Journal Articles

Prebiotics help mice fight melanoma by activating anti-tumor immunity

ABSTRACT: Growing evidence supports the importance of gut microbiota in the control of tumor growth and response to therapy. Here, we select prebiotics that can enrich bacterial taxa that promote anti-tumor immunity. Addition of the prebiotics inulin or mucin to the diet of C57BL/6 mice induces anti-tumor immune responses and inhibition of BRAF mutant melanoma growth in a subcutaneously implanted syngeneic mouse model. Mucin fails to inhibit tumor growth in germ-free mice, indicating that the gut microbiota is required for the activation of the anti-tumor immune response. Inulin and mucin drive distinct changes in the microbiota, as inulin, but not mucin, limits tumor growth in synge-neic mouse models of colon cancer and NRAS mutant melanoma and enhances the efficacy of a MEK inhibitor against melanoma while delaying the emergence of drug resistance. We highlight the importance of gut microbiota in anti-tumor immunity and the potential therapeutic role for prebiotics in this process.

Author Info: (1) Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA; (2) Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE 68588, U

Author Info: (1) Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA; (2) Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE 68588, USA; (3) Technion Integrated Cancer Center, Faculty of Medicine, Technion, Haifa 3525433, Israel; (4) Present address: Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA; (5) These authors contributed equally; (6) Lead Contact *Correspondence: speterson@sbpdiscovery.org (S.N.P.), zeev@ronailab.net (Z.A.R.)

Toll-like Receptors from the Perspective of Cancer Treatment

Toll-like receptors (TLRs) represent a family of pattern recognition receptors that recognize certain pathogen-associated molecular patterns and damage-associated molecular patterns. TLRs are highly interesting to researchers including immunologists because of the involvement in various diseases including cancers, allergies, autoimmunity, infections, and inflammation. After ligand engagement, TLRs trigger multiple signaling pathways involving nuclear factor-kappaB (NF-kappaB), interferon-regulatory factors (IRFs), and mitogen-activated protein kinases (MAPKs) for the production of various cytokines that play an important role in diseases like cancer. TLR activation in immune as well as cancer cells may prevent the formation and growth of a tumor. Nonetheless, under certain conditions, either hyperactivation or hypoactivation of TLRs supports the survival and metastasis of a tumor. Therefore, the design of TLR-targeting agonists as well as antagonists is a promising immunotherapeutic approach to cancer. In this review, we mainly describe TLRs, their involvement in cancer, and their promising properties for anticancer drug discovery.

Author Info: (1) Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea. (2) Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea.

Author Info: (1) Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea. (2) Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea.

Ribonucleic Acid Engineering of Dendritic Cells for Therapeutic Vaccination: Ready 'N Able to Improve Clinical Outcome

Targeting and exploiting the immune system has become a valid alternative to conventional options for treating cancer and infectious disease. Dendritic cells (DCs) take a central place given their role as key orchestrators of immunity. Therapeutic vaccination with autologous DCs aims to stimulate the patient's own immune system to specifically target his/her disease and has proven to be an effective form of immunotherapy with very little toxicity. A great amount of research in this field has concentrated on engineering these DCs through ribonucleic acid (RNA) to improve vaccine efficacy and thereby the historically low response rates. We reviewed in depth the 52 clinical trials that have been published on RNA-engineered DC vaccination, spanning from 2001 to date and reporting on 696 different vaccinated patients. While ambiguity prevents reliable quantification of effects, these trials do provide evidence that RNA-modified DC vaccination can induce objective clinical responses and survival benefit in cancer patients through stimulation of anti-cancer immunity, without significant toxicity. Succinct background knowledge of RNA engineering strategies and concise conclusions from available clinical and recent preclinical evidence will help guide future research in the larger domain of DC immunotherapy.

Author Info: (1) Laboratory of Experimental Hematology, VAXINFECTIO, Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Antwerp, Belgium. (2) Laboratory of Experimental Hemato

Author Info: (1) Laboratory of Experimental Hematology, VAXINFECTIO, Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Antwerp, Belgium. (2) Laboratory of Experimental Hematology, VAXINFECTIO, Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Antwerp, Belgium. (3) Center for Oncological Research, Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Antwerp, Belgium. (4) Laboratory of Experimental Hematology, VAXINFECTIO, Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Antwerp, Belgium. (5) Laboratory of Experimental Hematology, VAXINFECTIO, Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Antwerp, Belgium. Center for Oncological Research, Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Antwerp, Belgium.

Understanding the glioblastoma immune microenvironment as basis for the development of new immunotherapeutic strategies

Cancer immunotherapy by immune checkpoint blockade has proven its great potential by saving the lives of a proportion of late stage patients with immunogenic tumor types. However, even in these sensitive tumor types, the majority of patients do not sufficiently respond to the therapy. Furthermore, other tumor types, including glioblastoma, remain largely refractory. The glioblastoma immune microenvironment is recognized as highly immunosuppressive, posing a major hurdle for inducing immune-mediated destruction of cancer cells. Scattered information is available about the presence and activity of immunosuppressive or immunostimulatory cell types in glioblastoma tumors, including tumor-associated macrophages, tumor-infiltrating dendritic cells and regulatory T cells. These cell types are heterogeneous at the level of ontogeny, spatial distribution and functionality within the tumor immune compartment, providing insight in the complex cellular and molecular interplay that determines the immune refractory state in glioblastoma. This knowledge may also yield next generation molecular targets for therapeutic intervention.

Author Info: (1) Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium. Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium. (

Author Info: (1) Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium. Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium. (2) Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium. Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium. (3) Department of Neurosurgery, UZ Brussels, Brussels, Belgium. (4) Department of Medical Oncology, UZ Brussels, Brussels, Belgium. (5) Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium. Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium. (6) Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium. Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium.

Analysis of the association between prior chemotherapy regimens and outcomes of subsequent anti-PD-(L)1 monotherapy in advanced non-small cell lung cancer

Background: Immune checkpoint inhibitor (ICI) monotherapy targeting PD-1/PD-L1 has been a prominent option for the patients with advanced non-small cell lung cancer (NSCLC), which is now commonly used in second- or later-line settings after the failure of conventional chemotherapy. Chemotherapy can modulate tumor immunity in drug-dependent manner, suggesting pre-ICI chemotherapeutic regimens might influence the efficacy of immunotherapy. Therefore, it is of interest to investigate the associations between the types of pre-ICI chemotherapy and the outcomes of patients receiving ICIs treatment. Methods: The data from NSCLC patients who received anti-PD-1/PD-L1 ICI monotherapy after the failure of first-line chemotherapy were retrospectively reviewed. Clinical outcomes of the patients following ICIs monotherapy were compared according to different pre-ICI chemotherapeutic regimens. Results: Eighty-nine cases receiving ICI monotherapy immediately after the failure of first-line chemotherapy were included into final analysis. The patients in Gem group had the longest PFS (median: 6.50 m) following ICIs treatment (P=0.031), compared to Pem group and Tax group (median: 3.49 and 3.30 m, respectively). Pre-ICI chemotherapy with Gem retained independently associated with favorable PFS (P=0.014, HR 0.52; 95% CI, 0.31-0.88) in multivariate analysis after adjusting for other covariates. The patients in Gem group also achieved better objective response rate (ORR) (P=0.046) and disease control rate (DCR) (P=0.005) following ICIs treatment compared to those in Pem/Tax group. The differences in depth of response to ICIs between Gem and Pem/Tax groups were also compared. Of the 48 patients who achieved controlled disease and had >/=1 measurable target lesion during ICIs treatment, no greater tumor shrinkage was observed in Gem group (P=0.374), however, Gem group trended to have shorter TTM (P=0.074). Conclusions: Prior-line chemotherapy regimens might influence outcomes of the following ICIs monotherapy. Patients received pre-ICI gemcitabine-containing chemotherapy are significantly correlated with longer PFS and better response to ICIs treatment.

Author Info: (1) Department of Medical Oncology, Shanghai Pulmonary Hospital &Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai 200433, China. (2) Department of Medical

Author Info: (1) Department of Medical Oncology, Shanghai Pulmonary Hospital &Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai 200433, China. (2) Department of Medical Oncology, Shanghai Pulmonary Hospital &Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai 200433, China. (3) Department of Medical Oncology, Shanghai Pulmonary Hospital &Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai 200433, China. (4) Department of Lung Cancer and Immunology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China. (5) Department of Lung Cancer and Immunology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China. (6) Department of Medical Oncology, Shanghai Pulmonary Hospital &Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai 200433, China. (7) Department of Medical Oncology, Shanghai Pulmonary Hospital &Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai 200433, China. (8) Department of Medical Oncology, Shanghai Pulmonary Hospital &Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai 200433, China. (9) Department of Medical Oncology, Shanghai Pulmonary Hospital &Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai 200433, China. (10) Department of Medical Oncology, Shanghai Pulmonary Hospital &Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai 200433, China.

PSCA is a target of chimeric antigen receptor T cells in gastric cancer

Background: Gastric cancer is a deadly malignancy and is a prognostically unfavorable entity with restricted therapeutic strategies available. Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein widely expressed in bladder, prostate, and pancreatic cancers. Existing studies have thoroughly recognized the availability of utilizing anti-PSCA CAR-T cells in the treatment of metastatic prostate cancer and non-small-cell lung cancer. However, no previous study has investigated the feasibility of using anti-PSCA CAR-T cells to treat gastric cancer, irrespective of the proven expression of PSCA on the gastric cancer cell surface. Methods: We determined the expression of PSCA in several primary tumor tissues and constructed third-generation anti-PSCA CAR-T cells. We then incubated anti-PSCA CAR-T cells and GFP-T cells with target tumor cell lines at E:T ratios of 2:1, 1:1, 1:2, and 1:4 to evaluate the therapeutic efficacy of anti-PSCA CAR-T cells in vitro. We also assayed canonical T cell activation markers after coculturing anti-PSCA CAR-T cells with target cell lines by flow cytometry. The detection of a functional cytokine profile was carried out via enzyme-linked immunosorbent assays. We then evaluated the antitumor activity of anti-PSCA CAR-T cells in vivo by establishing two different xenograft GC mouse models. Results: Anti-PSCA CAR-T cells exhibited upregulated activation markers and increased cytokine production profiles related to T cell cytotoxicity in an antigen-dependent manner. Moreover, anti-PSCA CAR-T cells exhibited robust anti-tumor cytotoxicity in vitro. Importantly, we demonstrated that anti-PSCA CAR-T cells delivered by peritumoral injection successfully stunted tumor progression in vivo. However, intravenous administration of anti-PSCA CAR-T cells failed to reveal any therapeutic improvements. Conclusions: Our findings corroborated the feasibility of anti-PSCA CAR-T cells and their efficacy against gastric cancer, implicating the potential of applying anti-PSCA CAR-T cells to treat GC patients in the clinic.

Author Info: (1) 1School of Life Sciences, University of Science and Technology of China, Hefei, 230027 China.0000000121679639grid.59053.3a 2Key Laboratory of Regenerative Biology, South China

Author Info: (1) 1School of Life Sciences, University of Science and Technology of China, Hefei, 230027 China.0000000121679639grid.59053.3a 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 (2) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 4University of Chinese Academy of Sciences, Shijingshan District, Beijing, 100049 China.0000 0004 1797 8419grid.410726.6 (3) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 5Institute of Hematology, Medical College, Jinan University, Guangzhou, 510632 China.0000 0004 1790 3548grid.258164.c (4) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 4University of Chinese Academy of Sciences, Shijingshan District, Beijing, 100049 China.0000 0004 1797 8419grid.410726.6 (5) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 4University of Chinese Academy of Sciences, Shijingshan District, Beijing, 100049 China.0000 0004 1797 8419grid.410726.6 (6) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 4University of Chinese Academy of Sciences, Shijingshan District, Beijing, 100049 China.0000 0004 1797 8419grid.410726.6 (7) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 (8) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 (9) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 (10) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 (11) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 6Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 (12) 2Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3 3Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.0000 0004 1798 2725grid.428926.3

Exploiting Human NK Cells in Tumor Therapy

NK cells play an important role in the innate defenses against tumor growth and metastases. Human NK cell activation and function are regulated by an array of HLA class I-specific inhibitory receptors and activating receptors recognizing ligands expressed de novo on tumor or virus-infected cells. NK cells have been exploited in immunotherapy of cancer, including: (1) the in vivo infusion of IL-2 or IL-15, cytokines inducing activation and proliferation of NK cells that are frequently impaired in cancer patients. Nonetheless, the significant toxicity experienced, primarily with IL-2, limited their use except for combination therapies, e.g., IL-15 with checkpoint inhibitors; (2) the adoptive immunotherapy with cytokine-induced NK cells had effect on some melanoma metastases (lung), while other localizations were not affected; (3) a remarkable evolution of adoptive cell therapy is represented by NK cells engineered with CAR-targeting tumor antigens (CAR-NK). CAR-NK cells complement CAR-T cells as they do not cause GvHD and may be obtained from unrelated donors. Accordingly, CAR-NK cells may represent an "off-the-shelf" tool, readily available for effective tumor therapy; (4) the efficacy of adoptive cell therapy in cancer is also witnessed by the alphabetaT cell- and B cell-depleted haploidentical HSC transplantation in which the infusion of donor NK cells and gammadeltaT cells, together with HSC, sharply reduces leukemia relapses and infections; (5) a true revolution in tumor therapy is the use of mAbs targeting checkpoint inhibitors including PD-1, CTLA-4, the HLA class I-specific KIR, and NKG2A. Since PD-1 is expressed not only by tumor-associated T cells but also by NK cells, its blocking might unleash NK cells playing a crucial effector role against HLA class I-deficient tumors that are undetectable by T cells.

Author Info: (1) Immunology Research Area, IRCCS Bambino Gesu Pediatric Hospital, Rome, Italy. (2) UOC Immunology, IRCCS Ospedale Policlinico San Martino, Genoa, Italy. Department of Experiment

Author Info: (1) Immunology Research Area, IRCCS Bambino Gesu Pediatric Hospital, Rome, Italy. (2) UOC Immunology, IRCCS Ospedale Policlinico San Martino, Genoa, Italy. Department of Experimental Medicine (DIMES), Universita di Genova, Genoa, Italy. (3) Immunology Research Area, IRCCS Bambino Gesu Pediatric Hospital, Rome, Italy. (4) Department of Pathology, IRCCS Sacro Cuore Don Calabria, Negrar, Italy. (5) UOC Immunology, IRCCS Ospedale Policlinico San Martino, Genoa, Italy. Department of Experimental Medicine (DIMES), Center of Excellence for Biomedical Research, Universita di Genova, Genoa, Italy. (6) Immunology Research Area, IRCCS Bambino Gesu Pediatric Hospital, Rome, Italy.

PD-1+ Tim3+ tumor-infiltrating CD8 T cells sustain the potential for IFN-gamma production, but lose cytotoxic activity in ovarian cancer

Persistent exposure to tumor antigens results in exhausted tumor-infiltrating T cells (TILs) that express the immune checkpoint molecules, PD-1 and Tim3, and lack anti-tumor immunity. To examine the exhausted status of TILs in ovarian cancer, the potential for cytokine production, proliferation, and cytotoxicity by purified PD-1+ Tim3+ CD8 TILs was assessed. The production of IFN-gamma and TNF-alpha by PD-1+ Tim3+ CD8 TILs remained the same in an intracellular cytokine staining assay and was higher in a cytokine catch assay than that by PD-1- Tim3- and PD-1+ Tim3- CD8 TILs. %Ki67+ was higher in PD-1+ Tim3+ CD8 TILs than in PD-1- Tim3- CD8 TILs. However, patients with high PD-1+ Tim3+ CD8 TILs had a poor prognosis. The potential for cytotoxicity was then examined. %Perforin+ and %granzyme B+ were lower in PD-1+ Tim3+ CD8 TILs than in PD-1- Tim3- and PD-1+ Tim3- CD8 TILs. To observe the potential for direct cytotoxicity by T cells, a target cell line expressing membrane-bound anti-CD3scFv was newly established and a cytotoxic assay targeting these cells was performed. The cytotoxicity of PD-1+ Tim3+ CD8 TILs was significantly lower than that of PD-1- Tim3- and PD-1+ Tim3- CD8 TILs. Even though PD-1+ Tim3+ CD8 TILs in ovarian cancer showed a sustained potential for cytokine production and proliferation, cytotoxicity was markedly impaired, which may contribute to the poor prognosis of patients with ovarian cancer. Among the impaired functions of exhausted TILs, cytotoxicity may be an essential target for cancer immunotherapy.

Author Info: (1) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan. Department of Obstetrics and Gynecology, Osaka University Gradu

Author Info: (1) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan. Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan. (2) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan. Drug Discovery & Disease Research Laboratory, Shionogi & Co., Ltd., Toyonaka, Japan. (3) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan. Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan. (4) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan. Drug Discovery & Disease Research Laboratory, Shionogi & Co., Ltd., Toyonaka, Japan. (5) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan. (6) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan. (7) Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan. (8) Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan. (9) Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan. (10) Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan. (11) Department of Urology, Osaka University Graduate School of Medicine, Osaka, Japan. (12) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan. Drug Discovery & Disease Research Laboratory, Shionogi & Co., Ltd., Toyonaka, Japan. (13) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan. (14) Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan. (15) Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan. (16) Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan. (17) Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine, Osaka, Japan.

Endoplasmic Reticulum Aminopeptidase 1 beyond Antigenic Peptide-Processing Enzyme in the Endoplasmic Reticulum

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is well known as a processing enzyme of antigenic peptides, which are presented to major histocompatibility complex (MHC) class I molecules in the lumen of endoplasmic reticulum. Besides antigen processing, ERAP1 performs multiple functions in various cells depending on its intracellular and extracellular localization. Of note is the secretion of ERAP1 into the extracellular milieu in response to inflammatory stimuli, which further activates immune cells including macrophages and natural killer cells. Furthermore, secreted ERAP1 enhances the expression of pro-inflammatory cytokines like tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6. Such findings indicate that ERAP1 plays a significant role in the field of innate and acquired immunity. This review summarizes the functional analyses of ERAP1 that support our current understanding of its role as more than an antigenic peptide-processing enzyme, specifically emphasizing on its secretory form.

Author Info: (1) Faculty of Pharmaceutical Sciences, Teikyo Heisei University. (2) Faculty of Pharmaceutical Sciences, Teikyo Heisei University. (3) Faculty of Pharmaceutical Sciences, Teikyo H

Author Info: (1) Faculty of Pharmaceutical Sciences, Teikyo Heisei University. (2) Faculty of Pharmaceutical Sciences, Teikyo Heisei University. (3) Faculty of Pharmaceutical Sciences, Teikyo Heisei University. (4) Faculty of Pharmaceutical Sciences, Teikyo Heisei University.

Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.

Author Info: (1) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (2) Cytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Bi

Author Info: (1) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (2) Cytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium. (3) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (4) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (5) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (6) Department of Internal Medicine and Pediatrics, Ghent University, 9000 Ghent, Belgium. (7) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (8) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (9) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (10) Cytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium. (11) Cytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium. (12) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (13) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. (14) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium. Department of Internal Medicine and Pediatrics, Ghent University, 9000 Ghent, Belgium. (15) Cytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium. (16) Department of Diagnostic Sciences, Ghent University, 9000 Ghent, Belgium.

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