Sabine Hoves

Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy

Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for these macrophages is macrophage colony-stimulating factor 1 (CSF-1). We generated a monoclonal antibody (RG7155) that inhibits CSF-1 receptor (CSF-1R) activation. In vitro RG7155 treatment results in cell death of CSF-1-differentiated macrophages. In animal models, CSF-1R inhibition strongly reduces F4/80(+) tumor-associated macrophages accompanied by an increase of the CD8(+)/CD4(+) T cell ratio. Administration of RG7155 to patients led to striking reductions of CSF-1R(+)CD163(+) macrophages in tumor tissues, which translated into clinical objective responses in diffuse-type giant cell tumor (Dt-GCT) patients.

ISCOMATRIX Adjuvant Combines Immune Activation with Antigen Delivery to Dendritic Cells In Vivo Leading to Effective Cross-Priming of CD8(+) T Cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α+ DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α+ DCs in VDLNs, whereas Langerin+ DCs and CD8α− DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α+ DCs in vivo for efficient induction of CTL responses.

The battlefield of perforin/granzyme cell death pathways

A pore‐forming protein, PRF, and serine proteases, Grz, are key effector molecules of CL. These toxins are stored within secretory granules, which exocytose their contents in response to immune synapse formation between the CL and virus‐infected or transformed target cell. There, PRF and Grz synergize to induce various apoptotic death pathways and to maintain immune homeostasis. Mechanistic aspects of the synergy and apoptotic mechanisms are still not fully understood, and the current review will address some of the hotly debated controversies in the field.

Monocyte-Derived Human Macrophages Mediate Anergy in Allogeneic T Cells and Induce Regulatory T Cells

Activation of alloreactive T cells by APCs such as dendritic cells (DC) has been implicated as crucial step in transplant rejection. In contrast, it has been proposed that macrophages (Mφ) maintain tolerance toward alloantigens. It was therefore the aim of this study to further analyze the T cell-stimulatory capacity of mature DC and Mφ in vitro using the model of allogeneic MLR. There was a strong proliferative response in T cells cocultured with DC, which was further increased upon restimulation in a secondary MLR. In contrast, T cells did not proliferate in cocultures with Mφ despite costimulation with anti-CD28 and IL-2. Cytokine analysis revealed considerable levels of IL-10 in cocultures of T cells with Mφ, whereas high amounts of IL-2 and IFN-γ were present in cocultures with DC. There was only minimal T cell proliferation in a secondary MLR when T cells were rescued from primary MLR with Mφ and restimulated with DC of the same donor, or DC of an unrelated donor (third party), whereas a strong primary proliferative response was observed in resting T cells, demonstrating induction of T cell anergy by Mφ. Functional analysis of T cells rescued from cocultures with Mφ demonstrated that anergy was at least partly mediated by IL-10-producing regulatory T cells induced by Mφ. These results demonstrate that Mφ drive the differentiation of regulatory T cells and mediate anergy in allogeneic T cells, supporting the concept that Mφ maintain peripheral tolerance in vivo.

CSF-1/CSF-1R targeting agents in clinical development for cancer therapy.

Macrophage infiltration has been identified as an independent poor prognostic factor for several cancer entities. In mouse tumor models macrophages orchestrate various tumor-promoting processes. This observation sparked an interest to therapeutically target these plastic innate immune cells. To date, blockade of colony stimulating factor-1 or its receptor represents the only truly selective approach to manipulate macrophages in cancer patients. Here, we discuss the currently available information on efficacy and safety of various CSF-1/CSF-1R inhibitors in cancer patients and highlight potential combination partners emerging from preclinical studies while considering the differences between mouse and human macrophage biology.

Presentation of tumour antigens by dendritic cells and challenges faced

The use of dendritic cells (DCs) for the generation of anti-tumour immunity has been the focus of a vast array of scientific and clinical studies. The ability of DCs to present protein tumour antigens (T-Ags) to CD4(+) and CD8(+) T cells is pivotal to the success of therapeutic cancer vaccines. DCs specialised capacity to cross-present exogenous Ags onto major histocompatibility (MHC) class I molecules for the generation of T-Ag-specific cytotoxic T lymphocytes (CTLs) has made these cells the focal point of vaccine-based immunotherapy of cancer. However, although DC-based strategies can induce T cell responses in cancer patients, recent reviews of clinical studies demonstrate that DC-based approaches have essentially failed to meet their clinical end points. These findings highlight the need to re-evaluate the DC-based vaccine strategies and incorporate recent advancements in DC biology and tumour immunology. The current review considers the issues related to how best to target the Ag-processing pathway of DCs, the role of adjuvants, the appropriate conditioning of the DCs and strategies to overcome tumour-mediated immune escape.

Mature But Not Immature Fas Ligand (CD95L)-Transduced Human Monocyte-Derived Dendritic Cells Are Protected from Fas-Mediated Apoptosis and Can Be Used as Killer APC

Several in vitro and animal studies have been performed to modulate the interaction of APCs and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells in an Ag-specific manner. However, due to the difficulties in vector generation and low transduction frequencies, similar studies with primary human APC are still lacking. To evaluate whether Fas ligand (FasL/CD95L) expressing killer APC could be generated from primary human APC, monocyte-derived dendritic cells (DC) were transduced using the inducible Cre/Loxp adenovirus vector system. Combined transduction of DC by AdLoxpFasL and AxCANCre, but not single transduction with these vectors, resulted in dose- and time-dependent expression of FasL in >70% of mature DC (mDC), whereas <20% of immature DC (iDC) expressed FasL. In addition, transduction by AdLoxpFasL and AxCANCre induced apoptosis in >80% of iDC, whereas FasL-expressing mDC were protected from FasL/Fas (CD95/Apo-1)-mediated apoptosis despite coexpression of Fas. FasL-expressing mDC eliminated Fas+ Jurkat T cells as well as activated primary T cells by apoptosis, whereas nonactivated primary T cells were not deleted. Induction of apoptosis in Fas+ target cells required expression of FasL in DC and cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. Induction of apoptosis in Fas+ target cells required expression of FasL in DC, cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. The present results demonstrate that FasL-expressing killer APC can be generated from human monocyte-derived mDC using adenoviral gene transfer. Our results support the strategy to use killer APCs as immunomodulatory cells for the treatment of autoimmune disease and allograft rejection.

Elimination of activated but not resting primary human CD4+ and CD8+ T cells by Fas ligand (FasL/CD95L)-expressing Killer-dendritic cells.

Dendritic cells (DC) genetically engineered to express high levels of Fas ligand (FasL/CD95L) have been demonstrated to delete T cells in an antigen specific manner in several different animal models in vivo. However, the immunomodulatory capacity of primary human FasL-expressing Killer-DC has not been determined. Therefore, human Killer-DC were generated from mature monocyte-derived DC using the inducible CRE/LoxP adenoviral vector system, and the immunoregulatory capacity of these cells was analyzed in cocultures with primary human T cells in vitro. Combined transductions of DC by AdloxPFasL and AxCANCre resulted in FasL expression in > 70% of DC without affecting the mature phenotype. Proliferation of activated primary human T cells was inhibited up to 80% in cocultures with FasL-expressing DC but not EGFP-transduced DC, which was due to induction of apoptosis in activated but not resting CD4+ and CD8+ T cells. Apoptosis induced by Killer-DC could be blocked by an anti-FasL-antibody in a dose dependent fashion. The present results demonstrate that FasL-expressing Killer-DC eliminate activated but not resting primary human CD4+ and CD8+ T cells by induction of Fas-mediated apoptosis supporting the concept to apply Killer-DC as a novel strategy for the treatment of T cell-dependent autoimmune disease and allograft rejection in humans.

A Critical Role for Granzymes in Antigen Cross-Presentation through Regulating Phagocytosis of Killed Tumor Cells

Granzymes A and B (GrAB) are known principally for their role in mediating perforin-dependent death of virus-infected or malignant cells targeted by CTL. In this study, we show that granzymes also play a critical role as inducers of Ag cross-presentation by dendritic cells (DC). This was demonstrated by the markedly reduced priming of naive CD8+ T cells specific for the model Ag OVA both in vitro and in vivo in response to tumor cells killed in the absence of granzymes. Reduced cross-priming was due to impairment of phagocytosis of tumor cell corpses by CD8α+ DC but not CD8α− DC, demonstrating the importance of granzymes in inducing the exposure of prophagocytic “eat-me” signals on the dying target cell. Our data reveal a critical and previously unsuspected role for granzymes A and B in dictating immunogenicity by influencing the mode of tumor cell death and indicate that granzymes contribute to the efficient generation of immune effector pathways in addition to their well-known role in apoptosis induction.

Human and mouse perforin are processed in part through cleavage by the lysosomal cysteine proteinase cathepsin L

The pore‐forming protein perforin is synthesized as an inactive precursor in natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), and becomes active when a short C‐terminal peptide is cleaved within acidic lysosome‐like cytotoxic granules. Although it was shown more than a decade ago that this cleavage is pH dependent and can be inhibited by the generic cysteine cathepsin inhibitor E‐64d, no protease capable of processing the perforin C terminus has been identified. Neither is it known whether a single protease is responsible or the processing has inbuilt redundancy. Here, we show that incubation of human NK cells and primary antigen‐restricted mouse CTLs with the cathepsin L (CatL) inhibitor L1 resulted in a marked inhibition of perforin‐dependent target cell death and reduced perforin processing. In vitro, CatL preferentially cleaved a site on full‐length recombinant perforin close to its C terminus. The NK cells of mice deficient in CatL showed a reduction but not a complete absence of processed perforin, indicating that cysteine proteases other than CatL are also able to process perforin. We conclude that granule‐bound cathepsins are essential for processing perforin to its active form, and that CatL is an important, but not exclusive, participant in this process.