Combination of HPLC and solid-phase binding assay for isolation and purification of MHC class I and associated peptides using a bladder tumour cell line

2001 ◽  
Vol 15 (1) ◽  
pp. 18-24
Author(s):  
N. Torabi-Pour ◽  
A. M. E. Nouri ◽  
D. Perrett ◽  
R. T. D. Oliver
Keyword(s):  
Cell Line ◽  
Mhc Class I ◽  
Tumour Cell ◽  
Binding Assay ◽  
Solid Phase ◽  
Bladder Tumour ◽  
Class I ◽  
Tumour Cell Line ◽  
Isolation And Purification ◽  
Solid Phase Binding

Identification and Quantification of Antigens Associated with HLA Class I Molecules on a Bladder Tumour Cell Line

2003 ◽  
Vol 70 (3) ◽  
pp. 154-160 ◽  
Author(s):  
Nasir Torabi-Pour ◽  
W. John W. Morrow ◽  
Roghieh Saffie ◽  
Prashant G. Gowda ◽  
D. Perrett ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumour Cell ◽  
Bladder Tumour ◽  
Hla Class I ◽  
Class I ◽  
Tumour Cell Line ◽  
Hla Class I Molecules ◽  
Identification And Quantification

scholarly journals Evidence of widespread binding of HLA class I molecules to peptides.

1990 ◽  
Vol 172 (3) ◽  
pp. 827-834 ◽  
Author(s):  
J A Frelinger ◽  
F M Gotch ◽  
H Zweerink ◽  
E Wain ◽  
A J McMichael
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Assay ◽  
Solid Phase ◽  
Hla Class I ◽  
Class I ◽  
T Cell Epitopes ◽  
Single Class ◽  
Hla Class I Molecules ◽  
Solid Phase Binding ◽  
Hiv 1

We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein.

608 Immunodominant listeria epitopes compete with vaccine-directed cd8+ t-cell responses rescued by peptide-MHC stabilizing modifications

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A644-A644
Author(s):  
John Flickinger ◽  
Jagmohan Singh ◽  
Yanki Yarman ◽  
Robert Carlson ◽  
Scott Waldman ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
T Cell ◽  
Cell Line ◽  
Mhc Class I ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Class I ◽  
Peptide Ligand ◽  
Altered Peptide Ligand ◽  
Cell Responses

BackgroundThe Gram-positive bacterium Listeria monocytogenes (Lm) is a promising vector for cancer immunotherapy due to its ability to directly infect antigen-presenting cells, induce potent CD8+ T-cell immunity, and remodel immunosuppressive tumor microenvironments.1 Recent clinical trials have demonstrated safety and immunogenicity of Lm-based cancer vaccines in lung, cervical, pancreatic, and other cancers. In colorectal cancer, the transmembrane receptor guanylyl cyclase C (GUCY2C) is an emerging target for immunotherapy.2 Here, we examined the immunogenicity of a recombinant strain of Listeria monocytogenes secreting GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination induced robust Lm-specific CD8+ T-cell immunity but failed to prime GUCY2C-specific CD8+ T-cell responses. These studies explore the hypothesis that immunodominant Lm antigens suppress primary immunity to subdominant GUCY2C epitopes in Lm-GUCY2CMethodsLm-GUCY2C expresses the extracellular domain of mouse GUCY2C23-429 downstream of an ActA promoter integrated into the genome of the live, attenuated delta actA delta inlB Lm strain. Altered peptide ligands were designed based on NetMHCpan 4.0 peptide-MHC binding algorithms and similarly cloned into Lm. Peptide-MHC class I complex stability was quantified by FACS-based surface peptide-MHC dissociation on the TAP-deficient cell line, RMA-S H-2Kd.3In vivo efficacy studies employed IFNγ-ELISpot quantification of T-cell responses and tumor challenge studies with the CT26 colorectal cancer cell line. Adenovirus expressing GUCY2C was used as a positive control.2 4ResultsLm-GUCY2C vaccination of BALB/c mice generated Lm-specific CD8+ T-cell responses but an absence of GUCY2C-specific immunity. Peptide-MHC stability studies revealed poor stability of the dominant GUCY2C254-262 epitope complexed with H-2Kd compared to H-2Kd-restricted Lm epitopes derived from the LLO and p60 Lm antigens. Mutation of the GUCY2C254-262 peptide at critical anchoring residues for binding H-2Kd revealed that the altered peptide ligand with an F255Y mutation significantly improved the stability of the GUCY2C254-262-H-2Kd complex. Similarly, vaccination of mice with recombinant Lm-GUCY2C expressing the altered peptide ligand (Lm-GUCY2CF255Y) restored GUCY2C immunogenicity and antitumor immunity.ConclusionsImmunodominant Lm antigens may interfere with immune responses directed to the vaccine target antigen GUCY2C by competing with GUCY2C epitope for MHC class I binding and presentation. Moreover, use of a substituted GUCY2C -peptide ligand with enhanced peptide-MHC class I stability restored GUCY2C-specific immunity in the context of Lm-GUCY2C, an approach that can be translated to patients. Importantly, these studies also suggest that ongoing Lm-based vaccine development programs targeting a variety of antigens in other cancer types may be similarly limited by the immunodominance of Lm epitopes.AcknowledgementsThe authors thank Dr. Peter Lauer for providing the pPL2 integration vector used in cloning Lm-GUCY2C and Dr. Sean Murphy for providing the RMA-S H-2Kd cell line.Ethics ApprovalStudies were approved by the Thomas Jefferson University IACUC (Protocol # 01956).ReferencesFlickinger JC, Rodeck U, Snook AE. Listeria monocytogenes as a Vector for Cancer Immunotherapy: Current Understanding and Progress. Vaccines (Basel) 2018;6. doi:10.3390/vaccines6030048.Snook AE, Baybutt TR, Xiang B, Abraham TS, Flickinger JC, Hyslop T, et al. Split tolerance permits safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients. J Immunother Cancer 2019;7:104. doi:10.1186/s40425-019-0576-2.Müllbacher A, Lobigs M, Kos FJ, Langman R. Alloreactive cytotoxic T-cell function, peptide nonspecific. Scand J Immunol 1999;49:563–9.Flickinger J. JC, Singh J, Carlson R, Leong E, Baybutt T, Barton J, et al. Chimeric Ad5.F35 vector evades anti-adenovirus serotype 5 neutralization opposing GUCY2C-targeted antitumor immunity. J Immunother Cancer 2020.

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