From part of the patients peripheral blood was collected on the day of resection. culture. Baseline proliferation (= % of CFSElow T cells in the presence of eGFP\electroporated B cells) was normalized to 100% for each tested patient. Proliferation in response to tumor antigen is shown as percentage of CFSElow cells compared to baseline proliferation in response to eGFP. For those patients whose TIL responded to both tumor antigens, the average response to GPC3\ and MAGEC2\electroporated B cells was depicted. Bars show mean percentages in cultures derived from n?= 8 patients with SEM. IJC-145-1111-s002.tif (8.2M) GUID:?14E1D47B-16CC-475D-BDDB-5991CFF7BD87 Supplementary Table 1: Anti\human antibodies used for flow Rabbit Polyclonal to OR8J3 cytometry. IJC-145-1111-s003.docx (16K) GUID:?3919F5F7-5C7C-4C64-A711-F3091AD43CA4 Abstract No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti\PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co\inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co\stimulatory receptors might be able to stimulate anti\tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co\stimulatory receptor GITR could reinvigorate functional responses of tumor\infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor\free liver tissues, and studied the effects of combined GITR and PD1 targeting Budesonide on TIL responses. In all three tissue compartments, CD4+FoxP3+ regulatory T cells (Treg) showed higher GITR?expression than effector T\cell subsets. The highest expression of GITR was Budesonide found on CD4+FoxP3hiCD45RA? activated Treg in tumors. Recombinant GITR\ligand as well as a humanized agonistic anti\GITR antibody enhanced proliferative responses of CD4+ and CD8+ TIL to tumor antigens presented by mRNA\transfected autologous B\cell blasts, and also reinforced proliferation, IFN\ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti\PD1 antibody nivolumab further enhanced tumor antigen\specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC. Keywords: GITR, CD357, TNFRSF18, Treg, HCC, cancer immunotherapy, PD1 Short abstract What’s new? Therapeutic antibodies that block interaction of the T cell co\inhibitory receptor PD1 can unleash pre\existing anti\cancer T cell responses in hepatocellular carcinoma (HCC). However, whether agonistic targeting of co\stimulatory receptors could stimulate anti\tumor immunity remains unknown. This study is the first to show that agonistic targeting of the co\stimulatory receptor GITR can reinforce the functionality of tumor\infiltrating T cells isolated from human tumors. Combined targeting of PD1 and GITR exerts Budesonide additive stimulatory effects on functionality of tumor\infiltrating T cells from HCC patients. Targeting of GITR thus emerges as a promising strategy for single or combinatorial immunotherapy in HCC. AbbreviationsCFSEcarboxyfluorescein diacetate succinimidyl esterGITRLGITR ligandGPC3glypican 3HCChepatocellular carcinomaMAGEC2MAGE family member C2PBMCperipheral blood mononuclear cellsPD1programmed cell death 1Tregregulatory T cellsThT helper cellsGITRTNF receptor superfamily member 18TFLtumor\free liverTILtumor\infiltrating lymphocytes Introduction Liver cancer is the second most common cause of cancer\related mortality worldwide, with approximately 750,000 deaths per year. The most common primary liver cancer is hepatocellular carcinoma Budesonide (HCC), an aggressive malignancy derived from hepatocytes.1, 2 Surgical resection and liver transplantation are curative therapies for patients with early stage disease. However, about 50% of HCC patients present with advanced disease at diagnosis and can only be offered systemic therapies which provide limited survival advantage.3, 4 Therefore, novel therapies for HCC are urgently needed. Immune checkpoint antibodies are a new class of cancer immune therapeutics. T cells are activated upon antigen recognition their T\cell receptor and engagement of their co\stimulatory immune checkpoint receptors with corresponding ligands on other cells, while they are suppressed upon interaction of their co\inhibitory immune checkpoint receptors with their ligands. Therapeutic antibodies that block interaction of the co\inhibitory receptor PD1 with its ligands can unleash pre\existing anti\cancer T\cell responses within tumors, and have resulted in recent breakthroughs in clinical treatment of several types of advanced cancer.5, 6, 7, 8, 9, 10, 11, 12, 13 In HCC, a recent trial showed significant tumor load reduction (objective response) in response to anti\PD1 antibody (nivolumab) therapy in about 20% of advanced HCC patients, and disease control with stable disease for 6 months in another 17% of patients.14 Nevertheless, more than 50% of advanced HCC patients.
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