

In vitro induction of myeloid leukemia-specific CD4 and CD8 T cells by CD40 ligand-activated B cells gene modified to express primary granule proteins.

The primary granule proteins (PGP) of myeloid cells are a source of multiple antigens with immunotherapeutic potential for myeloid leukemias. Therefore, we developed a method to induce T-cell responses to PGP protein sequences. We found that gene-transfected antigen-presenting cells efficiently expand functionally competent PGP-specific CD4 and CD8 T cells. The system was optimized using T-cell responses to autologous CD40-activated B cells (CD40-B) transfected with a cytomegalovirus pp65-encoding expression vector. To generate leukemia-specific T cells, expression vectors encoding the PGP proteinase 3 (PR3), human neutrophil elastase, and cathepsin-G were transfected into CD40-B cells to stimulate post-allogeneic stem cell transplantation T cells from five patients with myeloid and three with lymphoid leukemias. T-cell responses to PGP proteinase 3 and human neutrophil elastase were observed in CD8+ and CD4+ T cells only in patients with myeloid leukemias. T-cell responses against cathepsin-G occurred in both myeloid and lymphoblastic leukemias. T cells from a patient with chronic myelogenous leukemia (CML) and from a posttransplant CML patient, expanded against PGP, produced IFN-gamma or were cytotoxic to the patient's CML cells, demonstrating specific antileukemic efficacy. This study emphasizes the clinical potential of PGP for expansion and adoptive transfer of polyclonal leukemia antigen-specific T cells to treat leukemia.