NK cells in peripheral blood carry trogocytosed tumor antigens from solid cancer cells.

The innate immune lymphocyte lineage natural killer (NK) cell infiltrates tumor environment where it can recognize and eliminate tumor cells. NK cell tumor infiltration is linked to patient prognosis. However, it is unknown if some of these antitumor NK cells leave the tumor environment. In blood-borne cancers, NK cells that have interacted with leukemic cells are recognized by the co-expression of two CD45 isoforms (CD45RARO cells) and/or the plasma membrane presence of tumor antigens (Ag), which NK cells acquire by trogocytosis. We evaluated solid tumor Ag uptake by trogocytosis on NK cells by performing co-cultures in vitro. We analyzed NK population subsets by unsupervised dimensional reduction techniques in blood samples from breast tumor (BC) patients and healthy donors (HD). We confirmed that NK cells perform trogocytosis from solid cancer cells in vitro. The extent of trogocytosis depends on the target cell and the antigen, but not on the amount of Ag expressed by the target cell or the sensitivity to NK cell killing. We identified by FlowSOM (Self-Organizing Maps) several NK cell clusters differentially abundant between BC patients and HD, including anti-tumor NK subsets with phenotype CD45RARO+CD107a+. These analyses showed that bona-fide NK cells that have degranulated were increased in patients and, additionally, these NK cells exhibit trogocytosis of solid tumor Ag on their surface. However, the frequency of NK cells that have trogocytosed is very low and much lower than that found in hematological cancer patients, suggesting that the number of NK cells that exit the tumor environment is scarce. To our knowledge, this is the first report describing the presence of solid tumor markers on circulating NK subsets from breast tumor patients. This NK cell immune profiling could lead to generate novel strategies to complement established therapies for BC patients or to the use of peripheral blood NK cells in the theranostic of solid cancer patients after treatment.

Transfer of mesenchymal stem cell mitochondria to CD4+ T cells contributes to repress Th1 differentiation by downregulating T-bet expression.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) are multipotent cells with strong tissue repair and immunomodulatory properties. Due to their ability to repress pathogenic immune responses, and in particular T cell responses, they show therapeutic potential for the treatment of autoimmune diseases, organ rejection and graft versus host disease. MSCs have the remarkable ability to export their own mitochondria to neighboring cells in response to injury and inflammation. However, whether mitochondrial transfer occurs and has any role in the repression of CD4+ Th1 responses is unknown. METHODS AND RESULTS: In this report we have utilized CD4+ T cells from HNT TCR transgenic mice that develop Th1-like responses upon antigenic stimulation in vitro and in vivo. Allogeneic bone marrow-derived MSCs reduced the diabetogenic potential of HNT CD4+ T cells in vivo in a transgenic mouse model of disease. In co-culture experiments, we have shown that MSCs were able to reduce HNT CD4+ T cell expansion, expression of key effector markers and production of the effector cytokine IFNγ after activation. This was associated with the ability of CD4+ T cells to acquire mitochondria from MSCs as evidenced by FACS and confocal microscopy. Remarkably, transfer of isolated MSC mitochondria to CD4+ T cells resulted in decreased T cell proliferation and IFNγ production. These effects were additive with those of prostaglandin E2 secreted by MSCs. Finally, we demonstrated that both co-culture with MSCs and transfer of isolated MSC mitochondria prevent the upregulation of T-bet, the master Th1 transcription factor, on activated CD4+ T cells. CONCLUSION: The present study demonstrates that transfer of MSC mitochondria to activated CD4+ T cells results in the suppression of Th1 responses in part by downregulating T-bet expression. Furthermore, our studies suggest that MSC mitochondrial transfer might represent a general mechanism of MSC-dependent immunosuppression.

A safe and highly efficient tumor-targeted type I interferon immunotherapy depends on the tumor microenvironment.

Despite approval for the treatment of various malignancies, clinical application of cytokines such as type I interferon (IFN) is severely impeded by their systemic toxicity. AcTakines (Activity-on-Target cytokines) are optimized immunocytokines that, when injected in mice, only reveal their activity upon cell-specific impact. We here show that type I IFN-derived AcTaferon targeted to the tumor displays strong antitumor activity without any associated toxicity, in contrast with wild type IFN. Treatment with CD20-targeted AcTaferon of CD20+ lymphoma tumors or melanoma tumors engineered to be CD20+, drastically reduced tumor growth. This antitumor effect was completely lost in IFNAR- or Batf3-deficient mice, and depended on IFN signaling in conventional dendritic cells. Also the presence of, but not the IFN signaling in, CD8+ T lymphocytes was critical for proficient antitumor effects. When combined with immunogenic chemotherapy, low-dose TNF, or immune checkpoint blockade strategies such as anti-PDL1, anti-CTLA4 or anti-LAG3, complete tumor regressions and subsequent immunity (memory) were observed, still without any concomitant morbidity, again in sharp contrast with wild type IFN. Interestingly, the combination therapy of tumor-targeted AcTaferon with checkpoint inhibiting antibodies indicated its ability to convert nonresponding tumors into responders. Collectively, our findings demonstrate that AcTaferon targeted to tumor-specific surface markers may provide a safe and generic addition to cancer (immuno)therapies.

Delivering Type I Interferon to Dendritic Cells Empowers Tumor Eradication and Immune Combination Treatments.

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I IFN has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1,000-fold more potent on target cells, allowing specific signaling in selected cell types only. Type I IFN-derived AcTaferon (AFN)-targeting Clec9A+ dendritic cells (DC) displayed strong antitumor activity in murine melanoma, breast carcinoma, and lymphoma models and against human lymphoma in humanized mice without any detectable toxic side effects. Combined with immune checkpoint blockade, chemotherapy, or low-dose TNF, complete tumor regression and long-lasting tumor immunity were observed, still without adverse effects. Our findings indicate that DC-targeted AFNs provide a novel class of highly efficient, safe, and broad-spectrum off-the-shelf cancer immunotherapeutics with no need for a tumor marker.Significance: Targeted type I interferon elicits powerful antitumor efficacy, similar to wild-type IFN, but without any toxic side effects. Cancer Res; 78(2); 463-74. ©2017 AACR.

AMPK/HuR-Driven IL-20 Post-Transcriptional Regulation in Psoriatic Skin.

IL-20 is involved in the development of skin psoriasis. The molecular mechanisms underlying IL-20 overexpression in psoriatic epidermis remain to be elucidated. We showed that IL-20 was primarily upregulated in psoriatic skin at the post-transcriptional level. The RNA-binding protein HuR relocalized to the cytoplasm of keratinocytes (KCs) of psoriatic patients, suggesting that it stabilizes numerous transcripts, as observed in the human KC cell lines used to assess IL-20 mRNA. We characterized epidermal HuR RNA targets in psoriatic skin using ribonucleoprotein immunoprecipitation analyzed via high-throughput sequencing. Numerous transcripts that are upregulated in psoriasis were targeted by HuR, supporting the participation of HuR in pathogenic processes such as morphological changes, innate and adaptive immune responses, and metabolic inflammatory responses. Finally, we identified the metabolic sensor AMP-activated protein kinase (AMPK) as being responsible for HuR cytoplasmic relocalization because its activity was severely impaired in human psoriatic epidermis, and in vivo drug-mediated AMPK inhibition in mouse epidermis promoted HuR cytoplasmic localization, IL-20 overproduction, acanthosis, and hyperkeratosis. These results provide insights into the molecular links between metabolism and post-transcriptional networks during chronic inflammation.

High efficiency cell-specific targeting of cytokine activity.

Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This 'activity-by-targeting' concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

Differential activity of type I interferon subtypes for dendritic cell differentiation.

The type I interferon (IFN) family comprises 15 cytokines (in human 13α, 1ß, 1ω), which exert several cellular functions through binding to a common receptor. Despite initial activation of the same Jak/Stat signalling pathway, the cellular response may differ depending on type I IFN subtype. We investigated the activity of six type I IFN subtypes - IFNα1, α2, α8, α21, ω and ß- to promote the differentiation of dendritic cells (DC). Transcriptome analyses identified two distinct groups, the IFNα/ω-DC and the IFNß-DC. In addition, the expression level of seven chemokines and several cell surface markers characteristic of DC distinguished IFNα-DC and IFNß-DC. These differences are unlikely to impact the efficacy of T cell functional response since IFNα2-DC and IFNß-DC were equipotent in inducing the proliferation and the polarization of allogenic naïve CD4 T cells into Th1 cells and in stimulating autologous antigen specific CD4 or CD8 T cells. Of the functional parameters analysed, the only one that showed a modest differential was the phagocytic uptake of dead cells which was higher for IFNα2-DC.

Constitutive expression of MC1R in HaCaT keratinocytes inhibits basal and UVB-induced TNF-alpha production.

Alpha-melanocyte stimulating hormone (alpha-MSH) binds to melanocortin-1 receptor (MC1R) on melanocytes to stimulate pigmentation and modulate various cutaneous inflammatory responses. MC1R expression is not restricted to melanocytic cells and may be induced in keratinocytes after UVB exposure. We hypothesized that MC1R signaling in keratinocytes, wherein basal conditions are barely expressed, may modulate mediators of inflammation, such as nuclear factor-kappa B (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha). Therefore, we generated HaCaT cells that stably express human MC1R or the Arg151Cys (R151C) nonfunctional variant. We demonstrate that: (1) the constitutive activity of MC1R results in elevated intracellular cAMP level, reduced NF-kappaB activity and decreased TNF-alpha transcription; (2) binding of alpha-MSH to MC1R and the subsequent increase in cAMP production do not inhibit TNFalpha-mediated NF-kappaB activation; (3) MC1R signaling is sufficient to strongly inhibit UVB-induced TNF-alpha expression and this inhibitory effect is further enhanced by alpha-MSH stimulation. Our findings suggest that the constitutive activity of the G-protein-coupled MC1R in keratinocytes may contribute to the modulation of inflammatory events and immune response induced by UV light.

Cell surface expression of melanocortin-1 receptor on HaCaT keratinocytes and alpha-melanocortin stimulation do not affect the formation and repair of UVB-induced DNA photoproducts.

Ultraviolet (UV) exposure induces an up-regulation of melanocortin-1 receptor (MC1R) expression in human skin and the alpha-melanocyte-stimulating hormone (alpha-MSH) may reduce UVB-induced DNA damage in normal human melanocytes. Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of DNA lesions in UVB-irradiated HaCaT cells stably transfected with the wild type MC1R gene (HaCaT-MC1R). Similar levels of 8 bipyrimidine photoproducts including cyclobutane pyrimidine dimers (CPDs) (T<>T, T<>C, C<>T), (6-4) photoproducts ((6-4)PPs) (TT-(6-4)PPs, TC-(6-4)PPs) and their Dewar valence isomers together with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were found to be generated in both non-transfected and HaCaT-MC1R cells after UVB exposure. Time-course studies of DNA photoproduct yields indicated that the DNA repair ability depended upon radiation doses. It was shown that (6-4)PPs were removed from the DNA of UVB-irradiated cells much more efficiently than CPDs. The repair efficiency of 8-oxodGuo, CPDs and (6-4)PPs was relatively similar in both cell lines and was not modified by stimulation with alpha-MSH before UVB-exposure. In conclusion, cell surface-enforced expression of MC1Rs on HaCaT keratinocytes and alpha-MSH stimulation do not affect the formation of UVB-induced DNA photoproducts and their subsequent repair.

On-line synthesis of pseudopeptide library incorporating a benzodiazepinone turn mimic: biological evaluation on MC1 receptors.

Alpha melanocyte stimulating hormone (alpha-MSH) is a widely distributed hormone. This tridecapeptide exhibits various biological activities mediated through different receptors. alpha-MSH binds to the melanocortin-1 receptor (MC1-R), mainly expressed in keratinocytes and melanocytes, inducing melanogenesis and anti-inflammatory processes. The central His-Phe-Arg-Trp tetrapeptide sequence of alpha-MSH is known to form a turn in the bioactive conformation. To find new potent analogs of alpha-MSH, we decided to introduce non-peptide building blocks in the alpha-MSH sequence. Molecular modeling studies showed that two amino acids of the central core sequence could be replaced by the benzodiazepinone building block without loosing the beta-turn conformation. Benzodiazepines are well-known pharmacophores exhibiting a wide scope of biological activities and are described as constrained dipeptide mimics templates. Although numerous synthetic pathways leading to benzodiazepinones have been described in literature, no methodology has 1,4-benzodiazepine-2,5-diones building blocks bearing a free carboxylic acid function and a protected amino function suitable for incorporation into peptide sequences. In this study, we report the synthesis of peptides with a benzodiazepinone moiety obtained directly during the course of solid-phase peptide synthesis (SPPS). This "on-line" strategy leads to the generation of a 54-member pseudo-peptide library of alpha-MSH analogs. After LC/MS purification, binding assays were performed on the MC1 receptor leading to the discovery of several micromolar ligands.