ERK5 modulates IL-6 secretion and contributes to tumor-induced immune suppression.

Tumors exhibit a variety of strategies to dampen antitumor immune responses. With an aim to identify factors that are secreted from tumor cells, we performed an unbiased mass spectrometry-based secretome analysis in lung cancer cells. Interleukin-6 (IL-6) has been identified as a prominent factor secreted by tumor cells and cancer-associated fibroblasts isolated from cancer patients. Incubation of dendritic cell (DC) cultures with tumor cell supernatants inhibited the production of IL-12p70 in DCs but not the surface expression of other activation markers which is reversed by treatment with IL-6 antibody. Defects in IL-12p70 production in the DCs inhibited the differentiation of Th1 but not Th2 and Th17 cells from naïve CD4+ T cells. We also demonstrate that the classical mitogen-activated protein kinase, ERK5/MAPK7, is required for IL-6 production in tumor cells. Inhibition of ERK5 activity or depletion of ERK5 prevented IL-6 production in tumor cells, which could be exploited for enhancing antitumor immune responses.

Novel anti-GARP antibody DS-1055a augments anti-tumor immunity by depleting highly suppressive GARP+ regulatory T cells.

Regulatory T (Treg) cells, which are essential for maintaining self-tolerance, inhibit anti-tumor immunity, consequently hindering protective cancer immunosurveillance, and hampering effective anti-tumor immune responses in tumor-bearing hosts. Here, we show that depletion of Treg cells via targeting glycoprotein A repetitions predominant (GARP) induces effective anti-tumor immune responses. GARP was specifically expressed by highly suppressive Treg cells in the tumor microenvironment (TME) of multiple cancer types in humans. In the periphery, GARP was selectively induced in Treg cells, but not in effector T cells, by polyclonal stimulation. DS-1055a, a novel afucosylated anti-human GARP monoclonal antibody, efficiently depleted GARP+ Treg cells, leading to the activation of effector T cells. Moreover, DS-1055a decreased FoxP3+CD4+ T cells in the TME and exhibited remarkable anti-tumor activity in humanized mice bearing HT-29 tumors. We propose that DS-1055a is a new Treg-cell-targeted cancer immunotherapy agent with augmentation of anti-tumor immunity.

In-Depth Immune-Oncology Studies of the Tumor Microenvironment in a Humanized Melanoma Mouse Model.

The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells-especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell-cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1-2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHCP using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model.

ERK3/MAPK6 is required for KRAS-mediated NSCLC tumorigenesis.

KRAS is one of the most frequently mutated oncogenes, especially in lung cancers. Targeting of KRAS directly or the downstream effector signaling machinery is of prime interest in treating lung cancers. Here, we uncover that ERK3, a ubiquitously expressed atypical MAPK, is required for KRAS-mediated NSCLC tumors. ERK3 is highly expressed in lung cancers, and oncogenic KRAS led to the activation and stabilization of the ERK3 protein. In particular, phosphorylation of serine 189 in the activation motif of ERK3 is significantly increased in lung adenocarcinomas in comparison to adjacent normal controls in patients. Loss of ERK3 prevents the anchorage-independent growth of KRAS G12C-transformed human bronchial epithelial cells. We further find that loss of ERK3 reduces the oncogenic growth of KRAS G12C-driven NSCLC tumors in vivo and that the kinase activity of ERK3 is required for KRAS-driven oncogenesis in vitro. Our results demonstrate an obligatory role for ERK3 in NSCLC tumor progression and suggest that ERK3 kinase inhibitors can be pursued for treating KRAS G12C-driven tumors.

Using immuno-PET imaging to monitor kinetics of T cell-mediated inflammation and treatment efficiency in a humanized mouse model for GvHD.

PURPOSE: Hematopoietic stem cell transplantation is the only curative treatment for several hematological malignancies and immune deficiency syndromes. Nevertheless, the development of graft-versus-host disease (GvHD) after transplantation is a severe complication with high morbidity and mortality. The aim of this study was to image human T cells during GvHD development and their migration into GvHD-related organs. By using a radiolabeled anti-human CD3 monoclonal antibody (mAb), we were able to visualize GvHD progression in a humanized mouse model. METHODS: Human peripheral blood mononuclear cells (PBMC) were transferred into immunodeficient mice (initially n = 11 mice/group) to induce GvHD. One group additionally received regulatory T cells (Treg) for prevention of GvHD. T cell migration was visualized by sequential small animal PET/MRI using 89Zr-labeled anti-human CD3 mAb. Flow cytometry and immunohistochemistry were used to measure T cell frequencies in relevant organs at different time points after engraftment. RESULTS: Using radiolabeled anti-CD3 mAb, we successfully visualized human T cells in inflamed organs of mice by 89Zr-anti-CD3-PET/MRI. Upon GvHD progression, we observed increased numbers of CD3+ T cells in the liver (22.9% on day 3; 94.2% on day 10) and the spleen (4.4% on day 3; 58.8% on day 10) which correlated with clinical symptoms. The liver showed distinct spot-like lesions representing a strong focal accumulation of T cells. Administration of Treg prior GvHD induction reduced T cell accumulation in the liver from 857 ± 177 CD3+ cells/mm2 to 261 ± 82 CD3+ cells/mm2 and thus prevented GvHD. CONCLUSION: 89Zr-labeled anti-human CD3 mAb can be used as a proof of concept to detect the exact spatio-temporal distribution of GvHD-mediating T cells. In the future, radiolabeled T cell-specific mAb could be employed as a predictive early biomarker during the course of GvHD maybe even before clinical signs of the disease become evident. Furthermore, monitoring T cell migration and proliferation might improve tailored GvHD therapy.

RAF kinases are stabilized and required for dendritic cell differentiation and function.

RAF kinases (ARAF, BRAF, and CRAF) are highly conserved enzymes that trigger the RAF-MEK1/2-ERK1/2 (MAPK) pathway upon activation of RAS. Despite enormous clinical interest, relatively little is known on the role of RAFs in mediating immune responses. Here, we investigated the role of RAF kinases and MEK1/2 in dendritic cells (DCs), the central regulators of T cell-mediated antitumor immune responses and the adaptive immune system. We demonstrate that RAF kinases are active and stabilized at their protein levels during DC differentiation. Inhibition of RAF kinases but not MEK1/2 impaired the activation of DCs in both mice and human. As expected, DCs treated with RAF inhibitors show defects in activating T cells. Further, RAF and MEK1/2 kinases are directly required for the activation and proliferation of CD4+ T cells. Our observations suggest that RAF and MEK1/2 have independent roles in regulating DC function that has important implications for administering RAF-MAPK inhibitors in the clinics.

Humanized mice in cutaneous leishmaniasis-Suitability analysis of human PBMC transfer into immunodeficient mice.

Humanized mice represent a suitable preclinical test system for example therapeutic interventions in various disease settings, including infections. Here, we intended to establish such system for cutaneous leishmaniasis by infecting T, B and NK cell-deficient mice adoptively transferred with human peripheral blood mononuclear cells (PBMC). L major infection led to the establishment of parasite lesions harbouring viable parasites and human T cells, but parasite elimination was not seen due to a species-specific activity of T cell-derived human IFNγ. In addition, up to 50% of infected mice succumbed to severe graft-versus-host disease. In summary, even though long-term disease outcome assessments are impossible, this model of humanized mice can be used for studying lesion development and generation of oligoclonal anti-parasite human T cell responses in vivo.

Novel Concept of CD4-Mediated Activation of Regulatory T Cells for the Treatment of Graft-Versus-Host Disease.

Allogeneic hematopoietic stem cell transplantation is the only curative treatment option for several hematological malignancies and immune deficiency syndromes. Nevertheless, the development of a graft-versus-host disease (GvHD) after transplantation is a high risk and a severe complication with high morbidity and mortality causing therapeutic challenges. Current pharmacological therapies of GvHD lead to generalized immunosuppression followed by severe adverse side effects including infections and relapse of leukemia. Several novel cell-based immunomodulatory strategies for treatment or prevention of GvHD have been developed. Herein, thymus-derived regulatory T cells (tTreg), essential for the maintenance of peripheral immunologic tolerance, are in the focus of investigation. However, due to the limited number of tTreg in the peripheral blood, a complex, time- and cost-intensive in vitro expansion protocol is necessary for the production of an efficient cellular therapeutic. We demonstrated that activation of tTreg using the CD4-binding human immunodeficiency virus-1 protein gp120 leads to a substantially increased suppressor activity of tTreg without the need for additional expansion. Gp120-activated tTreg prevent GvHD development in a preclinical humanized mouse model. In addition, gp120 is not only effective in prevention but also in therapy of GvHD by suppressing all clinical symptoms and improving survival of treated mice. These data indicate that tTreg activation by gp120 is a feasible and potent strategy for significant functional improvement of tTreg as cellular therapeutic for GvHD treatment without the need of complicated, time-intensive, and expensive in vitro expansion of isolated tTreg.

TCR signalling network organization at the immunological synapses of murine regulatory T cells.

Regulatory T (Treg) cells require T-cell receptor (TCR) signalling to exert their immunosuppressive activity, but the precise organization of the TCR signalling network compared to conventional T (Tconv) cells remains elusive. By using accurate mass spectrometry and multi-epitope ligand cartography (MELC) we characterized TCR signalling and recruitment of TCR signalling components to the immunological synapse (IS) in Treg cells and Tconv cells. With the exception of Themis which we detected in lower amounts in Treg cells, other major TCR signalling components were found equally abundant, however, their phosphorylation-status notably discriminates Treg cells from Tconv cells. Overall, this study identified 121 Treg cell-specific phosphorylations. Short-term triggering of T cell subsets via CD3 and CD28 widely harmonized these variations with the exception of eleven TCR signalling components that mainly regulate cytoskeleton dynamics and molecular transport. Accordingly, conjugation with B cells indeed caused variant cellular morphology and revealed a Treg cell-specific recruitment of TCR signalling components such as PKCθ, PLCγ1 and ZAP70 as well as B cell-derived CD86 into the IS. Together, results from this study support the existence of a Treg cell-specific IS and suggest Treg cell-specific cytoskeleton dynamics as a novel determinant for the unique functional properties of Treg cells.

Immunization with a Synthetic Human MUC1 Glycopeptide Vaccine against Tumor-Associated MUC1 Breaks Tolerance in Human MUC1 Transgenic Mice.

Breaking tolerance is crucial for effective tumor immunotherapy. We showed that vaccines containing tumor-associated human MUC1 glycopeptides induce strong humoral antitumor responses in mice. The question remained whether such vaccines work in humans, in systems where huMUC1 is a self-antigen. To clarify the question, mice transgenic in expressing huMUC1, mimicking the self-tolerant environment, and wild-type mice were vaccinated with a synthetic vaccine. This vaccine comprised STn and Tn antigens bound to a MUC1 tandem repeat peptide coupled to tetanus toxoid. The vaccine induced strong immune responses in wild-type and huMUC1-transgenic mice without auto-aggressive side effects. All antisera exhibited almost equivalent binding to human breast tumor cells. Similar increases of activated B-, CD4+ T-, and dendritic cells was found in the lymph nodes. The results demonstrate that tumor-associated huMUC1 glycopeptides coupled to tetanus toxoid are promising antitumor vaccines.

Dendritic Mesoporous Silica Nanoparticles for pH-Stimuli-Responsive Drug Delivery of TNF-Alpha.

Tumor necrosis factor-alpha (TNF-α) is a pleiotropic immune stimulatory cytokine and natural endotoxin that can induce necrosis and regression in solid tumors. However, systemic administration of TNF-α is not feasible due to its short half-life and acute toxicity, preventing its widespread use in cancer treatment. Dendritic mesoporous silica nanoparticles (DMSN) are used coated with a pH-responsive block copolymer gate system combining charged hyperbranched polyethylenimine and nonionic hydrophilic polyethylenglycol to encapsulate TNF-α and deliver it into various cancer cell lines and dendritic cells. Half-maximal effective concentration (EC50 ) for loaded TNF-α is reduced by more than two orders of magnitude. Particle stability and premature cargo release are assessed with enzyme-linked immunosorbent assay. TNF-α-loaded particles are stable for up to 5 d in medium. Tumor cells are grown in vitro as 3D fluorescent ubiquitination-based cell cycle indicator spheroids that mimic in vivo tumor architecture and microenvironment, allowing real-time cell cycle imaging. DMSN penetrate these spheroids, release TNF-α from its pores, preferentially affect cells in S/G2/M phase, and induce cell death in a time- and dose-dependent manner. In conclusion, DMSN encapsulation is demonstrated, which is a promising approach to enhance delivery and efficacy of antitumor drugs, while minimizing adverse side effects.

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7.

A fundamental mechanism of the innate immune system is the recognition, via extra- and intracellular pattern-recognition receptors, of pathogen-associated molecular patterns. A prominent example is represented by foreign nucleic acids, triggering the activation of several signaling pathways. Among these, the endosomal toll-like receptor 7 (TLR7) is known to be activated by single-stranded RNA (ssRNA), which can be specifically influenced through elements of sequence structure and posttranscriptional modifications. Furthermore, small molecules TLR7 agonists (smTLRa) are applied as boosting adjuvants in vaccination processes. In this context, covalent conjugations between adjuvant and vaccines have been reported to exhibit synergistic effects. Here, we describe a concept to chemically combine three therapeutic functions in one RNA bioconjugate. This consists in the simultaneous TLR7 stimulation by ssRNA and smTLRa as well as the therapeutic function of the RNA itself, e.g., as a vaccinating or knockdown agent. We have hence synthesized bioconjugates of mRNA and siRNA containing covalently attached smTLRa and tested their function in TLR7 stimulation. Strikingly, the bioconjugates displayed decreased rather than synergistically increased stimulation. The decrease was distinct from the antagonistic action of an siRNA bearing a Gm motive, as observed by direct comparison of the effects in the presence of otherwise stimulatory RNA. In summary, these investigations showed that TRL7 activation can be impeded by bioconjugation of small molecules to RNA.

Dimethyl Fumarate Therapy Significantly Improves the Responsiveness of T Cells in Multiple Sclerosis Patients for Immunoregulation by Regulatory T Cells.

Multiple sclerosis (MS) is a chronic autoimmune disease caused by an insufficient suppression of autoreactive T lymphocytes. One reason for the lack of immunological control is the reduced responsiveness of T effector cells (Teff) for the suppressive properties of regulatory T cells (Treg), a process termed Treg resistance. Here we investigated whether the disease-modifying therapy of relapsing-remitting MS (RRMS) with dimethyl fumarate (DMF) influences the sensitivity of T cells in the peripheral blood of patients towards Treg-mediated suppression. We demonstrated that DMF restores responsiveness of Teff to the suppressive function of Treg in vitro, presumably by down-regulation of interleukin-6R (IL-6R) expression on T cells. Transfer of human immune cells into immunodeficient mice resulted in a lethal graft-versus-host reaction triggered by human CD4⁺ Teff. This systemic inflammation can be prevented by activated Treg after transfer of immune cells from DMF-treated MS patients, but not after injection of Treg-resistant Teff from therapy-naïve MS patients. Furthermore, after DMF therapy, proliferation and expansion of T cells and the immigration into the spleen of the animals is reduced and modulated by activated Treg. In summary, our data reveals that DMF therapy significantly improves the responsiveness of Teff in MS patients to immunoregulation.

Treg cells as potential cellular targets for functionalized nanoparticles in cancer therapy.

Treg cell-mediated immune suppression appears to represent a significant barrier to effective anticancer immune responses and their inactivation or removal is viewed as a potential therapeutic approach. Although suitable tools for selective Treg cell manipulation in man are missing, their number and function can be altered by a number of drugs and biologicals and by reprogramming tumor-infiltrating antigen presenting cells. Nanoparticles offer exceptional new options in drug and gene delivery by prolonging the circulation time of their cargo, protecting it from degradation and promoting its local accumulation in cells and tissues. In tumor therapy, the use of nanoparticles is expected to overcome limitations in drug delivery and provide novel means for cell-specific functional alteration. In this perspective, we summarize strategies suitable for interference with Treg-mediated suppression, discuss the potential use of nanoparticles for this purpose and identify additional, unexplored opportunities.

A key role of GARP in the immune suppressive tumor microenvironment.

In melanoma patients, one of the main reasons for tumor immune escape and therapy failure is the immunosuppressive tumor microenvironment. Herein, suppressive immune cells and inhibitory factors secreted by the tumor itself play a central role.In the present study we show that the Treg activation marker GARP (glycoprotein A repetitions predominant), known to induce peripheral tolerance in a TGF-ß dependent way, is also expressed on human primary melanoma. Interestingly, membrane bound GARP is shed from the surface of both, activated Treg and melanoma cells, and, in its soluble form (sGARP), not only induces peripheral Treg but also a tumor associated (M2) macrophage phenotype. Notably, proliferation of cytotoxic T cells and their effector function is inhibited in the presence of sGARP. GARP expression on Treg and melanoma cells is significantly decreased in the presence of agents such as IFN-α, thus explaining at least in part a novel mechanism of action of this adjuvant therapy.In conclusion, GARP in its soluble and membrane bound form contributes to peripheral tolerance in a multipronged way, potentiates the immunosuppressive tumor microenvironment and thus acts as a negative regulator in melanoma patients. Therefore, it may qualify as a promising target and a new checkpoint for cancer immunotherapy.

Kinome Profiling of Regulatory T Cells: A Closer Look into a Complex Intracellular Network.

Regulatory T cells (Treg) are essential for T cell homeostasis and maintenance of peripheral tolerance. They prevent activation of auto-reactive T effector cells (Teff) in the context of autoimmunity and allergy. Otherwise, Treg also inhibit effective immune responses against tumors. Besides a number of Treg-associated molecules such as Foxp3, CTLA-4 or GARP, known to play critical roles in Treg differentiation, activation and function, the involvement of additional regulatory elements is suggested. Herein, kinase activities seem to play an important role in Treg fine tuning. Nevertheless, our knowledge regarding the complex intracellular signaling pathways controlling phenotype and function of Treg is still limited and based on single kinase cascades so far. To gain a more comprehensive insight into the pathways determining Treg function we performed kinome profiling using a phosphorylation-based kinome array in human Treg at different activation stages compared to Teff. Here we have determined intriguing quantitative differences in both populations. Resting and activated Treg showed an altered pattern of CD28-dependent kinases as well as of those involved in cell cycle progression. Additionally, significant up-regulation of distinct kinases such as EGFR or CK2 in activated Treg but not in Teff not only resemble data we obtained in previous studies in the murine system but also suggest that those specific molecular activation patterns can be used for definition of the activation and functional state of human Treg. Taken together, detailed investigation of kinome profiles opens the possibility to identify novel molecular mechanisms for a better understanding of Treg biology but also for development of effective immunotherapies against unwanted T cell responses in allergy, autoimmunity and cancer.

Interferon-Beta Therapy of Multiple Sclerosis Patients Improves the Responsiveness of T Cells for Immune Suppression by Regulatory T Cells.

Multiple sclerosis (MS) is an inflammatory autoimmune disease characterized by imbalanced immune regulatory networks, and MS patient-derived T effector cells are inefficiently suppressed through regulatory T cells (Treg), a phenomenon known as Treg resistance. In the current study we investigated T cell function in MS patients before and after interferon-beta therapy. We compared cytokine profile, responsiveness for Treg-mediated suppression ex vivo and evaluated reactivity of T cells in vivo using a humanized mouse model. We found that CD4+ and CD8+ T cells of therapy-naive MS patients were resistant to Treg-mediated suppression. Treg resistance is associated with an augmented IL-6 production, enhanced IL-6 receptor expression, and increased PKB/c-Akt phosphorylation. These parameters as well as responsiveness of T cells to Treg-mediated suppression were restored after interferon-beta therapy of MS patients. Following transfer into immunodeficient mice, MS T cells induced a lethal graft versus host disease (GvHD) and in contrast to T cells of healthy volunteers, this aggressive T cell response could not be controlled by Treg, but was abolished by anti-IL-6 receptor antibodies. However, magnitude and lethality of GvHD induced by MS T cells was significantly decreased after interferon-beta therapy and the reaction was prevented by Treg activation in vivo. Our data reveals that interferon-beta therapy improves the immunoregulation of autoaggressive T effector cells in MS patients by changing the IL-6 signal transduction pathway, thus restoring their sensitivity to Treg-mediated suppression.