(I-322) Using ELISA to Identify Autoantigenic Peptides for Expansion of Pathogenic CD4s

Introduction:: Human Leukocyte Antigens (HLA) are found on the surfaces of most cells in the body and are used to signal immune cells to mount an immune response. CD4+ T cells, known as helper T cells, use HLA Class II molecules to present foreign peptides to cytotoxic T cells [1]. Pathogenic CD4+ T cells can recognize a self-peptide as an infection, leading to conditions like Rheumatoid arthritis[2]. Studies have found that the occurrence of autoimmune diseases is closely linked to a person’s genetics. The main controlling genetic factors are Class II HLAs; these are governed by three main isotypes: HLA-DR, -DP, and –DQ, leading to numerous combinations [3,4]. Genetics can influence how immunity develops in life, so having certain Class II alleles can predispose a patient to developing disease [2]. There have been efforts to identify these antigen-specific CD4+ T cells that lead to autoimmune disease, but there have been many challenges slowing the development. For example, strides in machine learning and database mining have helped identify many peptides linked to disease [5]. However, there is a lack of high throughput wetlab assays for screening to verify the results of these models. One challenge is identifying the autoantigenic peptides that trigger the autoimmune response before the aberrant CD4+ T cells activate and multiply. To meet this challenge, we have developed an ELISA assay to identify autoantigenic peptides and predict their binding strength to HLA-DR receptors. The identified peptides were then used to expand pathogenic CD4s from naïve populations.

Materials and Methods:: HLA-DR-CLIP molecules underwent thrombin cleavage in peptide exchange to remove the placeholder CLIP molecule from the binding site (Fig. 1A, top). The cleaved HLA-DR was added to a 96-well plate coated in 10 mg of the candidate peptide to bind at 4C overnight in peptide exchange buffer (50 mM sodium citrate pH 5.2, 1% octylglucoside, 100 mM NaCl, and 1X Protease Inhibitor Cocktail). The binding of HLA-DR molecules was detected using biotinylated anti-HLA-DR monoclonal antibody (LN3) and avidin conjugated peroxidase (Fig. 1A, bottom). Absorption was measured using a plate reader. The peptides used in the study were DR4 restricted influenza HA (306-318) and DP4 restricted peptide p30 of tetanus toxin (947-967) (TT).

Human CD4 T cells were cultured in T cell culture media containing peptide exchanged HLA Class II APCs loaded with TT p30 peptide for 21 days. At 0, 7, 14, and 21 days, tetramer staining was performed by incubating cells at 20C with Human TruStain FcX Fc Receptor Blocking Solution (Biolegend) in T cell culture medium for 5 minutes. Following this, 50 nM dasatinib (Axon Medchem) was added for a 20-minute incubation at 36C. Then, PE-labeled cognate and non-cognate tetramers was added for a 30-minute incubation at 37C. Cells were then washed in PBS, stained with the Live/Dead Fixable Green (Invitrogen) and surface marker antibodies(CD3-BV605, CD4-APC/Cy7, CD11b-PE/Cy7,CB11c-PE/Cy7, CD14-PE/Cy7, CD19-PE/Cy7, CD45RA-BV421, CD56-PE/Cy7, CD62L-APC) for 15 minutes at 4C. The cells were analyzed on the Attune NxT Flow Cytometer.


Results, Conclusions, and Discussions:: An ELISA assay was used to measure the binding affinity between the peptide-HLA complexes (Fig. 1A bottom)
To optimize assay conditions, the pH of the binding buffer used when introducing the cleaved HLA-DR4 complex to the HA coated plate varied from a neutral solution to an acidic buffer (Fig 1B, left, center). The peptide exchange buffer (pH = 5.2) was far more successful at binding the cleaved HLA-DR4 complex to the HA peptide with an significant window between the control group, the uncleaved DR4-CLIP, and the experimental group, the cleaved DR4m compared to PBSA (pH = 7.0). The experiment was repeated with a different peptide-HLA pair: TT and HLA-DP4 (Fig. 1B, right). The significant window indicated the ELISA method is agnostic to the peptide-HLA combination used.

To validate the assay in cells, aAPCs were loaded with TT peptide and co-incubated with naive CD4+ T cells isolated from healthy patients. Over 21 days, antigen-specific populations were observed in all samples (169 depicted in Fig. 1C, left). To confirm that that expansion was from stimulation with the loaded TT peptide, a noncognate tetramer stain (CLIP-PE) was used to determine the effect CLIP had on expansion. The CLIP stimulation was insignificant, indicating that the predominant source of expansion was the TT peptide stimulation (Fig. 1D, top). Over the 21-day period, the TT specific CD4+ T cell population expanded from 1% to over 35%, and the number of these antigen specific CD4+ T cells increased 2-fold (Fig. 1D, bottom).

The next step for this project would be to perform competitive binding assays using the target restricted peptide and a competitor peptide to determine the relationship between concentration and binding. This will in turn help make a standardized method of comparing peptide binding strength in absence of a standard curve. This is essential because this ELISA method will be used to screen candidate peptides to identify the autoantigenic peptides that bind to HLA complexes.



Acknowledgements (Optional): : Dr. Jamie Spangler and Aliyah Silver provided the HLA-DR and HLA-DP monomers and dimers used in this study, as well as the original ELISA protocol that was modified for this study. We would like to thank Dr. Eleni Tiniakou and Dr. Erika Darrah for their support and guidance with this project and for providing myositis patient samples and candidate peptide sequences for future work with the assay described in this study.

References (Optional): : [1] Wosen Jonathan E., Mukhopadhyay Dhriti, Macaubas Claudia, Mellins Elizabeth D., Epithelial MHC Class II Expression and Its Role in Antigen Presentation in the Gastrointestinal and Respiratory Tracts. Front Immunol. 2018; 10.3389/fimmu.2018.02144

[2] Cruz-Tapias P. Autoimmunity: From Bench to Bedside [Internet]. El Rosario University Press; 2013.

[3] Fiorillo Maria Teresa, Paladini Fabiana, Tedeschi Valentina, Sorrentino Rosa, HLA Class I or Class II and Disease Association: Catch the Difference If You Can. Front Immunol. 2017; 10.3389/fimmu.2017.01475

[4] MacLachlan BJ, Dolton G, Papakyriakou A, Greenshields-Watson A, Mason GH, Schauenburg A, Besneux M, Szomolay B, Elliott T, Sewell AK, Gallimore A, Rizkallah P, Cole DK, Godkin A., Human leukocyte antigen (HLA) class II peptide flanking residues tune the immunogenicity of a human tumor-derived epitope. J Biol Chem. 2019; 10.1074/jbc.RA119.009437

[5] Wendorff M, Garcia Alvarez HM, Østerbye T, ElAbd H, Rosati E, Degenhardt F, Buus S, Franke A, Nielsen M., Unbiased Characterization of Peptide-HLA Class II Interactions Based on Large-Scale Peptide Microarrays; Assessment of the Impact on HLA Class II Ligand and Epitope Prediction. Front Immunol. 2020; 10.3389/fimmu.2020.01705