<p>S1. Schematic diagram of specific primers for the detection of PD-L1 variants. S2. Generation of recombinant fusion proteins of PD-L1 variants. S3. Inhibitory effects of membrane-bound PD-L1 on proliferation of antigen specific CD8+ T cells. S4. Effects of cytokines on expression of PD-L1 and cell proliferation in melanoma cell lines. S5. Associations between sPD-L1 secretion and PD-L1 expression in presence or absence of cytokine. S6. Secretion of sPD-L1 and expression of membrane-bound PD-L1 in response to sodium azide. S7. Specific and different recognitions of sPD-L1 variants by anti-PD-L1 antibody clone 29E.12B1 and 130021. S8. A. Schema on recognition regions of anti-PD-L1 antibodies. B. Detection of PD-L1-HIS (Amino acid 19-239) and PD-L1-3/Ig (Amino acid 19-178) by SDS-PAGE and immunoblotting assay with 130021 antibody. S9. Impact of serum or plasma matrices in quantitative detection of sPD-L1. S10. Kinetic changes of sPD-L1all and sPD-L1L in plasma of melanoma patients receiving ipilimumab plus bevacizumab. S11. Kinetic changes of cytokines in plasma of melanoma patients receiving ipilimumab plus bevacizumab. S12. sPD-L1 in plasma of melanoma patients receiving ipilimumab. S13. Kinetic changes of sPD-L1all and sPD-L1L in sera of melanoma patients receiving ipilimumab in ECOG 1608 trial. S14. Kinetic changes of sPD-L1all and sPD-L1L in plasma of melanoma patients receiving anti-PD-1 antibody. S15. Kinetic changes of cytokines in plasma of melanoma patients receiving anti-PD-1 antibody. S16. Associations of pretreatment levels of sPD-L1 with survival after check-point blockade. S17. Associations of long term or delayed increases in sPD-L1 with survival after check-point blockade. S18. Effects of cytokines on secretions of PD-L1 variants in DC. Supplemental Table 1. A. Amino acid regions of sPD-L1 variants. B. Amino acid regions of recombinant PD-L1 proteins. Supplemental Table 2. Associations of pretreatment levels of sPD-L1 with variables.</p>
CX3CL1 secreted in the tumor microenvironment serves as a chemoattractant playing a critical role in metastasis of CX3CR1 expressing cancer cells. CX3CR1 can be expressed in both cancer and immune-inhibitory myeloid cells to facilitate their migration. We generated a novel monoclonal antibody against mouse CX3CR1 that binds to CX3CR1 and blocks the CX3CL1-CX3CR1 interaction. We next explored the immune evasion strategies implemented by the CX3CL1-CX3CR1 axis and find that it initiates a resistance program in cancer cells that results in 1) facilitation of tumor cell migration, 2) secretion of soluble mediators to generate a pro-metastatic niche, 3) secretion of soluble mediators to attract myeloid populations, and 4) generation of tumor-inflammasome. The CX3CR1 monoclonal antibody reduces migration of tumor cells and decreases secretion of immune suppressive soluble mediators by tumor cells. In combination with anti-PD-1 immunotherapy, this CX3CR1 monoclonal antibody enhances survival in an immunocompetent mouse colon carcinoma model through a decrease in tumor-promoting myeloid populations. Thus, this axis is involved in the mechanisms of resistance to anti-PD-1 immunotherapy and the combination therapy can overcome a portion of the resistance mechanisms to anti-PD-1.
In the present study, follicular dendritic cells (FDCs) were purified to homogeneity in order to define the lineage and function of these cells. FDCs were identified by their characteristic morphology and by their expression of receptors for the third complement component, the myeloid-restricted antigen CD14, and the FDC antigen DRC-1. Unclustered FDCs displayed a unique antigenic phenotype since they expressed several B- and myeloid lineage-restricted antigens, but lacked T and NK cell antigens as well as the leukocyte common antigen. FDCs expressed adhesion molecules, including most of the VLA proteins, intercellular adhesion molecule 1 (ICAM-1), and CD11b. FDCs could be isolated to homogeneity by their intense staining with anti-CD14 using flow cytometric cell sorting. These highly purified FDCs expressed CD14 and CD21 but lacked CD20. This antigen pattern and characteristic morphology confirmed that these cells were, in fact, homogeneous FDC preparations. Analysis of polymerase chain reaction-amplified cDNA from highly purified FDCs showed no transcripts for IL-6. The isolation of homogeneous FDC populations will be important for the analysis of the functional role of FDCs within the lymphoid follicle.
// Senthil Kannan 1,2 , Raj K. Kurupati 2 , Susan A. Doyle 3 , Gordon J. Freeman 4 , Kenneth E. Schmader 3 and Hildegund C.J. Ertl 2 1 Biomedical Graduate Group, University of Pennsylvania, Philadelphia, PA, USA 2 The Wistar Institute, Philadelphia, PA, USA 3 GRECC, Durham VA Medical Center and Center for the Study of Aging and Human, Development and Division of Geriatrics, Department of Medicine, Duke University Medical Center, Durham, NC, USA 4 Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Correspondence to: Hildegund C.J. Ertl, email: // Keywords : BTLA, aging, HSV, immunosenescence, influenza Received : June 03, 2015 Accepted : June 11, 2015 Published : June 21, 2015 Abstract Virus-neutralizing antibody and B cell responses to influenza A viruses were measured in 35 aged and 28 middle-aged individuals following vaccination with the 2012 and 2013 trivalent inactivated influenza vaccines. Antibody responses to the vaccine strains were lower in the aged. An analysis of B cell subsets by flow cytometry with stains for immunoregulators showed that B cells of multiple subsets from the aged as compared to younger human subjects showed differences in the expression of the co-inhibitor B and T lymphocyte attenuator (BTLA). Expression of BTLA inversely correlated with age and appears to be linked to shifting the nature of the response from IgM to IgG. High BTLA expression on mature B cells was linked to higher IgG responses to the H1N1 virus. Finally, high BTLA expression on isotype switched memory B cells was linked to better preservation of virus neutralizing antibody titers and improved recall responses to vaccination given the following year.
592 Background: Colorectal cancer (CRC) is a major public health problem worldwide. Chemotherapy consisting of a fluoropyrimidine backbone constitutes a major therapeutic modality used in the treatment of CRC patients. However, cancer cells often develop resistance to such cytotoxic chemotherapy and this represents a major therapeutic challenge. B7-homolog 1 (B7-H1), also known as programmed death ligand-1 (PD-L1) is an immunoregulatory protein that belongs to the B7 family of T-cell co-regulatory molecules, and is overexpressed in several human tumors. Upregulation of PD-L1 expression is an important mechanism by which tumor cells can escape host T-cell immunity. Emerging evidence suggests that chemotherapeutic agents can regulate PD-L1 expression on cancer cells, which may have an impact on anti-tumor immunity and immune evasion. We performed this study to evaluate the effect of 5-Fluorouracil (5-FU) on PD-L1 expression in colon cancer cell lines, using an in vitro approach. Methods: Flow cytometry and western immunoblot analyses for PD-L1 expression were performed on human colon cancer cell lines (HCT116-WT, HCT116-p53 KO, SW480, HT29) upon treatment with IFN-ɣ and 5-FU. Results: We found that the tested human colon cancer cell lines rarely expressed PD-L1 protein on their cell surface at baseline, but a high level of expression was induced by treatment with IFN-ɣ. More importantly, we demonstrated that the chemotherapeutic agent 5-FU induces PD-L1 expression in colon cancer cells. Conclusions: It is therefore plausible that combining 5-FU with PD-1/PD-L1 blockade might help overcome some of the chemoresistance to 5-FU and thereby enhance its anti-cancer activity. Further experiments are being planned to formally test this hypothesis.
