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Human umbilical cord blood (UCB) represents a valuable source of hematopoietic stem cells, particularly for patients lacking a matching donor. UCB provides practical advantages, including a lower risk of graft-versus-host-disease and permissive human leukocyte antigen mismatching. These advantageous properties have so far been applied for stem cell, mesenchymal stromal cell, and chimeric antigen receptor T cell therapies. However, UCB-derived professional antigen-presenting cells are increasingly being utilized in the context of immune tolerance and regenerative therapy. Here, we review the cell-specific characteristics as well as recent advancements in UCB-based cell therapies focusing on dendritic cells, monocytes, B lymphocytes, innate lymphoid cells, and macrophages.

Checkpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E7 11-19 , 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential 'off-the-shelf' in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.

Despite significant advances in prevention and treatment of transplant rejection with immunosuppressive medications, we continue to face challenges of long-term graft survival, detrimental medication side effects to both the recipient and transplanted organ together with risks for opportunistic infections. Transplantation tolerance has so far only been achieved through hematopoietic chimerism, which carries with it a serious and life-threatening risk of graft versus host disease, along with variability in persistence of chimerism and uncertainty of sustained tolerance. More recently, numerous in vitro and in vivo studies have explored the therapeutic potential of silent clearance of apoptotic cells which have been well known to aid in maintaining peripheral tolerance to self. Apoptotic cells from a donor not only have the ability of down regulating the immune response, but also are a way of providing donor antigens to recipient antigen-presenting-cells that can then promote donor-specific peripheral tolerance. Herein, we review both laboratory and clinical evidence that support the utility of apoptotic cell-based therapies in prevention and treatment of graft versus host disease and transplant rejection along with induction of donor-specific tolerance in solid organ transplantation. We have highlighted the potential limitations and challenges of this apoptotic donor cell-based therapy together with ongoing advancements and attempts made to overcome them., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Husain and Luo.)

The delivery of anticancer agents via passive approaches such as the enhanced permeability and retention effect is unlikely to achieve sufficient concentrations throughout the tumor volume for effective treatment. Cell-based delivery approaches using tumor tropic cells have the potential to overcome the limitations of passive approaches. Specifically, this review focuses on the use of monocytes/macrophages for the delivery of a variety of anticancer agents, including nanoparticles, chemotherapeutics and gene constructs. The efficacy of this delivery approach, both as monotherapy and in combination with light-based phototherapy modalities, has been demonstrated in numerous in vitro and animal studies, however, its clinical potential remains to be determined.

Human γδ T lymphocytes play a role in the immune system defense against cancer. Their broad anti-cancer activity against different types of cancers makes them outstanding candidates for cancer immunotherapy. An issue of recent interest is whether their antigen presentation features are similar to mature dendritic cells. The antigen-presenting cell (APC)-like phenotype and function of γδ T lymphocytes have been confirmed in many clinical trials. In this study, to support the strong role played by Vγ9Vδ2 T cells against cancer, we provide evidence that Vγ9Vδ2 T cells activated with chronic myeloid leukemia (CML) cell lysate antigens can efficiently express an APC phenotype and function. Vγ9Vδ2 T cells derived from normal peripheral blood mononuclear cells were activated with tumor cell lysate, and the tumor-activated Vγ9Vδ2 T cells could recognize and kill CML through their cytotoxic activity. In conclusion, the Vγ9Vδ2 T cells activated by cancer cell lysate showed APC characteristics, and this may greatly increase interest in investigating their therapeutic potential in hematologic malignancies. Abbreviations: CML: chronic myeloid leukemia; APC: antigen-presenting cell; TCR: T cell receptor; MHC: major histocompatibility complex; N-BPs: nitrogen-containing bisphosphonates; IPP: isopentenyl pyrophosphate; PBMC: peripheral blood mononuclear cells; NKG2D: natural killer receptor group 2, member D; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand.

In cancer cells, cancer/testis (CT) antigens become epigenetically derepressed through DNA demethylation and constitute attractive targets for cancer immunotherapy. Here we report that activated CD4 + T helper cells treated with a DNA-demethylating agent express a broad repertoire of endogenous CT antigens and can be used as antigen-presenting cells to generate autologous cytotoxic T lymphocytes (CTLs) and natural killer cells. In vitro, activated CTLs induce HLA-restricted lysis of tumor cells of different histological types, as well as cells expressing single CT antigens. In a phase 1 trial of 25 patients with recurrent glioblastoma multiforme, cytotoxic lymphocytes homed to the tumor, with tumor regression ongoing in three patients for 14, 22, and 27 months, respectively. No treatment-related adverse effects were observed. This proof-of-principle study shows that tumor-reactive effector cells can be generated ex vivo by exposure to antigens induced by DNA demethylation, providing a novel, minimally invasive therapeutic strategy for treating cancer.

Human leukocyte antigens (HLAs) are essential immune molecules that affect transplantation and adoptive immunotherapy. When hematopoietic stem cells or organs are transplanted with HLA-mismatched recipients, graft-versus-host disease or graft rejection can be induced by allogeneic immune responses. The function of each HLA allele has been studied using HLA-deficient cells generated from mutant cell lines or by RNA interference, zinc finger nuclease, and the CRISPR/Cas9 system. To improve HLA gene editing, we attempted to generate an HLA class I null cell line using the multiplex CRISPR/Cas9 system by targeting exons 2 and 3 of HLA-A, HLA-B, and HLA-C genes simultaneously. Multiplex HLA editing could induce the complete elimination of HLA class I genes by bi-allelic gene disruption on target sites which was defined by flow cytometry and target-specific polymerase chain reaction. Furthermore, artificial antigen-presenting cells were generated by transfer of a single HLA class I allele and co-stimulatory molecules into this novel HLA class I null cell line. Artificial antigen-presenting cells showed HLA-restricted antigen presentation following antigen processing and were successfully used for the efficient generation of tumor antigen-specific cytotoxic T cells in vitro. The efficient editing of HLA genes may provide a basis for universal cellular therapies and transplantation.

While metallic biomaterials have led to an improvement in the quality of life, metal allergies, especially to palladium (Pd), has caused a recent increase in allergic patients. Metal allergy is known to be a T cell-mediated delayed-type hypersensitivity (DTH); however, the pathogenic T cell subsets and the specific T cell receptor (TCR) have not been identified. Therefore, we attempted to identify the pathogenic T cells responsible for Pd allergy. We found that activating CD8⁺ T cells significantly increased and that the TRAV (TCRα variable) 7-2*02 chain skewed in Pd allergic mice. Furthermore, adoptive transfer experiments revealed that in vitro-cultured Pd-stimulated antigen presenting cells (APCs) function as memory APCs with recipient mice developing Pd allergy and that the frequency of TRAV7-2*02 increases the same as conventional Pd allergic mice. In contrast, neither proliferation of CD8⁺ T cells nor increasing of TRAV7-2*02 was observed in major histocompatibility complex I (MHC I)-deficient Pd-APCs transferred to mice. Taken together, we revealed that TRAV7-2*02-expressing CD8⁺ T cells are the pathogenic T cells for the development of Pd allergy. We also identified the CDR3 consensus motif of pathogenic TCRs as CAAXSGSWQLIF in TRAV7-2*02/TRAJ (TCRα junction)22*01 positive cells. These results suggest that the specific TCRs represent novel targets for the development of diagnostics and treatments for metal allergy., Competing Interests: The authors declare no conflict of interest.

Background Aims: Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft-versus-host disease (GVHD). We previously completed a "first-in-human" clinical trial using in vitro expanded umbilical cord blood (UCB)-derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation., Methods: To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture., Results: aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced., Discussion: Expansion culture with cGMP aAPCs and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease., Competing Interests: The authors share a patent entitled “Methods to expand a T Regulatory Cell Master Cell Bank”; US Patent # 13/639,927., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on activated T cells and contributes to T-cell exhaustion. PD-L1 expressed on antigen-presenting cells (APCs) could be thought to inhibit the induction of Ag-specific cytotoxic T lymphocytes (CTLs) by transducing negative signal into T cells; however, the roles of PD-L1 on APCs have not yet been well examined. Therefore, we evaluated the roles of PD-L1 on APCs in the induction of Ag-specific CTLs. CD3 T cells isolated from cytomegalovirus (CMV)-seropositive healthy donors were stimulated with mature dendritic cells pulsed with CMV pp65-derived HLA-restricted peptides in the presence of anti-PD-L1 blocking antibody. Unexpectedly, PD-L1 blockade resulted in a less efficient induction of CMV-specific CTLs, suggesting that PD-L1 play a positive role in the induction of Ag-specific CTLs. For further evaluations and application to adoptive immunotherapy, we generated K562-based artificial APCs, which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1. K562/HLA+CD80/86+PD-L1 cells produced significantly higher induction of CMV-specific CTLs than K562/HLA or K562/HLA+CD80/86 cells without causing excessive differentiation or functional exhaustion of the induced CTLs, whereas PD-L1 itself did not have a stimulatory effect. Furthermore, only K562/HLA+CD80/86+PD-L1 cells pulsed with HLA-A*24:02-restricted Wilms tumor 1 (WT1) peptide clearly expanded WT1-specific CTLs from healthy donors. Our findings presumed that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of Ag-specific CTLs probably depending on fine-tuning excessive stimulation of CD80/86, and that K562/HLA+CD80/86+PD-L1 cells has therapeutic potential as a novel type of artificial APCs for adoptive immunotherapy.

Objective: Professional antigen-presenting dendritic cells (DCs) and cytokine-induced killer (CIK) cells, components of anti-cancer therapy, have shown clinical benefits and potential to overcome chemotherapeutic resistance. To evaluate whether DC-CIK cell-based therapy improves the clinical efficacy of chemotherapy, we reviewed the literature on DC-CIK cells and meta-analyzed randomized controlled trials (RCTs)., Methods: We searched several databases and selected studies using predefined criteria. RCTs that applied chemotherapy with and without DC-CIK cells separately in two groups were included. Odds ratio (OR) and mean difference (MD) were reported to measure the pooled effect., Results: Twelve reported RCTs (826 patients), which were all performed on Chinese patients, were included. Combination therapy exhibited better data than chemotherapy: 1-year overall survival (OS) (OR=0.22, P<0.01), 2-year OS (OR=0.28, P<0.01), 3-year OS (OR=0.41, P<0.01), 1-year disease-free survival (DFS) (OR=0.16, P<0.05), 3-year DFS (OR=0.32, P<0.01), objective response rate (ORR) (OR=0.54, P<0.01), and disease control rate (DCR) (OR=0.46, P<0.01). Moreover, the levels of CD3(+) T-lymphocytes (MD=-11.65, P<0.05) and CD4(+) T-lymphocytes (MD=-8.18, P<0.01) of the combination group were higher., Conclusions: Immunotherapy of DC-CIK cells may enhance the efficacy of chemotherapy on solid cancer and induces no specific side effect. Further RCTs with no publishing bias should be designed to confirm the immunotherapeutic effects of DC-CIK cells.

Acute graft-versus-host disease (GVHD) is induced by alloreactivity of donor T cells toward host antigens presented on antigen-presenting cells (APCs). Apoptotic cells are capable of inducing tolerance by altering APC maturation. Apoptosis can be induced by extracorporeal photopheresis (ECP). We demonstrate that the use of ECP as a prophylaxis prior to conditioning significantly improves survival (P < .0001) after bone marrow transplantation (BMT) by inhibiting the initiation phase of acute GVHD in a murine BMT model. ECP-treated autologous splenocytes resulted in immune tolerance in the host, including reduced dendritic cell activation with decreased nuclear factor-κB engagement, increased regulatory T-cell (Treg) numbers with enhanced expression of cytolytic T lymphocyte-associated antigen 4, potentiating their suppressive function. The protective effect required host production of interleukin-10 and host Tregs. Conventional T cells that entered this tolerant environment experienced reduced proliferation, as well as a reduction of tissue homing and expression of activation markers. The induction of this tolerant state by ECP was obviated by cotreatment with lipopolysaccharide, suggesting that the inflammatory state of the recipient prior to treatment would play a role in potential clinical translation. The use of prophylactic ECP may provide an alternative and safe method for immunosuppression in the bone marrow transplant setting., (© 2014 by The American Society of Hematology.)

It is known that inoculation of antigen into the anterior chamber (a.c.) of a mouse eye induces a.c.-associated immune deviation (ACAID), which is mediated in part by antigen-specific local and peripheral tolerance to the inciting antigen. ACAID can also be induced in vivo by intravenous (i.v.) inoculation of ex-vivo-generated tolerogenic antigen-presenting cells (TolAPC). The purpose of this study was to test if in-vitro-generated retinal antigen-pulsed TolAPC suppressed established experimental autoimmune uveitis (EAU). Retinal antigen-pulsed TolAPC were injected i.v. into mice 7 days post-induction of EAU. We observed that retinal antigen-pulsed TolAPC suppressed the incidence and severity of the clinical expression of EAU and reduced the expression of associated inflammatory cytokines. Moreover, extract of whole retina efficiently replaced interphotoreceptor retinoid-binding protein (IRBP) in the preparation of TolAPC used to induce tolerance in EAU mice. Finally, the suppression of EAU could be transferred to a new set of EAU mice with CD8⁺ but not with CD4⁺ regulatory T cells (T(reg)). Retinal antigen-pulsed TolAPC suppressed ongoing EAU by inducing CD8⁺ T(reg) cells that, in turn, suppressed the effector activity of the IRBP-specific T cells and altered the clinical symptoms of autoimmune inflammation in the eye. The ability to use retinal extract for the antigen raises the possibility that retinal extract could be used to produce autologous TolAPC and then used as therapy in human uveitis., (© 2013 The Authors. Clinical and Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society. for Immunology.)

A growing body of evidence shows that immune cells are pivotal in the fight against cancer. First, association studies have identified immune-protective immune response genes against cancer. Second, the presence of immune cells in the respective malignant tumor correlated with a better prognosis for the patients. Third, adoptive cell therapy (ACT) showed, in recent reports, an efficient reduction or even cure for malignant tumors. The focus of this review is a novel in vitro ACT technique, using the patient's cascade-primed immune cells. The cascade-priming procedure stimulates APCs from the peripheral blood. Stimulated APCs digest and present tumor material better and differentiate naive cytotoxic T-lymphocyte effector cells against the patient's cancer. The principle and the impressive results of the cascade-primed immune cell treatment in patient case series is compared with the ACT concepts of lymphokine-activated killer, macrophage-activated killer, macrophage-activated killer-dendritic cell, cytokine-induced killer and tumor-infiltrating lymphocyte methods.

MS is an inflammatory CNS disorder, which typically occurs in early adulthood and rarely in children. Here we tested whether functional maturation of innate immune cells may determine susceptibility to CNS autoimmune disease in EAE. Two-week-old mice were resistant to active EAE, which causes fulminant paralysis in adult mice; this resistance was associated with an impaired development of Th1 and Th17 cells. Resistant, young mice had higher frequencies of myeloid-derived suppressor cells and plasma-cytoid DCs. Furthermore, myeloid APCs and B cells from young mice expressed lower levels of MHC class II and CD40, produced decreased amounts of proinflammatory cytokines, and released enhanced levels of anti-inflammatory IL-10. When used as APCs, splenocytes from 2-week-old mice failed to differentiate naive T cells into Th1 and Th17 cells irrespective of the T-cell donor's age, and promoted development of Treg cells and Th2 cells instead. Adoptive transfer of adult APCs restored the ability of 2-week-old mice to generate encephalitogenic T cells and develop EAE. Collectively, these findings indicate that the innate immune compartment functionally matures during development, which may be a prerequisite for development of T-cell-mediated CNS autoimmune disease., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The use of anti-HER2 monoclonal antibodies (mAbs) has improved the clinical outcome of HER2-overexpressing breast cancers (BCs). Unfortunately, often these tumors tend to relapse and, when metastatic, the duration of clinical benefit is limited over time and almost invariably followed by tumor progression. Alternative approaches to this essentially passive immunotherapy are therefore needed in HER2-overexpressing BC patients. As HER2 is one of the most suitable targets for active immunotherapy in BC, manipulating the immune system is a highly attractive approach., Material and Methods: A computer-based literature search was carried out using PubMed (keywords: breast neoplasm, HER2 vaccine, immunology); data reported at international meetings were included., Results: This review provides a focus on the following active vaccinal approaches under clinical investigation against HER2-overexpressing BC: (i) peptide and protein based; (ii) DNA based; (iii) whole tumor cell based; (iv) dendritic cell based. Moreover, the review discuss future challenges in the field, trying to define the best setting for the development of this innovative strategy, considering both immunological and clinical aspects of HER2 targeting., Conclusions: Development of effective vaccines for BC remains a distinct challenge but is likely to become a substantial advance for patients with HER2-overexpressing BCs.

Local radiotherapy plus intratumoral syngeneic dendritic cell injection can mediate apoptosis/cell death and immunological tumor eradication in murine models. A novel method of coordinated intraprostatic, autologous dendritic cell injection together with radiation therapy was prospectively evaluated in five HLA-A2(+) subjects with high-risk, localized prostate cancer, using androgen suppression, 45 Gy external beam radiation therapy in 25 fractions over 5 weeks, dendritic cell injections after fractions 5, 15 and 25 and then interstitial radioactive seed placement. Serial prostate biopsies before and during treatment showed increased apoptotic cells and parenchymal distribution of CD8(+) cells. CD8(+) T-cell responses to test peptides were assessed using an enzyme-linked immunosorbent spot IFN-γ production assay, demonstrating some prostate cancer-specific protein-derived peptides associated with increased titer. In conclusion, the technique was feasible and well-tolerated and specific immune responses were observable. Future trials could further test the utility of this approach and improve on temporal coordination of intratumoral dendritic cell introduction with particular timelines of therapy-induced apoptosis.

Infections with a range of common community viruses remain a major cause of mortality and morbidity after allogeneic hematopoietic stem cell transplantation. T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenoviruses can safely prevent and infections with these three most common culprits, but the manufacture of individual T cell lines for each virus would be prohibitive in terms of time and cost. We have demonstrated that T cells specific for all three viruses can be manufactured in a single culture using monocytes and EBV-transformed B lymphoblastoid cell lines (LCLs), both transduced with an adenovirus vector expressing pp65 of CMV, as antigen-presenting cells. Trivirus-specific T cell lines produced from healthy stem cell donors could prevent and treat infections with all three viruses, not only in the designated recipient, but in unrelated, partially-HLA-matched third party recipients. We now provide the details and logistics of T cell manufacture.

The recent vigorous transnational migration of people and materials reflecting the development of transportation facilities, changes in social structure, and war disasters has increased the global spread of emerging and re-emerging infections. Once, as the 2009 pandemic influenza A (H1N1) virus, person-to-person transmission was achieved, the spread of pandemic cannot be contained in reality. Thus enhancement of the crisis-management structure against pandemic is critically important to maintain national function. On the basis of this social background, the development of vaccination, which is the only fundamental prophylaxis, is in attention, and earliest possible establishment of system that supply mass-vaccines in a short time is required. Even if, however, rapid manufacture of vaccine antigen is actualized, there are several problems that vaccine is not easily spread across the developing country and mass vaccination is not performed immediately at the time of the crisis, because conventional vaccination is performed mainly by injection. Our research group developed transcutaneous vaccine devices; a hydrogel patch and a dissolving microneedle array which delivered antigens to antigen-presenting cells in the epidermal layer. Our transcutaneous vaccination system receives a high evaluation as novel, easy-to-use, and less-invasive vaccination method against infections from home and abroad. In this review, we introduce the research progress resulted from our basic, preclinical, and clinical study for practical use.

Transplants of SBA-E- allogeneic marrow or G-CSF mobilized CD34+ (ISOLEX) E- peripheral blood progenitor cells which are adequately depleted of T-cells, when administered without post-transplant immunosuppression now induce consistent engraftment with low incidences of acute and chronic GVHD both in HLA matched and HLA disparate recipients. Furthermore, the incidence of relapse post transplant is not increased in patients transplanted for AML, MDS or ALL. In our series, the incidence of severe infections in HLA-matched recipients of such T-cell depleted grafts also does not differ from that detected following similarly matched unmodified grafts. However, in recipients of HLA-haplotype disparate T-cell depleted grafts, the risk of lethal viral infections is increased and prolonged. In many cases, this risk is closely correlated with failures of immunodominant virus-specific donor T-cells transferred in the graft to recognize infected host cells because they are restricted by HLA alleles not shared by the host. To address this limitation, we have developed a panel of artificial antigen presenting cells, each expressing a single prevalent HLA-allele. Using this panel, we are able to selectively generate virus-specific cytotoxic T-cells of desired HLA restriction, to insure their effectiveness in HLA haplotype-disparate transplant recipients. We have also shown that partially HLA-matched, third party-derived EBV-specific T-cells, selected from our bank of previously generated and characterized GMP-grade cell lines on the basis of their HLA restriction, can induce durable remissions of rituximab-refractory EBV lymphomas. These approaches may thus provide new, immediately accessible resources for the generation and broad application of immune cell therapies to treat and prevent severe viral diseases post transplant., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γc(null) mice under the influence of activated CLL-derived T lymphocytes. By co-transferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4(+) T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity.

Bone marrow derived cells, especially B lymphocytes, have been shown to function as tolerogenic antigen-presenting cells (APC's) both in vivo and in vitro. In addition, it is well established that immunoglobulins can function as potent tolerogenic carriers for associated epitopes. We have taken advantage of these properties to develop a gene therapy approach to induce unresponsiveness in a number of animal models for clinical diseases. In our system, we engineered target peptide-IgG constructs into retroviral vectors and transduced hematopoietic cells to create tolerogenic antigen-presenting cells. In this review, we discuss the strategies and mechanism of our gene therapy approach mediated by B cells, as well as by bone marrow cells, for tolerance acquisition in various mouse models for autoimmune disease and hemophilia A. Our results show that MHC class II and co-stimulatory molecules must be expressed on the tolerogenic antigen presenting cells for efficacy. This therapy requires regulatory T cells for both the induction and maintenance of tolerance. The putative role of epitopes provided by the IgG carrier in this process. Studies in non-human primates and with human T cell clones in vitro are in progress to transition this approach to the clinic. The use of stem cells and B cell-delivered gene therapy in human clinical diseases may soon become a reality.

Background: Antigen-presenting cells (APCs) play a crucial role in the induction of immune responses. However, the optimal administration route of tumor-specific APCs for inducing effective immunological responses via cancer immunotherapy remains to be elucidated. Human NKT cells are known to have strong anti-tumor activities and are activated by the specific ligand, namely, α-galactosylceramide (αGalCer)., Methods: Seventeen patients with head and neck squamous cell carcinoma (HNSCC) were enrolled in this study. Patients received an injection of αGalCer-pulsed APCs into the nasal, or the oral floor submucosa. Then total body image and single photon emission computed tomography (SPECT) images were examined. The immunological responses including the number of peripheral blood NKT cells, anti-tumor activities and the CD4(+) CD25(high) Foxp3(+) T cells (Tregs) induced following APCs were also compared., Results: APCs injected into the nasal submucosa quickly migrated to the lateral lymph nodes and those injected into the oral floor submucosa dominantly migrated to the submandibular nodes rather than the lateral lymph nodes. An increase in the absolute number of NKT cells and the IFN-γ producing cells was observed in peripheral blood after injection of the APCs into the nasal submucosa, however, these anti-tumor activities were not detected and the increased frequency of Treg cells were observed after administration into oral floor., Conclusions: These results indicate that a different administration route of APCs has the potential to bring a different immunological reaction. The submucosal administration of αGalCer into the oral submucosa tends to induce immunological suppression.

The desire to create a vaccine that might prevent or cure asthma is irresistibly attractive and widely regarded as one of the main goals in translational asthma research. Several broad vaccination strategies, including immunotherapy against allergens or immunodominant peptide epitopes, induction of immune deviation using vaccines, exploiting adjuvant-like immunomodulation, targeting of damaging cytokines or cells, provocation of directed cell-mediated cytotoxicity, modification of antigen-presenting cells to induce vaccine-like effects and targeting of common infectious pathogens to reduce exacerbations, have demonstrated proof-of-principal in model systems, and some of these concepts have been tested clinically with tantalizing results. However, a poor understanding of asthma endotypes (molecular heterogeneity of pathways determining treatment responses), flawed assumptions about underlying immunopathogenic pathways (over-reliance on the Th2 disease paradigm) and the lack of plasticity of well-established immuno/inflammatory effector mechanisms continue to frustrate efforts to develop an asthma vaccine.

Allogeneic hematopoietic stem cell transplantation (HSCT) is used effectively to treat a number of hematological malignancies. Its beneficial effects rely on donor-derived T cell-targeted leukemic cells, the so-called graft-versus-leukemia (GVL) effect. Induction of GVL is usually associated with concomitant development of graft-versus-host disease (GVHD), a major complication of allogeneic HSCT. The T cells that mediate GVL and GVHD are activated by alloantigen presented on host antigen-presenting cells of hematopoietic origin, and it is not well understood how alloantigen expression on non-hematopoietic cells affects GVL activity. Here we show, in mouse models of MHC-matched, minor histocompatibility antigen-mismatched bone marrow transplantation, that alloantigen expression on host epithelium drives donor T cells into apoptosis and dysfunction during GVHD, resulting in a loss of GVL activity. During GVHD, programmed death-1 (PD-1) and PD ligand-1 (PD-L1), molecules implicated in inducing T cell exhaustion, were upregulated on activated T cells and the target tissue, respectively, suggesting that the T cell defects driven by host epithelial alloantigen expression might be mediated by the PD-1/PD-L1 pathway. Consistent with this, blockade of PD-1/PD-L1 interactions partially restored T cell effector functions and improved GVL. These results elucidate a previously unrecognized significance of alloantigen expression on non-hematopoietic cells in GVL and suggest that separation of GVL from GVHD for more effective HSCT may be possible in human patients.

At present, the treatment of T-cell-dependent autoimmune diseases relies exclusively on strategies leading to nonspecific suppression of the immune systems causing a substantial reduced ability to control concomitant infections or malignancies. Furthermore, long-term treatment with most drugs is accompanied by several serious adverse effects and does not consequently result in cure of the primary immunological malfunction. By contrast, antigen-specific immunotherapy offers the potential to achieve the highest therapeutic efficiency in accordance with minimal adverse effects. Therefore, several studies have been performed utilizing antigen-presenting cells specifically engineered to deplete allo- or antigen-specific T cells ('guided missiles'). Many of these strategies take advantage of the Fas/Fas ligand signaling pathway to efficiently induce antigen-presenting cell-mediated apoptosis in targeted T cells. In this article, we discuss the advantages and shortcomings of a novel non-cell-based 'killer artificial antigen-presenting cell' strategy, developed to overcome obstacles related to current cell-based approaches for the treatment of T-cell-mediated autoimmunity.

Tumor-recruited CD11b myeloid cells, including myeloid-derived suppressor cells, play a significant role in tumor progression, as these cells are involved in tumor-induced immune suppression and tumor neovasculogenesis. On the other hand, the tumor-infiltrated CD11b myeloid cells could potentially be a source of immunostimulatory antigen-presenting cells (APCs), since most of these cells represent common precursors of both dendritic cells and macrophages. Here, we investigated the possibility of generating mature APCs from tumor-infiltrated CD11b myeloid cells. We demonstrate that in vitro exposure of freshly excised mouse tumors to DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (decitabine, AZA) results in selective elimination of tumor cells, but, surprisingly it also enriches CD45(+) tumor-infiltrated cells. The majority of "post-AZA" surviving CD45(+) tumor-infiltrated cells were represented by CD11b myeloid cells. A culture of isolated tumor-infiltrated CD11b cells in the presence of AZA and GM-CSF promoted their differentiation into mature F4/80/CD11c/MHC class II-positive APCs. These tumor-derived myeloid APCs produced substantially reduced amounts of immunosuppressive (IL-13, IL-10, PGE(2)), pro-angiogenic (VEGF, MMP-9) and pro-inflammatory (IL-1beta, IL-6, MIP-2) mediators than their precursors, freshly isolated tumor-infiltrated CD11b cells. Vaccinating naïve mice with ex vivo generated tumor-derived APCs resulted in the protection of 70% mice from tumor outgrowth. Importantly, no loading of tumor-derived APC with exogenous antigen was needed to stimulate T cell response and induce the anti-tumor effect. Collectively, our results for the first time demonstrate that tumor-infiltrated CD11b myeloid cells can be enriched and differentiated in the presence of DNA demethylating agent 5-aza-2'-deoxycytidine into mature tumor-derived APCs, which could be used for cancer immunotherapy.

We investigated the roles of specific subsets of donor APCs purified from bone marrow in donor T cell activation and graft-vs-leukemia (GvL) activity in murine models of hemopoietic stem cell transplantation. Lineage(-)CD11c(+) APC precursors were separated from donor bone marrow based on expression of CD11b. Transplanting lineage(-)CD11c(+)CD11b(-) APC (CD11b(-) APC) in combination with c-kit(+)Sca-1(+)lineage(-) hemopoietic stem cells (HSC) and congenic donor T cells led to increased donor CD4(+) and CD8(+) T cell proliferation and higher donor T cell chimerism than with transplanting grafts containing HSC, T cells, and lineage(-)CD11c(+)CD11b(+) APCs (CD11b(+) APC), or grafts containing only HSC and T cells. Transplanting CD11b(-) APCs induced Th1/type 1 cytotoxic T lymphocyte donor T cell immune polarization and enhanced GvL activity of donor T cells without increased graft-vs-host disease in both MHC- and minor histocompatibility Ag-mismatched murine hemopoietic stem cell transplantation models, whereas CD11b(+) APCs led to Th2/type 2 cytotoxic T lymphocyte donor T cell immune polarization. Donor CD11b(-) APCs were plasmacytoid dendritic cell progenitors (>90% CD317; PDCA-1(+)) and up-regulated CD80, CD86, and IL-12 during alloantigen presentation, whereas CD11b(+) APCs expressed Gr-1 and up-regulated expression of programmed death ligands-1 and 2 after activation. These results are the first to show that manipulation of the content of donor APCs in allogeneic HSC grafts can regulate donor T cell immunity and enhance GvL without increasing graft-vs-host disease activity.

Vaccines are attractive as consolidation therapy after autologous stem cell transplantation (ASCT) for multiple myeloma (MM). We report the results of a phase II trial of the immunotherapeutic, APC8020 (Mylovenge), given after ASCT for MM. We compared the results with that of other patients with MM who underwent ASCT at Mayo Clinic during the same time period. Twenty-seven patients were enrolled on the trial between July, 1998 and June, 2001, and the outcomes were compared to that of 124 consecutive patients transplanted during the same period, but not enrolled on the trial. The median (range) follow-up for patients still alive from the vaccine trial is 6.5 (2.9-8 years), and 7.1 (6-8 years) in the control group. The median age was 57.4 range (36.1-71.3) in the DB group and 56.4 (range, 30-69) in the trial group. Known prognostic factors including PCLI, B2M, and CRP were comparable between the groups. The median overall survival for the trial patients was 5.3 years (95% CI: 4.0 years-N/A) compared to 3.4 years (95% CI: 2.7-4.6 years) for the DB group (P = 0.02). The median time to progression and progression-free survival for the trial group was similar to the DB group. Although not a controlled trial, the vaccines given after ASCT appear to be associated with improved overall survival compared to historical controls. This approach warrants further investigation to confirm this and define the role of vaccine therapy in myeloma., ((c) 2009 Wiley-Liss, Inc.)

Interferon (IFN)-gamma is a pleiotropic cytokine with a central role in innate and adaptive immunity. As a potent pro-inflammatory and antitumor cytokine, IFN-gamma is conventionally thought to be responsible for driving cellular immune response. On the other hand, accumulating evidence suggests that IFN-gamma also has immunosuppressive activity. An important role for IFN-gamma in inhibiting graft-versus-host disease (GVHD) has been demonstrated in murine models, despite IFN-gamma being one of the key factors amplifying T cell activation during the process of acute GVHD (aGVHD), the major complication and cause of post-transplant mortality in allogeneic bone marrow transplantation (BMT). At the same time, IFN-gamma facilitates graft-versus-leukemia (GVL) activity. Dissociation of GVL effects from GVHD has been the ultimate goal of allogeneic BMT in the treatment of hematologic malignancies. This paradoxic role of IFN-gamma makes modulating its activity a promising strategy to maximize GVL while minimizing GVHD and improve clinical outcomes in BMT. In this review, the effects of IFN-gamma on GVHD and GVL are discussed with consideration of the mechanism of IFN-gamma action.

Dendritic cells (DCs) are potent antigen-presenting cells that have been used in cancer immunotherapy. To take advantage of the ability of DCs to acquire antigenic materials from their environment and generate primary as well as recall immune responses, 37 patients with advanced cancers were enrolled in a series of protocols based on direct intratumoral injection of immature DCs. To augment antigen uptake and antitumor immune response, DC injection was combined with radiotherapy or chemotherapy and/or injection of activated T cells. Treatments were well tolerated with no adverse reactions. Clinical responses were based on Response Evaluation Criteria in Solid Tumors, with the majority of patients showing stable disease. One of two patients who also received local radiation achieved a sustained complete response at injected and metastatic sites. The clinical responses observed in cancer patients with advanced disease suggest potential effectiveness of combination strategies and establish the basis for the current treatment protocol that is underway.

Cells have been previously used in experimental models for tolerance induction in organ transplantation and autoimmune diseases. One problem with the therapeutic use of cells is standardization of their preparation. We discuss an immunosuppressive strategy relying on cells irreversibly transformed by a chemotherapeutic drug. Dendritic cells (DCs) of transplant donors pretreated with mitomycin C (MMC) strongly prolonged rat heart allograft survival when injected into recipients before transplantation. Likewise, MMC-DCs loaded with myelin basic protein suppressed autoreactive T cells of MS patients in vitro and prevented experimental autoimmune encephalitis in mice. Comprehensive gene microarray analysis identified genes that possibly make up the suppressive phenotype, comprising glucocorticoid leucine zipper, immunoglobulin-like transcript 3, CD80, CD83, CD86, and apoptotic genes. Based on these findings, a hypothetical model of tolerance induction by MMC-treated DCs is delineated. Finally, we describe the first clinical application of MMC-treated monocyte-enriched donor cells in an attempt to control the rejection of a haploidentical stem cell transplant in a sensitized recipient and discuss the pros and cons of using MMC-treated antigen-presenting cells for tolerance induction. Although many questions remain, MMC-treated cells are a promising clinical tool for controlling allograft rejection and deleterious immune responses in autoimmune diseases.

Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.

Mucosal administration of Ag conjugated to cholera toxin B subunit (CTB) can efficiently induce peripheral immunologic tolerance, so-called oral tolerance, associated with development of Foxp3(+)CD25(+)CD4(+) regulatory T (Treg) cells. Using an established sublingual tolerization regimen with Ag(OVA)/CTB conjugate, wherein CTB mediates Ag uptake and presentation by most B lymphocytes irrespective of their Ag specificity, we have assessed the importance of B cells for induction of Ag-specific Treg cells and oral tolerance. We found that Treg cells are reduced in microMT(-/-) B cell-deficient mice compared with wild-type (WT) mice. After sublingual Ag/CTB treatment, Treg cells increased much more in WT than in microMT(-/-) mice; however, adoptive transfer of B cells before treatment normalized Treg cell development and functional oral tolerance. B cells from OVA/CTB-treated mice expressed more IL-10 and less CD86 than control B cells. Adoptive transfer of these cells before parenteral immunization with OVA led to efficient suppression of proliferation and to induction of apoptotic depletion of Ag-specific CD25(-)CD4(+) effector T cells associated with the expansion of Treg cells. However, also OVA/CTB-treated microMT(-/-) mice could suppress the immune response to parenteral immunization with OVA, which was associated with a strong increase in Foxp3(-)CD4(+) T cells expressing LAP/TGF-beta. Our results indicate that mucosal tolerance comprises at least two separate pathways: one being B cell dependent and associated with expansion of Treg cells and Treg-mediated suppression and depletion of effector T cells, and one being B cell independent and associated with development of Foxp3(-)LAP(+)TGF-beta(+) regulatory T cells.

Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease that frequently begins at infancy and the majority of them develop asthma and/or allergic rhinitis later, in which food and inhaled allergens play an important role. There is a murine model for AD that is induced by repeated epicutaneous (e.c.) exposure with ovalbumin (OVA). This model shares many characteristic features with AD, including development of asthma as well as dermatitis. Recently, it is reported that ocular tolerance or tolerance induced by intravenous administration of in vitro generated tolerogenic antigen-presenting cells (tol-APC), which can bypasses ocular tolerance, inhibits the immune response in a murine asthma model. The present study was designed to investigate whether tolerance induced by tol-APC and ocular tolerance inhibits AD-like dermatitis induced by repeated e.c. sensitization with OVA. BALB/c mice were given a total of three 1 week e.c. exposures to OVA with 2-week intervals between exposures. After second exposure to OVA, mice received the tol-APC or received OVA in the anterior chamber (AC) of the eye (ocular tolerance). Both groups of mice received the tol-APC and mice that received OVA in the AC of the eye showed weakened cellular infiltration in the skin including eosinophils and mast cells, lower levels of antigen-specific IgE, lower levels of transcripts of IL-4, IL-5, IL-13 in the skin and less production of Th1 and Th2 cytokine by regional lymph node cells, compared with those of mice that received sham treatment and mice that received the tol-APC treated with unrelated antigen after second e.c. exposure to OVA. These results indicate that antigen-specific tolerance induced by the tol-APC and ocular tolerance can inhibit the dermatitis and its related systemic immune response in the murine AD model. These types of tolerance might lead to a new therapeutic approach to allergic skin disease.

In vitro manipulated dendritic cells (DC) have increasingly been used as a promising vaccine formulation against cancer and infectious disease. However, improved understanding of the immune mechanisms is needed for the development of safe and efficacious mucosal DC immunization. We have developed a murine model of respiratory mucosal immunization by using a genetically manipulated DC vaccine. Within 24 h of intranasal delivery, the majority of vaccine DCs migrated to the lung mucosa and draining lymph nodes and elicited a significant level of T cells capable of IFN-gamma secretion and CTL in the airway lumen as well as substantial T cell responses in the spleen. And such T cell responses were associated with enhanced protection against respiratory mucosal intracellular bacterial challenge. In comparison, parenteral i.m. DC immunization did not elicit marked airway luminal T cell responses and immune protection regardless of strong systemic T cell activation. Although repeated mucosal DC delivery boosted Ag-specific T cells in the airway lumen, added benefits to CD8 T cell activation and immune protection were not observed. By using MHC-deficient vaccine DCs, we further demonstrated that mucosal DC immunization-mediated CD8 and CD4 T cell activation does not require endogenous DCs. By using IL-12-deficient vaccine DCs, we also observed that IL-12(-/-) DCs failed to migrate to the lymph nodes but remained capable of T cell activation. Our observations indicate that mucosal delivery of vaccine DCs represents an effective approach to enhance mucosal T cell immunity, which may operate independent of vaccine IL-12 and endogenous DCs.

A growing body of evidence has shown that professional antigen-presenting cells (APC) treated with transforming growth factor-beta (TGF-beta) can induce a systemic antigen (Ag)-specific tolerance, similar to anterior chamber-associated immune deviation (ACAID). However, the exact mechanism for immune tolerance induced by TGF-beta-treated APC has not been elucidated. In this study, we showed that intravenous injection of ovalbumin (OVA)-pulsed APC treated with TGF-beta(2) induced a peripheral tolerance as evidenced by an impaired delayed-type hypersensitivity (DTH) response. CD4(+) T cells from mice receiving an intravenous injection of TGF-beta(2)-treated APC pulsed with OVA could adoptively transfer a specific tolerance to naïve mice. An increased frequency of FoxP3-expressing CD4(+) CD25(+) T cells was observed in mice with tolerance. CD4(+ )CD25(+) T cells from TGF-beta(2)-treated APC-injected mice produced a large amount of TGF-beta(1) and exhibited an in vitro antigen-specific suppressive activity. CD4(+) CD25(+) T cells from TGF-beta(2)-treated APC-injected mice were able to inhibit the antigen-specific DTH response significantly when adoptively transferred to naïve mice. These results indicate that FoxP3-expressing CD4(+) CD25(+) T cells might be actively involved in the development of tolerance induced by TGF-beta(2)-treated APC.

CD4(+) helper T cells are critical for protective immune responses and yet suboptimally primed in response to tumors. Cell-based vaccination strategies are under evaluation in clinical trials but limited by the need to derive antigen-presenting cells (APC) from patients or compatible healthy donors. To overcome these limitations, we developed CD4(+) T cell-targeted synthetic microbead-based artificial APC (aAPC) and used them to activate CD4(+) T lymphocytes specific for a tumor-associated model antigen (Ag) directly from the naive repertoire. In vitro, aAPC specifically primed Ag-specific CD4(+) T cells that were activated to express high levels of CD44, produced mainly interleukin 2, and could differentiate into Th1-like or Th2-like cells in combination with polarizing cytokines. I.v. administration of aAPC led to Ag-specific CD4(+) T-cell activation and proliferation in secondary lymphoid organs, conferred partial protection against subcutaneous tumors, and prevented the establishment of lung metastasis. Taken together, our data support the use of cell-free, synthetic aAPC as a specific and versatile alternative to expand peptide-specific CD4(+) T cells in adoptive and active immunotherapy.

Background: Human Valpha24 natural killer T (NKT) cells are activated by the specific ligand, alpha-galactosylceramide (alpha-GalCer), in a CD1d-dependent manner. Potent anti-tumor activity of activated NKT cells has been previously demonstrated., Methods: We conducted a phase I study with alpha-GalCer-pulsed antigen presenting cells (APCs) administered in the nasal submucosa of patients with head and neck cancer, and evaluated the safety and feasibility of such a treatment. Nine patients with unresectable or recurrent head and neck cancer received two treatments 1 week apart, of 1 x 10(8) of alpha-GalCer-pulsed autologous APCs into the nasal submucosa., Results: During the clinical study period, no serious adverse events (Common Terminology Criteria for Adverse Events version 3.0 greater than grade 3) were observed. After the first and the second administration of alpha-GalCer-pulsed APCs, an increased number of NKT cells was observed in four patients and enhanced natural killer activity was detected in the peripheral blood of eight patients., Conclusion: The administration of alpha-GalCer-pulsed APCs into the nasal submucosa was found to be safe and induce anti-tumor activity in some patients.

Transmembrane signaling adaptor DAP12 has increasingly been recognized for its important role in innate responses. However, its role in the regulation of antimicrobial T cell responses has remained unknown. In our current study, we have examined host defense, T cell responses, and tissue immunopathology in models of intracellular infection established in wild-type and DAP12-deficient mice. During mycobacterial infection, lack of DAP12 leads to pronounced proinflammatory and Th1 cytokine responses, overactivation of Ag-specific CD4 and CD8 T cells of type 1 phenotype, and heightened immunopathology both in the lung and lymphoid organs. DAP12-deficient airway APC display enhanced NF-kappaB activation and cytokine responses upon TLR stimulation or mycobacterial infection in vitro. Of importance, adoptive transfer of Ag-loaded DAP12-deficient APC alone could lead to overactivation of transferred transgenic or endogenous wild-type T cells in vivo. We have further found that the immune regulatory role by DAP12 is not restricted only to intracellular bacterial infection, since lack of this molecule also leads to uncontrolled type 1 T cell activation and severe immunopathology and tissue injury during intracellular viral infection. Our study thus identifies DAP12 as an important novel immune regulatory molecule that acts, via APC, to control the level of antimicrobial type 1 T cell activation and immunopathology.

In order to achieve sufficient therapeutic potency, it has been proposed that vaccine therapy with dendritic cells needs to be combined with manipulation of immunological checkpoints, such as inhibition of regulatory T cells and blockade of negative signals, and enhancement of T cell trafficking to tumor sites. In the combinatorial cancer immunotherapy, use of matured/activated dendritic cells (DCs) with more potent antigen presenting capacity seems to be essential for eliciting anti-tumor immune responses. We herein established an ex vivo induction strategy for activated DCs capable of eliciting efficient tumor antigen-specific cytotoxic T lymphocytes (CTLs) from patients with metastatic cancer as well as healthy donors. Immature DCs were matured by 48-h culture in the presence of anti-CD40 antibody and penicillin-killed streptococcus pyogenes (OK432). Supplementation with both anti-CD40 and OK432 resulted in induction of activated DCs with higher surface expression of CD80, CD83, CD86 and major histocompatibility complex class II antigens, compared with other mature DCs that were induced by the combination of anti-CD40 with tumor necrosis factor-alpha or lipopolysaccharide. In analysis of the produced cytokine profiles, the activated DCs produced the highest T-helper 1-type cytokines for at least 72 h. Furthermore, the activated DCs, pulsed with tumor-associated antigen peptide, elicited in vitro tumor-specific CTLs, but DCs activated with other combinations did not in cancer patients. Therefore, we suggest that the activated DCs studied here might be used as a basic element for the combinatorial cancer immunotherapy.

Adaptive T regulatory (T(R)) cells mediate the suppression of donor-specific, delayed-type hypersensitivity (DTH) in tolerant organ transplant recipients. We hypothesized that cells belonging to the CD4(+)CD25(+) T cell subset but distinct from natural T(R) cells may fulfill this role. To test this hypothesis, PBMC and biopsy samples from two tolerant kidney transplant recipients (K1 and K2) were analyzed. When transferred with recipient APC into a SCID mouse footpad, CD4(+) T cells were hyporesponsive in DTH to donor type HLA-B Ags and derivative allopeptides. However, anti-human TGF-beta1 Ab revealed a response to immunodominant allopeptides in both patients, suggesting that CD4(+) T effector (T(E)) cells coexisted with suppressive, TGF-beta1-producing CD4(+) T(R) cells. During in vitro culture, allopeptide stimulation induced both IFN-gamma-producing and surface TGF-beta1(+) T cells. The relative strength of the latter response in patient K1 was inversely correlated with the level of systemic anti-donor DTH, which varied over a 6-year interval. Allopeptide-induced surface TGF-beta1 expression was found primarily in Forkhead box P3 (FoxP3)-negative CD4(+)CD25(low) T cells, which could adoptively transfer suppression of donor-specific DTH. Biopsy samples contained numerous surface TGF-beta1(+) mononuclear cells that costained for CD4 and, less frequently CD25, but were negative for FoxP3. The CD4(+)TGF-beta1(+) T cells were localized primarily to the tubulointerstitium, whereas TGF-beta1(-)FoxP3(+)CD25(+) cells were found mainly in lymphoid aggregates. Thus, adaptive T(R) cells suppressing T(E) cell responses to donor allopeptides in two tolerant patients appear to be functionally and phenotypically distinct from CD4(+)CD25(high)FoxP3(+) T cells.

Purpose: Antitumor lymphocytes can be generated ex vivo unencumbered by immunoregulation found in vivo. Adoptive transfer of these cells is a promising therapeutic modality that could establish long-term antitumor immunity. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we sought to establish a clinical grade culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL)., Experimental Design: We created an off-the-shelf, standardized, and renewable artificial antigen-presenting cell (aAPC) line that coexpresses HLA class I, CD54, CD58, CD80, and the dendritic cell maturation marker CD83. We tested the ability of aAPC to generate tumor antigen-specific CTL under optimal culture conditions. The number, phenotype, effector function, and in vitro longevity of generated CTL were determined., Results: Stimulation of CD8(+) T cells with peptide-pulsed aAPC generated large numbers of functional CTL that recognized a variety of tumor antigens. These CTLs, which possess a phenotype consistent with in vivo persistence, survived ex vivo for prolonged periods of time. Clinical grade aAPC(33), produced under current Good Manufacturing Practices guidelines, generated sufficient numbers of CTL within a short period of time. These CTL specifically lysed a variety of melanoma tumor lines naturally expressing a target melanoma antigen. Furthermore, antitumor CTL were easily generated in all melanoma patients examined., Conclusions: With clinical grade aAPC(33) in hand, we are now poised for clinical translation of ex vivo generated antitumor CTL for adoptive cell transfer.

MHC class II (MHC II)-restricted T cell responses are a common driving force of autoimmune disease. Accordingly, numerous therapeutic strategies target CD4(+) T cells with the hope of attenuating autoimmune responses and restoring self-tolerance. We have previously reported that i.v. treatment with Ag-pulsed, ethylenecarbodiimide (ECDI)-fixed splenocytes (Ag-SPs) is an efficient protocol to induce Ag-specific tolerance for prevention and treatment of experimental autoimmune encephalomyelitis (EAE). Ag-SPs coupled with peptide can directly present peptide:MHC II complexes to target CD4(+) T cells in the absence of costimulation to induce anergy. However, Ag-SPs coupled with whole protein also efficiently attenuates Ag-specific T cell responses suggesting the potential contribution of alternative indirect mechanisms/interactions between the Ag-SPs and target CD4(+) T cells. Thus, we investigated whether MHC II compatibility was essential to the underlying mechanisms by which Ag-SP induces tolerance during autoimmune disease. Using MHC-deficient, allogeneic, and/or syngeneic donor Ag-SPs, we show that MHC compatibility between the Ag-SP donor and the host is not required for tolerance induction. Interestingly, we found that ECDI treatment induces apoptosis of the donor cell population which promotes uptake and reprocessing of donor cell peptides by host APCs resulting in the apparent MHC II-independent induction of tolerance. However, syngeneic donor cells are more efficient at inducing tolerance, suggesting that Ag-SPs induce functional Ag-SP tolerance via both direct and indirect (cross-tolerance) mechanisms leading to prevention and effective treatment of autoimmune disease.

Background: APC8015 (sipuleucel-T) is a cellular prostate cancer vaccine containing autologous antigen-presenting cells (APC) loaded with PA2024, a recombinant prostatic acid phosphatase/granulocyte-macrophage-colony-stimulating factor fusion protein, as the immunogen. Bevacizumab is a recombinant antibody against vascular endothelial growth factor, a proangiogenic protein with inhibitory effects on APC. A clinical trial was conducted to determine the prostate-specific antigen (PSA) and immunomodulatory effects of this combination immunotherapy., Methods: Patients with androgen-dependent prostate cancer who had received prior definitive therapy with nonmetastatic, recurrent disease as manifested by a rising PSA of between 0.4 ng/mL and 6.0 ng/mL were enrolled. APC8015 was given intravenously(i.v.) on Weeks 0, 2, and 4. Bevacizumab was given at a dose of 10 mg/kg i.v. on Weeks 0, 2, 4, and every 2 weeks thereafter until toxicity or disease progression. PSA changes were recorded and the PSA doubling time (PSADT) was calculated. Immune response versus PA2024 was measured at baseline and after treatment by T-cell proliferation and interferon-gamma enzyme-linked immunospot (ELISPOT) assays., Results: Twenty-two patients were treated. One patient achieved a > or =50% decrease in PSA. Nine patients exhibited some decrease in PSA from baseline, ranging from 6% to 72%, with the PSA of 3 patients decreasing at least 25%. The median pretreatment PSADT for the 20 evaluable patients was 6.9 months and the median posttreatment PSADT was 12.7 months (P = .01). All patients demonstrated induction of an immune response against PA2024., Conclusions: The combination of APC8015 and bevacizumab induces an immune response and modulates PSA in patients with biochemically recurrent prostate cancer., (Copyright 2006 American Cancer Society.)