CD4 Inhibits Helper T Cell Activation at Lower Affinity Threshold for Full-Length T Cell Receptors Than Single Chain Signaling Constructs

Deborah K Johnson,Jordan G Finnell,Taylor S Orton,K Scott Weber,Stephen P Persaud,Kenneth A Christensen,Sheldon J Myers,Wyatt Magoffin,John C Hancock

doi:10.3389/fimmu.2020.561889

Deborah K Johnson, Jordan G Finnell + Show 7 more

Open Access

https://doi.org/10.3389/fimmu.2020.561889

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Abstract

CD4+ T cells are crucial for effective repression and elimination of cancer cells. Despite a paucity of CD4+ T cell receptor (TCR) clinical studies, CD4+ T cells are primed to become important therapeutics as they help circumvent tumor antigen escape and guide multifactorial immune responses. However, because CD8+ T cells directly kill tumor cells, most research has focused on the attributes of CD8+ TCRs. Less is known about how TCR affinity and CD4 expression affect CD4+ T cell activation in full length TCR (flTCR) and TCR single chain signaling (TCR-SCS) formats. Here, we generated an affinity panel of TCRs from CD4+ T cells and expressed them in flTCR and three TCR-SCS formats modeled after chimeric antigen receptors (CARs) to understand the contributions of TCR-pMHCII affinity, TCR format, and coreceptor CD4 interactions on CD4+ T cell activation. Strikingly, the coreceptor CD4 inhibited intermediate and high affinity TCR-construct activation by Lck-dependent and -independent mechanisms. These inhibition mechanisms had unique affinity thresholds dependent on the TCR format. Intracellular construct formats affected the tetramer staining for each TCR as well as IL-2 production. IL-2 production was promoted by increased TCR-pMHCII affinity and the flTCR format. Thus, CD4+ T cell therapy development should consider TCR affinity, CD4 expression, and construct format.

Highlights

CD4+ T cells are critical for tumor elimination through both indirect and direct mechanisms
To further elucidate the effects of T cell receptor (TCR)-pMHCII affinity on CD4+ T cell activation, the variable regions of LLO56 and LLO118 (Figure 1A) were used as templates for generating a panel of single-chain TCRs with low, intermediate, and high affinities. Single chain T cell receptor (scTCR) libraries generated by random mutagenesis and expressed via yeast surface display (Figure 1B) were selected for protein folding stability through magnetic column sorting (Figure 1C). scTCR expression levels vary according to yeast cell cycle stage and can result in multiple peaks
We engineered and characterized a panel of MHCIIspecific TCRs with increasing pMHC affinity in order to interrogate the relationships between TCR format, TCRpMHCII affinity, and the coreceptor CD4 on CD4+ T cell activation

Summary

Introduction

CD4+ T cells are critical for tumor elimination through both indirect and direct mechanisms. The presence of tumor-specific CD4+ T cells is CD4 Inhibits High Affinity TCR Activation correlated with improved patient survival following vaccination with cancer-associated peptides whether or not they are directly involved in tumor suppression [6,7,8]. CD4+ T cells can sustain an immune response when CD8+-specific antigens are lost which otherwise might result in tumor escape [9]. Despite these clear benefits, only one published clinical study [10] focuses on the immunotherapeutic benefits of CD4+ T cell receptors (TCRs) [10, 11]

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