Kerstin Wennhold

Profiling of anti-fibrotic signaling by hepatocyte growth factor in renal fibroblasts

Hepatocyte growth factor (HGF) is a multifunctional growth factor affecting cell proliferation and differentiation. Due to its mitogenic potential, HGF plays an important role in tubular repair and regeneration after acute renal injury. However, recent reports have shown that HGF also acts as an anti-inflammatory and anti-fibrotic factor, affecting various cell types such as renal fibroblasts and triggering tubulointerstitial fibrosis of the kidney. The present study provides evidence that HGF stimulation of renal fibroblasts results in the activation of both the Erk1/2 and the Akt pathways. As previously shown, Erk1/2 phosphorylation results in Smad-linker phosphorylation, thereby antagonizing cellular signals induced by TGFbeta. By siRNA mediated silencing of the Erk1/2-Smad linkage, however, we now demonstrate that Akt signaling acts as an auxiliary pathway responsible for the anti-fibrotic effects of HGF. In order to define the anti-fibrotic function of HGF we performed comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridization. Functional cluster analyses and quantitative PCR assays indicate that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix proteins, various chemokines, and members of the CCN family.

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

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

Immune checkpoints programmed death 1 ligand 1 and cytotoxic T lymphocyte associated molecule 4 in gastric adenocarcinoma

ABSTRACT Remarkable efficacy of immune checkpoint inhibition has been reported for several types of solid tumors and early studies in gastric adenocarcinoma are promising. A detailed knowledge about the natural biology of immune checkpoints in gastric adenocarcinoma is essential for clinical and translational evaluation of these drugs. This study is a comprehensive analysis of cytotoxic T lymphocyte associated molecule 4 (CTLA-4) and programmed death 1 ligand 1 (PD-L1) expression in gastric adenocarcinoma. PD-L1 and CTLA-4 were stained on tumor sections of 127 Caucasian patients with gastric adenocarcinoma by immunohistochemistry (IHC) and somatic mutation profiling was performed using targeted next-generation sequencing. Expression of PD-L1 and CTLA-4 on lymphocytes in tumor sections, tumor-draining lymph nodes (TDLN) and peripheral blood were studied by flow-cytometry and immune-fluorescence microscopy in an additional cohort. PD-L1 and CTLA-4 were expressed in 44.9% (57/127) and 86.6% (110/127) of the analyzed gastric adenocarcinoma samples, respectively. Positive tumor cell staining for PD-L1 or CTLA-4 was associated with inferior overall survival. Somatic mutational analysis did not reveal a correlation to expression of PD-L1 or CTLA-4 on tumor cells. Expression of PD-1 (52.2%), PD-L1 (42.2%) and CTLA-4 (1.6%) on tumor infiltrating T cells was significantly elevated compared to peripheral blood. Of note, PD-1 and PD-L1 were expressed far higher by tumor-infiltrating lymphocytes than CTLA-4. In conclusion, specific immune checkpoint-inhibitors should be evaluated in this disease and the combination with molecular targeted therapies might be of benefit. An extensive immune monitoring should accompany these studies to better understand their mode of action in the tumor microenvironment.

CD30-targeted therapy with brentuximab vedotin and DLI in a patient with T-cell posttransplantation lymphoma: induction of clinical remission and cellular immunity.

Posttransplantation lymphoproliferative disorder (PTLD) is a major complication after organ or allogeneic hematopoietic stem cell transplantation (alloSCT) (1). More than 90% of PTLD arise from B cells and are often associated with Epstein-Barr virus (EBV). Here, rituximab-containing treatment regimens have improved outcomes (2). T-cell PTLD constitutes a rare subgroup (2%Y6%) with a poor prognosis resulting in a median, subtype-dependent survival of 5 to 18 months (3, 4). Although 36% of the published T-cell PTLD patients were EBV positive, the exact pathomechanisms are still unclear and a number of other viruses have been associated with this condition (e.g., cytomegalovirus, human herpes virus, and human T lymphotropic virus) (4). In contrast to T-cell lymphoma in HIV-infected patients of which 18% to 28% show a CD30 anaplastic largecell phenotype, the expression of CD30 in T-cell PTLD has only been occasionally

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

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

Characterization of tumor-associated T-lymphocyte subsets and immune checkpoint molecules in head and neck squamous cell carcinoma

The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors (n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa (n = 7) of 34 treatment-naïve HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA−/CCR7−). Naïve T cells (CD45RA+/CCR7+) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4+/CD25+/CD127low and CD4+/CD39+) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the ‘Immunoscore’ (CD3+ and CD8+ cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance.

Targeting Tumor-Infiltrating B Cells in Cutaneous T-Cell Lymphoma

Introduction The tumor microenvironment and infiltrating immune cells are important for cancer biology and progression which has been exploited therapeutically in recent years. Whereas infiltrating T cells, macrophages, or natural-killer (NK) cells have been extensively analyzed, recent studies also suggest a role for B cells in cancer biology. Primary cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas of which the most common subtype is mycosis fungoides (MF). At early stages ( IIA; European Organisation for Research and Treatment of Cancer [EORTC]/International Society for Cutaneous Lymphomas [ISCL]), MF usually runs an indolent course with almost normal life expectancy. However, advanced-stage MF ( IIB), the folliculotropic MF subtype (FMF) and also Sézary syndrome (SS) are more aggressive (median survival, 13-48 months). Preclinical data suggest that chronic inflammation promotes CTCL progression with a critical role for macrophages and mast cells. Occasionally, B-cell infiltrations have been reported in CTCL and although a well-established CTCL model is lacking, B-cell deficient mice exhibited significant regressions of EL4-T-cell lymphoma grafts. Therefore, we analyzed diagnostic skin biopsies of 33 consecutively treated CTCL patients for infiltrating B cells (CD20/ CD79a) and reviewed respective clinical data from 1979 to 2011 (Table 1). Psoriasis and eczema tissues served as controls. Staging followed ISCL/EORTC criteria. Lymphoma-tissues containing 50 B-cells/mm of lymphoma-infiltrate were assigned as B-cell positive. The study was approved by the institutional review board (No. 08-144). Immunohistochemistry revealed remarkable differences of CD20 B-cell infiltrates between CTCL subtypes (Fig 1A) and additional flow-cytometric analysis in available frozen samples supported the B-cell nature of CD20 cells (Fig 3A). Eighteen out of 33 CTCL patients (MF [n 25], FMF [n 5], SS [n 3]) had significantly increased median B-cell numbers within the lymphoma infiltrate, whereas controls (psoriasis [n 5], eczema [n 5]) were B-cell negative (Fig 1B and Table 1). Remarkably, all FMF and SS cases showed significantly increased median B-cell numbers compared with classic MF. However, in classic MF 40% of the cases were B-cell positive. Given the fact that FMF and SS represent more aggressive subtypes, we asked whether B-cell infiltrations correspond with the clinical stage and behavior. Comparison of B-cell infiltrates in early ( IIB) versus advanced ( IIB) stages revealed a significant correlation of B-cell positivity with advanced stages. This correlation not only applied to the entire CTCL cohort but also to classic MF cases solely (Fig 1C). Moreover, clinical data analyses revealed a significant correlation of B-cell infiltrates with progression-free survival (PFS). As shown in Figure 1D, patients with B-cell positive lymphoma had a significantly shortened median PFS, ie, 50 months (entire cohort) and 126 months (classic MF only). In contrast, median PFS in B-cell negative cases was not reached at the end of observation (360 months).

Inhibition of Protein Geranylgeranylation Specifically Interferes with CD40-Dependent B Cell Activation, Resulting in a Reduced Capacity To Induce T Cell Immunity

Ab-independent effector functions of B cells, such as Ag presentation and cytokine production, have been shown to play an important role in a variety of immune-mediated conditions such as autoimmune diseases, transplant rejection, and graft-versus-host disease. Most current immunosuppressive treatments target T cells, are relatively unspecific, and result in profound immunosuppression that places patients at an increased risk of developing severe infections and cancer. Therapeutic strategies, which interfere with B cell activation, could therefore be a useful addition to the current immunosuppressive armamentarium. Using a transcriptomic approach, we identified upregulation of genes that belong to the mevalonate pathway as a key molecular event following CD40-mediated activation of B cells. Inhibition of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme of the mevalonate pathway, by lipophilic statins such as simvastatin and atorvastatin resulted in a specific inhibition of B cell activation via CD40 and impaired their ability to act as stimulatory APCs for allospecific T cells. Mechanistically, the inhibitory effect resulted from the inhibition of protein geranylgeranylation subsequent to the depletion of mevalonate, the metabolic precursor for geranylgeranyl. Thus, inhibition of geranylgeranylation either directly through geranylgeranyl transferase inhibitors or indirectly through statins represents a promising therapeutic approach for the treatment of diseases in which Ag presentation by B cells plays a role.

Results of a Phase II clinical trial with Id-protein-loaded dendritic cell vaccine in multiple myeloma: encouraging or discouraging?

Recently gained insight into the role of dendritic cells (DCs) as APCs has attracted the attention of many researchers who hope to use them as a tool in immunotherapy for the induction of tumor-specific immunity in cancer settings. Despite high expectations, in multiple myeloma patients the results of DC-based vaccines in terms of clinical response have been disappointing. The findings of Zahradova et al. in a Phase II clinical trial with multiple myeloma patients corroborated these results. Although no clinical responses were observed, the investigators induced immunity after vaccination with Id-protein-loaded DC vaccine in some patients. These immunological results showed a trend towards a longer duration of stable disease in those patients that received the vaccination. Moreover, this study showed that Id-protein-loaded DC vaccines are safe and nontoxic and that they are able to induce immunity in some patients. Therefore, standardization of vaccination protocols appears to be the key to achieving a better clinical outcome.

In vitro and in vivo imaging of initial B-T-cell interactions in the setting of B-cell based cancer immunotherapy

There has been a growing interest in the use of B cells for cancer vaccines, since they have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated B (CD40B) cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo. Dynamic interactions of human antigen-presenting cells (APCs) and T cells were observed by time-lapse video microscopy. The migratory and chemoattractant potential of CD40B cells was analyzed in vitro and in vivo using flow cytometry, standard transwell migration assays, and imaging of fluorescently labeled murine CD40B cells. Murine CD40B cells show migratory features similar to human CD40B cells. They express important lymph node homing receptors which were functional and induced chemotaxis of T cells in vitro. Striking differences were observed with regard to interactions of human APCs with T cells. CD40B cells differ from DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived and sequential interactions with T cells. In vivo, CD40B cells are home to the secondary lymphoid organs where they accumulate in the B cell zone before traveling to the B/T cell boundary. Moreover, intravenous (i.v.) administration of murine CD40B cells induced an antigen-specific cytotoxic T cell response. Taken together, this data show that CD40B cells home secondary lymphoid organs where they physically interact with T cells to induce antigen-specific T cell responses, thus underscoring their potential as cellular adjuvant for cancer immunotherapy.