Competition-Based Cell Assay Employing Soluble T Cell Receptors to Assess MHC Class II Antigen Processing and Presentation

Abstract

Accurate assessment of antigen-specific immune responses is critical in the development of safe and efficacious biotherapeutics and vaccines. Endosomal processing of a protein antigen followed by presentation on major histocompatibility complex (MHC) class II constitute necessary steps in the induction of CD4+ T cell immune responses. Current preclinical methods for assessing immunogenicity risk consist of in vitro cell-based assays and computational prediction tools. Cell-based assays are time and labor-intensive while in silico methodologies have limitations. Here, we propose a novel cell-based assay capable of investigating an antigen's endosomal processing and MHC class II presentation capabilities. This novel assay relies on competition between epitopes for MHC class II binding and employs labeled soluble T cell receptors (sTCRs) as detectors of epitope presentation.

Keywords: APC; HLA-DR; MHC; TCR; antigen presentation.

Fig. 1

Soluble T cell receptor construct and assay design. a Construct design of truncated soluble TCRs: a single ORF encoding TCRα and TCRβ variable and constant domains and leucine zipper followed by a flexible (G4S)4 linker for interchain stability, native signal peptide (SP) was used. Picornavirus ribosomal skipping 2A sequence allows for equimolar chain production (2A). b Assay design: cell-based assay to assess antigen presentation. Left panel: cells are pulsed with reference epitope only. Right panel: cells are pulsed with reference epitope and a test peptide/protein. Efficient test compound presentation is quantified by decreased sTCR binding signal to the reference antigen

Fig. 2

sTCRs specifically bind the reference peptide and MHC class II complex. a F24 staining of 9036 HLA-DRB1*11:01 and 1055 *15:01 cells pulsed with an increasing amount of p24 epitope (X-axis). b F11 staining of 1111 HLA-DRB1*04:01 and 1124 *01:01 cells pulsed with an increasing amount of the HA epitope (X-axis). c F24 staining of 9036 HLA-DRB1*11:01 and 1055 *15:01 cells pulsed with 1200 μM of the reference epitope (p24) or a negative control (HA). d F11 staining of 1111 HLA-DRB1*04:01 and 1124 *01:01 cells pulsed with 1200 μM of the reference epitope (HA) or a negative control (p24). The data on Y-axis is expressed as the fold MFI of sTCR fluorescence in peptide-treated cells of mock-treated cells: MFI_{(reference epitope)} / median MFI_(mock). The one-way ANOVA test with multiple comparisons was used to determine significance, p > 0.05 not significant (ns)

Fig. 3

Competition-mediated detection of test epitope presentation on MHC-II. a F24 staining of 1055 HLA-DRB1*15:01 and 9036 *11:01 pulsed with p24 (reference epitope) alone or with an increasing concentration of HA test antigen (test peptide). b F11 staining of 1111 HLA-DRB1*04:01 and 1124 *01:01 cells pulsed with HA (reference epitope) alone or with an increasing concentration p24 test antigen (test peptide) (c, d). F24 staining of 9036 HLA-DRB1*11:01 and 1055 *15:01 cells was pulsed with p24 alone or in the presence of Omomyc (c) or influenza A H5N1 HA protein (d). The data on Y-axis is expressed as the percent sTCR florescence signal (MFI) relative to cells pulsed with reference epitope: MFI_{(test compound + reference epitope)} / median MFI_{(reference epitope alone).} The one-way ANOVA with multiple comparisons was used to determine significance, p > 0.05 not significant (ns). For statistical comparisons in a and b, samples treated with test peptide were compared to no test epitope; for c and d, samples treated with reference peptide + protein were compared to the reference epitope alone

Fig. 4

Detection of MHC class II epitope containing regions by the sTCR antigen presentation assay. a Omomyc protein sequence was divided into 9 peptides of 25AA in length with 10AA overlap; core binding epitopes are highlighted. b F24 sTCR binding to B-LCLs pulsed with p24 reference epitope alone or in combination with each of the 9 Omomyc overlapping peptides. c Output of four HLA-DR binding prediction algorithms and comparison with the in vitro sTCR binding data. Algorithm-predicted unique core binding epitopes or “hits” for each peptide are indicated (highlighted in yellow). d Representation of MAPPs detected peptides in B-LCLs expressing HLA-DRB1 types: *03:01 (1152), *01:01 (1124), *07:01 (9050), or *15:01 (1055) pulsed with full length Omomyc. Graph represents sum of unique MAPPs detected peptide containing the amino acid and is a compilation of four cell lines. e Position of presented Omomyc peptides by sTCR analysis within Omomyc sequence. In silico core binding epitopes or “hits” for each peptide are indicated (highlighted in yellow). The data on Y-axis is expressed as the percent sTCR florescence signal (MFI) relative to cells pulsed with reference epitope: MFI_{(test compound + reference epitope)} / median MFI_{(reference epitope alone).} The one-way ANOVA with multiple comparisons was used to determine significance comparing cells treated with reference epitope + Omomyc peptide to reference epitope alone, p > 0.05 not significant (ns)

Fig. 5

Minimum peptide length required for endosomal processing. a F24 binding to p24/MHC-II complex in the presence full length Omomyc in untreated B-LCLs or B-LCLs treated with chloroquine. b F24 binding to p24/MHC-II complex in B-LCLs pulsed with Omomyc peptides of increasing lengths in the presence or absence of chloroquine. The data on Y-axis is expressed as the percent sTCR florescence signal (MFI) relative to cells pulsed with reference epitope: MFI_{(test compound + reference epitope)} / median MFI_{(reference epitope alone).} The one-way ANOVA with multiple comparisons was used to determine significance, p > 0.05 not significant (ns)