Tomasz Rygiel

Immune Cells in Cancer Therapy and Drug Delivery

Recent studies indicate the critical role of tumour associated macrophages, tumour associated neutrophils, dendritic cells, T lymphocytes, and natural killer cells in tumourigenesis. These cells can have a significant impact on the tumour microenvironment via their production of cytokines and chemokines. Additionally, products secreted from all these cells have defined specific roles in regulating tumour cell proliferation, angiogenesis, and metastasis. They act in a protumour capacity in vivo as evidenced by the recent studies indicating that macrophages, T cells, and neutrophils may be manipulated to exhibit cytotoxic activity against tumours. Therefore therapy targeting these cells may be promising, or they may constitute drug or anticancer particles delivery systems to the tumours. Herein, we discussed all these possibilities that may be used in cancer treatment.

Effects of cannabinoids and their receptors on viral infections

Cannabinoids, the active ingredient in marijuana, and their derivatives have received remarkable attention in the last two decades because they can affect tumor growth and metastasis. There is a large body of evidence from in vivo and in vitro models showing that cannabinoids and their receptors influence the immune system, viral pathogenesis, and viral replication. The present study reviews current insights into the role of cannabinoids and their receptors on viral infections. The results reported here indicate that cannabinoids and their receptors have different sequels for viral infection. Although activation or inhibition of cannabinoid receptors in the majority of viral infections are proper targets for development of safe and effective treatments, caution is required before using pharmaceutical cannabinoids as a treatment agent for patients with viral infections. J. Med. Virol. 88:1–12, 2016.

Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.

Use of an anti-CD200 antibody for prolonging the survival of allografts: a patent evaluation of WO2012106634A1

Background: Allograft rejection continues to be the biggest hurdle in successful organ transplantation. This application (WO2012106634A1) claims the use of blocking CD200 antibody to achieve long-term survival of allografts. CD200 is the ligand for the inhibitory CD200 receptor (CD200R). Methods: In mouse allograft transplantation models, a blocking CD200 antibody was used to improve renal and cardiac graft survival. Similarly, in humans a blocking CD200 antibody would be administered to the organ recipient in combination with currently used immunosuppressive drugs or even as a monotherapy. Results: In the presented animal experiments, anti-CD200 antibody application to the allograft recipient decreases SHIP expression in splenocytes. This is accompanied by a significant increase in renal or cardiac graft survival. Furthermore, anti-CD200 antibody has an immunosuppressive effect manifested by an increased production of T regulatory and myeloid-derived suppressor cells (MDSC) and a decrease in B cells and activated T cells. Conclusion: In vivo administration of anti-CD200 antibody has a remarkable positive effect on allograft survival. However, since this finding contradicts all previous effects of in vivo CD200 manipulation described in transplantation settings, future development of this invention is highly uncertain.

Neutrophil-mediated Suppression of Influenza-induced Pathology Requires CD11b/CD18 (MAC-1)

&NA; Severe influenza virus infection can lead to life‐threatening pathology through immune‐mediated tissue damage. In various experimental models, this damage is dependent on T cells. There is conflicting evidence regarding the role of neutrophils in influenza‐mediated pathology. Neutrophils are often regarded as cells causing tissue damage, but, in recent years, it has become clear that a subset of human neutrophils is capable of suppressing T cells, which is dependent on macrophage‐1 antigen (CD11b/CD18). Therefore, we tested the hypothesis that immune suppression by neutrophils can reduce T cell‐mediated pathology after influenza infection. Wild‐type (WT) and CD11b−/− mice were infected with A/HK/2/68 (H3N2) influenza virus. Disease severity was monitored by weight loss, leukocyte infiltration, and immunohistochemistry. We demonstrated that CD11b−/− mice suffered increased weight loss compared with WT animals upon infection with influenza virus. This was accompanied by increased pulmonary leukocyte infiltration and lung damage. The exaggerated pathology in CD11b−/− mice was dependent on T cells, as it was reduced by T cell depletion. In addition, pathology in CD11b−/− mice was accompanied by higher numbers of T cells in the lungs early during infection compared with WT mice. Importantly, these differences in pathology were not associated with an increased viral load, suggesting that pathology was immune‐mediated rather than caused by virus‐induced damage. In contrast to adoptive transfer of CD11b−/− neutrophils, a single adoptive transfer of WT neutrophils partly restored protection against influenza‐induced pathology, demonstrating the importance of neutrophil CD11b/CD18. Our data show that neutrophil CD11b/CD18 limits pathology in influenza‐induced, T cell‐mediated disease.

Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages

Established cell lines are widely used in research, however an appealing question is the comparability of the cells between various laboratories, their characteristics and stability in time. Problematic is also the cell line misidentification, genetic and phenotypic shift or Mycoplasma contamination which are often forgotten in research papers. The monocyte/macrophage-like cell line RAW 264.7 has been one of the most commonly used myeloid cell line for more than 40 years. Despite its phenotypic and functional stability is often discussed in literature or at various scientific discussion panels, their stability during the consecutive passages has not been confirmed in any solid study. So far, only a few functional features of these cells have been studied, for example their ability to differentiate into osteoclasts. Therefore, in the present paper we have investigated the phenotype and functional stability of the RAW 264.7 cell line from passage no. 5 till passage no. 50. We found out that the phenotype (expression of particular macrophage-characteristic genes and surface markers) and functional characteristics (phagocytosis and NO production) of RAW 264.7 cell line remains stable through passages: from passage no. 10 up to passage no. 30. Overall, our results indicated that the RAW 264.7 cell line should not be used after the passage no. 30 otherwise it may influence the data reliability.

Immune Cells As Targets and Tools For Cancer Therapy

Katarzyna Tonecka1#, Zofia Pilch1#, Kavita Ramji1, Bartlomiej Taciak2, Lukasz Kiraga2, Magdalena Krol2, and Tomasz P Rygiel1* 1Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, Warsaw, Poland 2Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland *Corresponding author: Tomasz P Rygiel, Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, Banacha 1a, 02-097 Warsaw, Poland, E-mail: tomasz.rygiel@wum.edu.pl

Low dose of GRP78-targeting subtilase cytotoxin improves the efficacy of photodynamic therapy in vivo

Photodynamic therapy (PDT) exerts direct cytotoxic effects on tumor cells, destroys tumor blood and lymphatic vessels and induces local inflammation. Although PDT triggers the release of immunogenic antigens from tumor cells, the degree of immune stimulation is regimen-dependent. The highest immunogenicity is achieved at sub-lethal doses, which at the same time trigger cytoprotective responses, that include increased expression of glucose-regulated protein 78 (GRP78). To mitigate the cytoprotective effects of GRP78 and preserve the immunoregulatory activity of PDT, we investigated the in vivo efficacy of PDT in combination with EGF-SubA cytotoxin that was shown to potentiate in vitro PDT cytotoxicity by inactivating GRP78. Treatment of immunocompetent BALB/c mice with EGF-SubA improved the efficacy of PDT but only when mice were treated with a dose of EGF-SubA that exerted less pronounced effects on the number of T and B lymphocytes as well as dendritic cells in mouse spleens. The observed antitumor effects were critically dependent on CD8+ T cells and were completely abrogated in immunodeficient SCID mice. All these results suggest that GRP78 targeting improves in vivo PDT efficacy provided intact T-cell immune system.

Engineered ferritin for lanthanide binding.

Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295–375 nm, fluorescence excitation.

Antitumor Activity of TLR7 Is Potentiated by CD200R Antibody Leading to Changes in the Tumor Microenvironment

Treatments that alter the immune composition of the tumor microenvironment affect antitumor activity. Stimulating TLR7 in combination with an agonistic mAb to CD200R improved the antitumor effects of TLR7 signaling. Subsequent changes affected myeloid cell composition and activation. Stimulation of Toll-like receptor 7 (TLR7) activates myeloid cells and boosts the immune response. Previously, we have shown that stimulation of the inhibitory CD200 receptor (CD200R) suppresses TLR7 signaling and that the absence of CD200R signaling leads to a decreased number of papillomas in mice. Here, we investigated the effects of agonistic anti-CD200R on the antitumor activity of a TLR7 agonist (R848) in a syngeneic mouse tumor model. Intratumoral administration of R848 inhibited the growth of the CT26 colon carcinoma and simultaneously decreased CD200R expression in tumor-infiltrating immune cells. The antitumor effects of R848 were potentiated by anti-CD200R. Successfully treated mice were resistant to rechallenge with the same tumor cells. However, the immediate antitumor effects were independent of lymphocytes, because treatment efficacy was similar in wild-type and Rag1tm1Mom mice. Administration of R848, particularly in combination with anti-CD200R, changed the phenotype of intratumoral myeloid cells. The infiltration with immature MHC-II+ macrophages decreased and in parallel monocytes and immature MHC-II− macrophages increased. Combined treatment decreased the expression of the macrophage markers F4/80, CD206, CD86, CD115, and the ability to produce IL1β, suggesting a shift in the composition of intratumor myeloid cells. Adoptively transferred CD11b+ myeloid cells, isolated from the tumors of mice treated with R848 and anti-CD200R, inhibited tumor outgrowth in recipient mice. We conclude that administration of agonistic anti-CD200R improves the antitumor effects of TLR7 signaling and changes the local tumor microenvironment, which becomes less supportive of tumor progression. Cancer Immunol Res; 6(8); 930–40. ©2018 AACR.