Determining if T cell antigens are naturally processed and presented on HLA class I molecules

Jay Friedman,Clint T Allen,Christian Hinrichs,Maxwell Lee,Ke Bai,Sreenivasulu Gunti

doi:10.1186/s12865-022-00478-4

Abstract

BackgroundDetermining T cell responses to naturally processed and presented antigens is a critical immune correlate to determine efficacy of an investigational immunotherapeutic in clinical trials. In most cases, minimal epitopes and HLA restriction elements are unknown.ResultsHere, we detail the experimental use of ex vivo expanded autologous B cells as antigen presenting cells to overcome the limitation of unknown HLA restriction, and the use of electroporated full length mRNA encoding full length parental proteins to ensure that any observed T cell responses are specific for antigens that are naturally processed and presented. ConclusionsThis technique can serve as useful experimental approach to determine the induction or enhancement of specific responses to naturally processed and presented antigens on HLA class I molecules in peripheral blood or tumor infiltrating T cells.

Highlights

Determining T cell responses to naturally processed and presented antigens is a critical immune correlate to determine efficacy of an investigational immunotherapeutic in clinical trials
B cells used for Antigen processing cells (APC) efficiently expanded in culture In this experimental system, B cells to be used as APCs were isolated via positive magnetic selection from deidentified, cryopreserved human leukocyte antigen (HLA)-A*02 positive healthy donor peripheral blood mononuclear cells (PBMC) from the National Institutes of Health (NIH) Clinical Center Blood Bank
As a positive control for electroporation efficiency, separate B cells were electroporated with mRNA encoding GFP and analyzed 24 h later by fluorescent imaging and flow cytometry for green fluorescent protein (GFP) fluorescence

Summary

Introduction

Determining T cell responses to naturally processed and presented antigens is a critical immune correlate to determine efficacy of an investigational immunotherapeutic in clinical trials. The use of antigen presenting cells (APCs) loaded with overlapping peptides spanning the candidate protein from which the minimal epitope may be derived is a common approach to assess such T cell responses. The exogenous application of peptides 15 amino acids in length (15mer) overlapping by 11 amino acids can induce CD4+ and CD8+ T lymphocyte responses [4, 5] This indicates that APCs can cross present human leukocyte antigen (HLA) class I-restricted antigens after endocytosis of exogenous peptides. Use of 15mer peptides allows the determination of putative minimal epitopes responsible for observed T cell responses This approach may bypass one or more steps in the natural processing and presentation of antigen derived from an endogenous intracellular full-length protein, potentially leading to falsely positive results [8]

Methods

Results

Discussion

Conclusion