Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7.

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

Multidimensional Analysis Integrating Human T-Cell Signatures in Lymphatic Tissues with Sex of Humanized Mice for Prediction of Responses after Dendritic Cell Immunization.

Mice transplanted with human cord blood-derived hematopoietic stem cells (HSCs) became a powerful experimental tool for studying the heterogeneity of human immune reconstitution and immune responses in vivo. Yet, analyses of human T cell maturation in humanized models have been hampered by an overall low immune reactivity and lack of methods to define predictive markers of responsiveness. Long-lived human lentiviral induced dendritic cells expressing the cytomegalovirus pp65 protein (iDCpp65) promoted the development of pp65-specific human CD8+ T cell responses in NOD.Cg-Rag1 tm1Mom -Il2rγ tm1Wj humanized mice through the presentation of immune-dominant antigenic epitopes (signal 1), expression of co-stimulatory molecules (signal 2), and inflammatory cytokines (signal 3). We exploited this validated system to evaluate the effects of mouse sex in the dynamics of T cell homing and maturation status in thymus, blood, bone marrow, spleen, and lymph nodes. Statistical analyses of cell relative frequencies and absolute numbers demonstrated higher CD8+ memory T cell reactivity in spleen and lymph nodes of immunized female mice. In order to understand to which extent the multidimensional relation between organ-specific markers predicted the immunization status, the immunophenotypic profiles of individual mice were used to train an artificial neural network designed to discriminate immunized and non-immunized mice. The highest accuracy of immune reactivity prediction could be obtained from lymph node markers of female mice (77.3%). Principal component analyses further identified clusters of markers best suited to describe the heterogeneity of immunization responses in vivo. A correlation analysis of these markers reflected a tissue-specific impact of immunization. This allowed for an organ-resolved characterization of the immunization status of individual mice based on the identified set of markers. This new modality of multidimensional analyses can be used as a framework for defining minimal but predictive signatures of human immune responses in mice and suggests critical markers to characterize responses to immunization after HSC transplantation.

Dendritic-tumor cell hybrids induce tumor-specific immune responses more effectively than the simple mixture of dendritic and tumor cells.

BACKGROUND AIMS: Dendritic cell (DC)-tumor cell hybrids have been used clinically in cancer immunotherapy, but their advantage over the simple mixture of tumor cells and DCs is still a matter of controversy. In this study, we compared DC-tumor cell hybrids with the non-fused mixture of DC and tumor cells directly in their ability to induce a specific immune response. METHODS: Hybrids were obtained by electrofusion of tumor cells and monocyte-derived DCs. Cell phenotype was evaluated by flow cytometry and antigen-presenting ability by co-culture with syngeneic T cells followed by tetramer analysis and interferon (IFN)-γ ELISPOT. RESULTS: Less than half the cells in the mixture expressed DC co-stimulatory molecules. Furthermore, DCs in the mixture had significantly lower expression of MHC class I molecules than DCs in the fusion. Conversely, nearly all CD11c(+)Her2/neu(+) hybrids expressed CD80, CD86, CD83, HLA-DR and MHC class I from both tumor cells and DCs. Using tumor cells constitutively expressing a cytomegalovirus (CMV) antigen, we show that expansion of CMV-specific cytotoxic T lymphocytes (CTLs) restricted by DCs' MHC class I molecules was higher when DC-tumor hybrids were the stimulators. Furthermore, only hybrids stimulated CTLs to produce IFN-γ in response to CMV-positive target cells. CONCLUSIONS: These data show the superiority of DC-tumor cell hybrids over their simple mixture as T-cell stimulators. Hybrids expressed more co-stimulatory and MHC molecules, induced higher antigen-specific T-cell expansion and were the only cells able to induce IFN-γ-producing antigen-specific T cells. Thus, these data offer further support for cancer immunotherapeutic approaches using DC-tumor cell hybrids.

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation.

BACKGROUND: Reactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs ("SmyleDCpp65") that accelerated antigen-specific T cell development. METHODS: Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed. RESULTS: Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated. CONCLUSION: These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.

Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV.

Human cytomegalovirus (HCMV) harmfully impacts survival after peripheral blood hematopoietic stem cell transplantation (PB-HSCT). Delayed immune reconstitution after cord blood (CB)-HSCT leads to even higher HCMV-related morbidity and mortality. Towards a feasible dendritic cell therapy to accelerate de novo immunity against HCMV, we validated a tricistronic integrase-defective lentiviral vector (coexpressing GM-CSF, IFN-α, and HCMV pp65 antigen) capable to directly induce self-differentiation of PB and CB monocytes into dendritic cells processing pp65 ("SmyleDCpp65"). In vitro, SmyleDCpp65 resisted HCMV infection, activated CD4(+) and CD8(+) T cells and expanded functional pp65-specific memory cytotoxic T lymphocytes (CTLs). CD34(+) cells obtained from PB and CB were transplanted into irradiated NOD.Rag1(-/-).IL2γc(-/-) mice. Donor-derived SmyleDCpp65 administration after PB-HSCT stimulated peripheral immune effects: lymph node remodeling, expansion of polyclonal effector memory CD8(+) T cells in blood, spleen and bone marrow, and pp65-reactive CTL and IgG responses. SmyleDCpp65 administration after CB-HSCT significantly stimulated thymopoiesis. Expanded frequencies of CD4(+)/CD8(+) T cell precursors containing increased levels of T-cell receptor excision circles in thymus correlated with peripheral expansion of effector memory CTL responses against pp65. The comparative in vivo modeling for PB and CB-HSCT provided dynamic and spatial information regarding human T and B cell reconstitution. In vivo potency supports future clinical development of SmyleDCpp65.

Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation.

De novo regeneration of immunity is a major problem after allogeneic hematopoietic stem cell transplantation (HCT). HCT modeling in severely compromised immune-deficient animals transplanted with human stem cells is currently limited because of incomplete maturation of lymphocytes and scarce adaptive responses. Dendritic cells (DC) are pivotal for the organization of lymph nodes and activation of naive T and B cells. Human DC function after HCT could be augmented with adoptively transferred donor-derived DC. In this study, we demonstrate that adoptive transfer of long-lived human DC coexpressing high levels of human IFN-α, human GM-CSF, and a clinically relevant Ag (CMV pp65 protein) promoted human lymphatic remodeling in immune-deficient NOD.Rag1(-/-).IL-2rγ(-/-) mice transplanted with human CD34(+) cells. After immunization, draining lymph nodes became replenished with terminally differentiated human follicular Th cells, plasma B cells, and memory helper and cytotoxic T cells. Human Igs against pp65 were detectable in plasma, demonstrating IgG class-switch recombination. Human T cells recovered from mice showed functional reactivity against pp65. Adoptive immunotherapy with engineered DC provides a novel strategy for de novo immune reconstitution after human HCT and a practical and effective tool for studying human lymphatic regeneration in vivo in immune deficient xenograft hosts.

Monitoring dendritic cell and cytokine biomarkers during remission prior to relapse in patients with FLT3-ITD acute myeloid leukemia.

Relapse occurs frequently after treatment of acute myeloid leukemia (AML) patients with the FMS-like tyrosine kinase 3-internal tandem duplication (ITD) mutation. The availability of immunologic biomarkers to predict patients at high risk could allow clinicians to accelerate alternative treatments such as stem cell transplantation, immunotherapy, or novel drugs. We have previously reported that first diagnostic (FD) ITD(+) AML showed immunophenotypic and functional characteristics of arrested dendritic cell (DC) precursors. In this study, we show that the high frequency of precursor DCs in 16 FD ITD(+) AML samples (Lin(-)/HLA-DR(+)/CD11c(+)/CD123(+)) was associated with a lack of terminal DCs (myeloid DCs: BDCA-1(+) or BDCA-3(+); plasmacytoid DC: BDCA-2(+)). We further evaluated prospectively the peripheral blood complete remission (CR) samples obtained from 11 ITD(+) AML patients after chemotherapy regarding the frequency of DCs and their pattern of cytokine production. Whereas the aberrant frequencies of precursor and terminal plasmacytoid DCs resolved during remission, the myeloid DC compartment did not fully recover. For an available cohort of patients (n = 4) who could be monitored over a period of >15 months after FD, we identified IL-10, TNF-α, IL-6, and IL-1ß as cytokines produced by the CR samples at high levels a few months prior to relapse. Cell-free supernatant of an FD ITD(+) AML sample stimulated monocytes obtained from two healthy donors to secrete IL-10, TNF-α, IL-6, and IL-1ß. Thus, we hypothesize that ITD(+) AML minimal residual disease can act directly as dysfunctional antigen-presenting cells or indirectly by production of factors that convert monocytes into myeloid-derived suppressor cells secreting cytokines that promote immune evasion. Monitoring these immunologic biomarkers could improve prediction of relapse.

Lentivirus-induced dendritic cells for immunization against high-risk WT1(+) acute myeloid leukemia.

Wilms' tumor 1 antigen (WT1) is overexpressed in acute myeloid leukemia (AML), a high-risk neoplasm warranting development of novel immunotherapeutic approaches. Unfortunately, clinical immunotherapeutic use of WT1 peptides against AML has been inconclusive. With the rationale of stimulating multiantigenic responses against WT1, we genetically programmed long-lasting dendritic cells capable of producing and processing endogenous WT1 epitopes. A tricistronic lentiviral vector co-expressing a truncated form of WT1 (lacking the DNA-binding domain), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) was used to transduce human monocytes ex vivo. Overnight transduction induced self-differentiation of monocytes into immunophenotypically stable "SmartDC/tWT1" (GM-CSF(+), IL-4(+), tWT1(+), IL-6(+), IL-8(+), TNF-α(+), MCP-1(+), HLA-DR(+), CD86(+), CCR2(+), CCR5(+)) that were viable for 3 weeks in vitro. SmartDC/tWT1 were produced with peripheral blood mononuclear cells (PBMC) obtained from an FLT3-ITD(+) AML patient and surplus material from a donor lymphocyte infusion (DLI) and used to expand CD8(+) T cells in vitro. Expanded cytotoxic T lymphocytes (CTLs) showed antigen-specific reactivity against WT1 and against WT1(+) leukemia cells. SmartDC/tWT1 injected s.c. into Nod.Rag1(-/-).IL2rγc(-/-) mice were viable in vivo for more than three weeks. Migration of human T cells (huCTLs) to the immunization site was demonstrated following adoptive transfer of huCTLs into mice immunized with SmartDC/tWT1. Furthermore, SmartDC/tWT1 immunization plus adoptive transfer of T cells reactive against WT1 into mice resulted in growth arrest of a WT1(+) tumor. Gene array analyses of SmartDC/tWT1 demonstrated upregulation of several genes related to innate immunity. Thus, SmartDC/tWT1 can be produced in a single day of ex vivo gene transfer, are highly viable in vivo, and have great potential for use as immunotherapy against malignant transformation overexpressing WT1.

Integrase-defective lentiviral vectors encoding cytokines induce differentiation of human dendritic cells and stimulate multivalent immune responses in vitro and in vivo.

Integrase-defective lentiviral vectors (ID-LVs) show several hallmarks of conventional lentiviral vectors such as absence of cytotoxic effects and long-term expression in non-replicating target cells. The integration rate of ID-LVs into the genome of target cells is dramatically reduced, which enhances safety and opens perspectives for their use in vaccine development. ID-LVs have been shown to be effective vaccines in mouse models, but functional studies with human cells in vitro and in vivo are lacking. Here, we evaluated whether ID-LVs expressing combinations of cytokines (GM-CSF/IL-4 or GM-CSF/IFN-α) used to transduce human monocytes would result in functional "induced dendritic cells" (iDCs). Overnight transduction of monocytes with high titer ID-LVs generated highly viable (14 days) and immunophenotypically stable iDCs expressing GM-CSF/IL-4 ("SmartDCs") or GM-CSF/IFN-α ("SmyleDCs"). SmartDCs and SmyleDCs maintained in vitro continuously secreted the transgenic cytokines and showed up-regulation of several endogenously produced inflammatory cytokines (IFN-γ, IL-2, -5, -6, and -8). Both iDC types potently stimulated T cells in mixed lymphocyte reactions at levels comparable to conventional DCs (maintained with exogenous cytokines). A single in vitro stimulation of CD8(+) T cells with autologous SmartDCs or SmyleDCs pulsed with peptide pools of pp65 (a human cytomegalovirus antigen) resulted in high expansion of central memory and effector memory CTLs reactive against different pp65 epitopes. We further evaluated the effects of SmartDCs and SmyleDCs to expand anti-pp65 CTLs in vivo using immune deficient NOD/Rag1((-/-))/IL-2rγ((-/-)) (NRG) mice. NRG mice immunized subcutaneously with SmartDCs or SmyleDCs co-expressing the full-length pp65 were subsequently infused with autologous CD8(+) T cells. Both types of iDCs effectively stimulated human CTLs reactive against different pp65 antigenic determinants in vivo. Due to the simplicity of generation, robust viability and combined capacity to stimulate homeostatic, antigenic and multivalent responses, iDCs are promising vaccines to be explored in immunization of lymphopenic patients in the post-transplantation setting.

Identity, potency, in vivo viability, and scaling up production of lentiviral vector-induced dendritic cells for melanoma immunotherapy.

SmartDCs (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors) consist of highly viable dendritic cells (DCs) induced to differentiate with lentiviral vectors (LVs) after an overnight ex vivo transduction. Tricistronic vectors co-expressing cytokines (granulocyte-macrophage-colony stimulating factor [GM-CSF], interleukin [IL]-4) and a melanoma antigen (tyrosine related protein 2 [TRP2]) were used to transduce mouse bone marrow cells or human monocytes. Sixteen hours after transduction, the cells were dispensed in aliquots and cryopreserved for identity, potency, and safety analyses. Thawed SmartDCs readily differentiated into highly viable cells with a DC immunophenotype. Prime/boost subcutaneous administration of 1×10(6) thawed murine SmartDCs into C57BL/6 mice resulted into TRP2-specific CD8(+) T-cell responses and protection against lethal melanoma challenge. Human SmartDC-TRP2 generated with monocytes obtained from melanoma patients secreted endogenous cytokines associated with DC activation and stimulated TRP2-specific autologous T-cell expansion in vitro. Thawed human SmartDCs injected subcutaneously in NOD.Rag1(-/-).IL2rγ(-/-) mice maintained DC characteristics and viability for 1 month in vivo and did not cause any signs of pathology. For development of good manufacturing practices, CD14(+) monocytes selected by magnetic-activated cell separation were transduced in a closed bag system (multiplicity of infection of 5), washed, and cryopreserved. Fifty percent of the monocytes used for transduction were recovered for cryopreservation. Thawed SmartDCs produced in two independent runs expressed the endogenous cytokines GM-CSF and IL-4, and the resulting homogeneous SmartDCs that self-differentiated in vitro contained approximately 1.5-3.0 copies of integrated LVs per cell. Thus, this method facilitates logistics, standardization, and high recovery for the generation of viable genetically reprogrammed DCs for clinical applications.

Preconditioning therapy with lentiviral vector-programmed dendritic cells accelerates the homeostatic expansion of antigen-reactive human T cells in NOD.Rag1-/-.IL-2rγc-/- mice.

Dendritic cell (DC)-based immunization is a potent strategy to direct prompt and durable immune responses against viral reactivations after transplantations. Here, we show that overnight lentiviral vector (LV) gene transfer into human monocytes co-expressing granulocyte-macrophage colony stimulating factor and interleukin (IL)-4 induced self-differentiated DCs (SMART-DCs) with stable DC immunophenotype over weeks in culture and secreted several inflammatory cytokines. SMART-DCs injected subcutaneously in immunodeficient NOD.Rag1(-/-).IL2rγ(-/-) (NRG) mice 1 day after LV transduction were stable for a month in vivo. "Conventional" DCs (cDCs) and SMART-DCs were compared with regard to their potency to accelerate the expansion, biodistribution, and antigenic stimulation of autologous human T cells. Peripheral blood cells obtained from human cytomegalovirus (hCMV)-reactive donors and full-length hCMV pp65 antigenic protein or peptides were used. DCs loaded with pp65 were administered subcutaneously into NRG mice as a preconditioning treatment a week prior to intravenous infusion with T cells. Optical imaging analyses demonstrated that in mice preconditioned with SMART-DC-pp65, T cells were directly recruited to the immunization site and subsequently spread to the spleen and other organs. A dramatic expansion of both human CD8(+) and CD4(+) T cells could be observed within a few days after infusion, and this was associated with consistent measurable CD8(+) effector memory T-cell responses against different pp65 epitopes. Thus, this mouse model demonstrates the proof-of-principle for SMART-DCs to accelerate expansion of human lymphocytes, resulting in poly-functional and antigen-specific immune responses against hCMV-pp65.

Lentiviral vectors for immunization: an inflammatory field.

Lentiviruses are retroviruses that are able to transduce both dividing and nondividing cells. Dendritic cells are key players in the innate and adaptive immune responses, and are natural targets for lentiviruses. Lentiviral vectors (LVs) have recently reached the clinical gene therapy arena, prompting their use as clinical vaccines. In recent years, LVs have emerged as a robust and practical experimental platform for gene delivery and rational genetic reprogramming of dendritic cells. Here, we present the status quo of the LV system for protective or therapeutic vaccine development. This vector system has been extensively evaluated for ex vivo and in vivo (immuno)gene delivery. Improvements of the LV design in order to further grant a higher biosafety profile for vaccine development are presented.

Monocytes transduced with lentiviral vectors expressing hepatitis C virus non-structural proteins and differentiated into dendritic cells stimulate multi-antigenic CD8(+) T cell responses.

Halting the spread of hepatitis C virus (HCV) and also eradicating HCV in subjects with chronic infection are major goals for global health. To this end, several years of research on HCV vaccine development have led to the conclusion that multi-antigenic and multi-functional vaccine types are necessary for effectiveness against HCV infection. In this study, we evaluated lentiviral vectors (LV) expressing clusters of HCV structural (LV-HCV-S) and non-structural (LV-HCV-NS) genes for future vaccine development. Batches of high titer LV were used to transduce differentiated dendritic cells (DC) and monocytes. We report successful delivery of HCV gene clusters, particularly into monocytes, leading to >80% LV-HCV-NS and >70% LV-HCV-S and transduced cells, respectively. Intracellular expression of HCV proteins in monocyte-derived DC resulted in immunophenotypic changes, such as downregulation of CD83 and CD86. Monocytes expressing NS proteins and differentiated into DC stimulated allogeneic and autologous CD8(+) and CD4(+) T cells in vitro and resulted in antigen-specific CD8(+) T cell responses against NS3, NS4a and NS5b. Hence, lentiviral-mediated expression of the multi-antigenic HCV-NS cluster in monocytes subsequently differentiated into DC is a novel potential anti-HCV vaccine modality.