Single-cell transcriptomics identifies multiple pathways underlying antitumor function of TCR- and CD8αβ-engineered human CD4 ⁺ T cells

Abstract

<jats:p>Transgenic coexpression of a class I–restricted tumor antigen–specific T cell receptor (TCR) and CD8αβ (TCR8) redirects antigen specificity of CD4<jats:sup>+</jats:sup> T cells. Reinforcement of biophysical properties and early TCR signaling explain how redirected CD4<jats:sup>+</jats:sup> T cells recognize target cells, but the transcriptional basis for their acquired antitumor function remains elusive. We, therefore, interrogated redirected human CD4<jats:sup>+</jats:sup> and CD8<jats:sup>+</jats:sup> T cells by single-cell RNA sequencing and characterized them experimentally in bulk and single-cell assays and a mouse xenograft model. TCR8 expression enhanced CD8<jats:sup>+</jats:sup> T cell function and preserved less differentiated CD4<jats:sup>+</jats:sup> and CD8<jats:sup>+</jats:sup> T cells after tumor challenge. TCR8<jats:sup>+</jats:sup>CD4<jats:sup>+</jats:sup> T cells were most potent by activating multiple transcriptional programs associated with enhanced antitumor function. We found sustained activation of cytotoxicity, costimulation, oxidative phosphorylation– and proliferation-related genes, and simultaneously reduced differentiation and exhaustion. Our study identifies molecular features of TCR8 expression that can guide the development of enhanced immunotherapies.</jats:p>

Transgenic CD8αβ co-receptor rescues endogenous TCR function in TCR-transgenic virus-specific T cells

“… 13 Transfer of CD8αβ can redirect CD4+ T cells to class I-restricted antigens, and the transgenic CD4+ T cells become cytotoxic hybrid cells. 14 22–27 We have recently analyzed the transcriptional consequences of TCR8 expression in polyclonal CD4+ and CD8+ T cells by single cell RNA sequencing and experimental validation and showed that transgenic TCR8 has multiple advantages in both CD4+ and CD8+ lineages, promoting an overall enhanced anti-tumor function. 14 We now investigated the impact of transgenic CD8αβ on endogenous class I-restricted TCR function in CD8+ T cells as we hypothesized that combining CD8αβ co-receptor as a transgene with a tumor-targeted TCR is one potential means of overcoming the limitation of reduced anti-viral activity in TCR+ VSTs.…”

“… 2–4 6 Several features of the CD8αβ co-receptor most likely explain our findings, including its role in TCR-pMHC recognition and modulation of antigen-sensitivity, 28 recruitment of Lck to the immune synapse, and activation of signaling components that are crucial during early T-cell activation, 29 and, as recently demonstrated by single cell RNA sequencing, its impact on a variety of transcriptional pathways upon tumor challenge that support enhanced anti-tumor function. 14 …”

“…TCR8 expression was associated with a better preservation of less differentiated VSTs in the CD4+ compartment. 14 The only HLA restriction of the product is defined by the transgenic TCR used to redirect VSTs to the cancer. Feasibility and safety of our manufacturing have already been demonstrated in CAR+ VST clinical trials and are GMP compliant.…”

“…The retroviral vectors expressing the survivin-specific (s24) TCR and the combination of TCR and CD8αβ have previously been described. 14 15 Genes encoding for the human CD8α (Uniprot P01732) and CD8β isoform 1 (βM1, Uniprot P10966-1) chains, separated by a 2A sequence, were synthesized by Geneart (Invitrogen) and cloned into the SFG retroviral vector backbone ( figure 1A ) (In-Fusion HD Cloning Kit, Clontech). Transient retroviral supernatants were prepared by co-transfection of 293 T cells with RD114 and Pegpam plasmids and the SFG vector containing the gene of interest.…”

“…Incorporation of CD8αβ into the transgenic TCR vector enhances the function of polyclonal TCR+ T cells through multiple pathways, 14 and here we investigated if this strategy rescues endogenous class I restricted anti-viral TCR function. We used a CD8-dependent TCR targeting the tumor-associated antigen survivin in the context of HLA-A*02:01 and expressed the TCR alone (TCR) 15 or in combination with CD8αβ (TCR8) 14 in VSTs targeting cytomegalovirus (CMV) and Epstein-Barr virus (EBV). We consistently generated TCR+ and TCR8+ VSTs with a predominant central memory phenotype and showed that anti-viral reactivities were restored in TCR8+ VSTs while anti-tumor function was retained.…”