Alexander Michael Dohnal

Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines

Manufacturing procedures for cellular therapies are continuously improved with particular emphasis on product safety. We previously developed a dendritic cell (DC) cancer vaccine technology platform that uses clinical grade lipopolysaccharide (LPS) and interferon (IFN)‐y for the maturation of monocyte derived DCs. DCs are frozen after 6 hrs exposure at a semi‐mature stage (smDCs) retaining the capacity to secret interleukin (IL)‐12 and thus support cytolytic T‐cell responses, which is lost at full maturation. We compared closed systems for monocyte enrichment from leucocyte apheresis products from healthy individuals using plastic adherence, CD14 selection, or CD2/19 depletion with magnetic beads, or counter flow centrifugation (elutriation) using a clinical grade in comparison to a research grade culture medium for the following DC generation. We found that elutriation was superior compared to the other methods showing 36 ± 4% recovery, which was approximately 5‐fold higher as the most frequently used adherence protocol (8 ± 1%), and a very good purity (92 ± 5%) of smDCs. Immune phenotype and IL‐12 secretion (adherence: 1.4 ± 0.4; selection: 20 ± 0.6; depletion: 1 ±0.5; elutriation: 3.6 ± 1.5 ng/ml) as well as the potency of all DCs to stimulate T cells in an allogeneic mixed leucocyte reaction did not show statistically significant differences. Research grade and clinical grade DC culture media were equally potent and freezing did not impair the functions of smDCs. Finally, we assessed the functional capacity of DC cancer vaccines manufactured for three patients using this optimized procedure thereby demonstrating the feasibility of manufacturing DC cancer vaccines that secret IL‐12 (9.4 ± 6.4 ng/ml). We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing.

Immune Suppression by γδ T-cells as a Potential Regulatory Mechanism After Cancer Vaccination With IL-12 Secreting Dendritic Cells

In cancer patients undergoing immune therapy with lipopolysaccharides/interferon-γ activated interleukin (IL)-12 secreting dendritic cells (DCs) we observed enhanced proliferative capacity of pyrophosphate-responsive peripheral blood (PB) γδ T-cells. This was not noted before as in other clinical trials DCs were used that were not enabled for IL-12 secretion and mice do not have a corresponding subset of PB γδ T-cells. In vitro examination of IL-12 DC/PB γδ T-cell interactions revealed a potential of PB γδ T-cells to negatively regulate the proliferative capacity of CD4 and CD8 T-cells. We further demonstrate that IL-12 is critical in the activation of PB γδ T-cells. In contrast, the regulatory activity of PB γδ T-cells in immune responses against strong recall antigens or alloantigens did not require activated DCs, but depended on pyrophosphate activation of PB γδ T-cells. Depletion of PB γδ T-cells abrogated the regulatory activity in IL-12 DC/peripheral blood mononuclear cell cocultures; adding back graded numbers of PB γδ T-cells restored it. Our observations revealed a potential PB γδ T cell-mediated negative regulatory feedback mechanism triggered by IL-12 DCs, which may critically impact on the design of DC cancer vaccines.

Toll-Like Receptor 4 Engagement Drives Differentiation of Human and Murine Dendritic Cells from a Pro- into an Anti-Inflammatory Mode

The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs’ initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.

CD40 ligation restores type 1 polarizing capacity in TLR4-activated dendritic cells that have ceased interleukin-12 expression.

Inflammation triggered by microbial lipopolysaccharide (LPS) through Toll‐like receptor (TLR) 4 in the presence of interferon (IFN)‐γ induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation program. This DC differentiation is characterized not only by a dynamic immune activating but also by tolerance‐inducing phenotype associated with down‐modulation of cytokines previously considered to be irreversible. CD40L on activated T cells further modifies DC differentiation. Using DNA micro‐arrays, we showed down‐regulated mRNA levels of TLR signalling molecules, whereas CD40/CD40L signalling molecules were up‐regulated at a time when LPS/IFN‐γ‐activated DCs had ceased cytokine expression. Accordingly, we demonstrated that CD40/CD40L but not TLR4 or TLR3 signalling mediated by LPS or poly (cytidylic‐inosinic) acid (poly I:C) and dsRNA re‐established the capacity for secreting interleukin (IL)‐12 in primarily LPS/IFN‐γ‐activated DCs, which have exhausted their potential for cytokine secretion. The resulting TH1 polarizing DC phenotype – which lacked accompanying secretion of the crucial immune suppressive factor IL‐10 – maintained the potential for activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T‐cell‐mediated stimulus at an exhausted DC state, which prevents an immune tolerant DC phenotype. These findings impact on the rational design of TLR‐activated DC‐based cancer vaccines for the induction of anti‐tumoural CTL responses.

The MAPK-Activated Kinase MK2 Attenuates Dendritic Cell–Mediated Th1 Differentiation and Autoimmune Encephalomyelitis

Dendritic cell (DC)–mediated inflammation induced via TLRs is promoted by MAPK-activated protein kinase (MK)-2, a substrate of p38 MAPK. In this study we show an opposing role of MK2, by which it consolidates immune regulatory functions in DCs through modulation of p38, ERK1/2-MAPK, and STAT3 signaling. During primary TLR/p38 signaling, MK2 mediates the inhibition of p38 activation and positively cross-regulates ERK1/2 activity, leading to a reduction of IL-12 and IL-1α/β secretion. Consequently, MK2 impairs secondary autocrine IL-1α signaling in DCs, which further decreases the IL-1α/p38 but increases the anti-inflammatory IL-10/STAT3 signaling route. Therefore, the blockade of MK2 activity enables human and murine DCs to strengthen proinflammatory effector mechanisms by promoting IL-1α–mediated Th1 effector functions in vitro. Furthermore, MK2-deficient DCs trigger Th1 differentiation and Ag-specific cytotoxicity in vivo. Finally, wild-type mice immunized with LPS in the presence of an MK2 inhibitor strongly accumulate Th1 cells in their lymph nodes. These observations correlate with a severe clinical course in DC-specific MK2 knockout mice compared with wild-type littermates upon induction of experimental autoimmune encephalitis. Our data suggest that MK2 exerts a profound anti-inflammatory effect that prevents DCs from prolonging excessive Th1 effector T cell functions and autoimmunity.

Leukemia-associated antigenic isoforms induce a specific immune response in children with T-ALL

The potential immunogenicity of acute lymphoblastic leukemia of the T cell (T‐ALL), a small subgroup of childhood leukemia with increased risk for treatment failure and early relapse, was addressed by serological identification of leukemia‐derived antigens by recombinant expression cloning (SEREX). Thirteen antigens with homology to known genes that are involved in critical cellular processes were detected. Further characterization of the 4 novel isoforms revealed that 3 (HECTD1Δ, CX‐ORF‐15Δ and hCAP‐EΔ) had restricted mRNA expression in more than 70% of T‐ALLs (n = 22) and that specific antibodies against these isoforms were detected in up to 30% of patients (n = 16), with the highest frequency for HECTD1Δ. The latter protein was present at high abundance in T‐ALLs but not in normal hematopoietic tissues. Given that the leukemia‐associated antigens detected in this study have an intracellular localization, the generation of immune effector responses most likely requires antigen presentation. To test this assumption, dendritic cells were loaded with HECTD1Δ protein and used for T cell stimulation. A specific T cell response was induced in vitro in all 3 healthy donors studied, including a former T‐ALL patient. These data suggest that T‐ALL may induce a specific cellular and humoral antileukemia immune response in children, thereby supporting new approaches for immunotherapy.

Loss of Phosphatase and Tensin Homolog in APCs Impedes Th17-Mediated Autoimmune Encephalomyelitis

The PI3K signaling cascade in APCs has been recognized as an essential pathway to initiate, maintain, and resolve immune responses. In this study, we demonstrate that a cell type–specific loss of the PI3K antagonist phosphatase and tensin homolog (PTEN) in myeloid cells renders APCs toward a regulatory phenotype. APCs deficient for PTEN exhibit reduced activation of p38 MAPK and reduced expression of T cell–polarizing cytokines. Furthermore, PTEN deficiency leads to upregulation of markers for alternative activation, such as Arginase 1, with concomitant downregulation of inducible NO synthase in APCs in vitro and in vivo. As a result, T cell polarization was dysfunctional in PTEN−/− APCs, in particular affecting the Th17 cell subset. Intriguingly, mice with cell type–specific deletions of PTEN-targeting APCs were protected from experimental autoimmune encephalomyelitis, which was accompanied by a pronounced reduction of IL-17– and IL-22–producing autoreactive T cells and reduced CNS influx of classically activated monocytes/macrophages. These observations support the notion that activation of the PI3K signaling cascade promotes regulatory APC properties and suppresses pathogenic T cell polarization, thereby reducing the clinical symptoms and pathology of experimental autoimmune encephalomyelitis.

CD28 Blockade Ex Vivo Induces Alloantigen-Specific Immune Tolerance but Preserves T-Cell Pathogen Reactivity

Donor T-cells contribute to reconstitution of protective immunity after allogeneic hematopoietic stem cell transplantation (HSCT) but must acquire specific tolerance against recipient alloantigens to avoid life-threatening graft-versus-host disease (GvHD). Systemic immunosuppressive drugs may abrogate severe GvHD, but this also impedes memory responses to invading pathogens. Here, we tested whether ex vivo blockade of CD28 co-stimulation can enable selective T-cell tolerization to alloantigens by facilitating CD80/86-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) signaling. Treatment of human allogeneic dendritic cell/T-cell co-cultures with a human CD28 blocking antibody fragment (α-huCD28) significantly abrogated subsequent allospecific immune responses, seen by decreased T-cell proliferation and of type 1 cytokine (IFN-γ and IL-2) expression. Allo-tolerization persisted after discontinuation of CD28 blockade and secondary alloantigen stimulation, as confirmed by enhanced CTLA-4 and PD-1 immune checkpoint signaling. However, T-cells retained reactivity to pathogens, supported by clonotyping of neo-primed and cross-reactive T-cells specific for Candida albicans or third-party antigens using deep sequencing analysis. In an MHC-mismatched murine model, we tolerized C57BL/6 T-cells by ex vivo exposure to a murine single chain Fv specific for CD28 (α-muCD28). Infusion of these cells, after α-muCD28 washout, into bone marrow-transplanted BALB/c mice caused allo-tolerance and did not induce GvHD-associated hepatic pathology. We conclude that selective CD28 blockade ex vivo can allow the generation of stably allo-tolerized T-cells that in turn do not induce graft-versus-host reactions while maintaining pathogen reactivity. Hence, CD28 co-stimulation blockade of donor T-cells may be a useful therapeutic approach to support the immune system after HSCT.

Loss of MAPK-activated protein kinase 2 enables potent dendritic cell-driven anti-tumour T cell response.

Maintaining dendritic cells (DC) in a state of dysfunction represents a key mechanism by which tumour cells evade recognition and elimination by the immune system. Limited knowledge about the intracellular mediators of DC dysfunction restricts success of therapies aimed at reactivating a DC-driven anti-tumour immune response. Using a cell type-specific murine knock-out model, we have identified MAPK-activated protein kinase 2 (MK2) as a major guardian of a suppressive DC phenotype in the melanoma tumour microenvironment. MK2 deletion in CD11c+ cells led to an expansion of stimulatory CD103+ DCs, mounting a potent CD8+ T cell response that resulted in elimination of highly aggressive B16-F10 tumours upon toll-like receptor (TLR) activation in the presence of tumour antigen. Moreover, tumour infiltration by suppressive myeloid cells was strongly diminished. These insights into the regulation of DC functionality reveal MK2 as a targetable pathway for DC-centred immunomodulatory cancer therapies.

Mast Cells Are Abundant in Primary Cutaneous T-Cell Lymphomas: Results from a Computer-Aided Quantitative Immunohistological Study

Background Mast cells (MC) are bone marrow derived haematopoetic cells playing a crucial role not only in immune response but also in the tumor microenvironment with protumorigenic and antitumorigenic functions. The role of MC in primary cutaneous T-cell lymphomas (CTCL), a heterogeneous group of non-Hodgkin lymphomas with initial presentation in the skin, is largely unknown. Objective To gain more accurate information about presence, number, distribution and state of activation (degranulated vs. non-degranulated) of MC in CTCL variants and clinical stages. Materials and Methods We established a novel computer-aided tissue analysis method on digitized skin sections. Immunohistochemistry with an anti-MC tryptase antibody was performed on 34 biopsies of different CTCL subtypes and on control skin samples. An algorithm for the automatic detection of the epidermis and of cell density based CTCL areas was developed. Cells were stratified as being within the CTCL infiltrate, in P1 (a surrounding area 0–30 μm away from CTCL), or in P2 (30–60 μm away from CTCL) area. Results We found high MC counts within CTCL infiltrates and P1 and a decreased MC number in the surrounding dermis P2. Higher MC numbers were found in MF compared to all other CTCL subgroups. Regarding different stages of MF, we found significantly higher mast cell counts in stages IA and IB than in stages IIA and IIB. Regarding MC densities, we found a higher density of MC in MF compared to all other CTCL subgroups. More MC were non-degranulated than degranulated. Conclusion Here for the first time an automated method for MC analysis on tissue sections and its use in CTCL is described. Eliminating error from investigator bias, the method allows for precise cell identification and counting. Our results provide new insights on MC distribution in CTCL reappraising their role in the pathophysiology of CTCL.