Single Cell RNA Sequencing Identifies Transcriptional Programs That Enhance Anti-Tumor Function of Transgenic CD4+ T Cells Redirected with TCR and CD8αβ
Authors: Rath Jan A , Bajwa Gagan , Carreres Benoit , Gruber Isabelle , Hoyer Elisabeth , Wu Meng-Fen , Yu Yi-Ru , Ho Ping-Chih , Brenner Malcolm K. , Gfeller David , Arber Caroline 
Published: February 2020

Introduction:Transgenic co-expression of a major histocompatibility complex class I restricted tumor associated antigen specific TCR and CD8αβ (TCR8) has been previously proposed as a strategy to redirect CD4+ T cells to tumors. However, it is unknown whether forced TCR8 expression induces more fundamental transcriptional consequences in both CD4+ and CD8+ T cells, and whether T cell lineage origin affects this outcome. Here we deeply interrogate the effects of transgenic TCR and TCR8 in human CD4+ and CD8+ T cells upon leukemia challenge by single cell RNA sequencing (scRNAseq) and investigate T cell function in vitro and in vivo. We identify profound changes of gene expression that have significant functional consequences.
Methods:A previously characterized HLA-A*02:01 restricted survivin-specific TCR was used (Arber et al, JCI, 2015 Jan;125(1):157-68) and a new polycistronic vector with this TCR and CD8αβ was generated. CD4+ and CD8+ T cells were isolated and scRNAseq (25'474 cells in total) was performed on (1) freshly isolated cells, (2) retrovirally transduced (TCR or TCR8) expanded cells, and (3) TCR+CD8+, TCR8+CD8+ and TCR8+CD4+ T cells co-cultured with BV173 leukemia cells (HLA-A*02:01+survivin+). scRNAseq results were cross-validated in independent experiments with FACS analysis of selected markers, in vitro stress-killing assays, analysis of cytokine production, and assessment of anti-tumor function in vivo in xenograft mice.
Results:CD4+ T cells only killed BV173 leukemia cells when redirected with TCR8 but not with TCR alone (p=0.0004, n=7), while killing by TCR+CD8+ and TCR8+CD8+ T was comparable (p=NS). To explore some of the possible underlying mechanisms, we used dimensionality reduction and unsupervised clustering of the scRNASeq data and identified 19 distinct cell clusters. CD4+ and CD8+ lineage origin clearly separated the samples, but separation by transgene type only became apparent upon co-culture. Analyzing differentially expressed genes, we found that co-cultured samples contained clusters with high expression of cytotoxic markers but with significant differences between CD4+ and CD8+ lineages (e.g. transcription of GZMB in CD4+ T cells and GNLY, NKG7, GZMK in CD8+ T cells). Next, we analyzed which genes were upregulated from the expanded to co-cultured states. Co-cultured TCR8+CD4+ T cells had more upregulated genes with a broader diversity compared to TCR+CD8+ or TCR8+CD8+ T cells. Among these upregulated pathways were cytotoxicity, co-stimulation, oxidative phosphorylation, NFkB regulation, cell growth and transcription factors. TCR8+CD4+ T cells also retained a less differentiated phenotype (e.g. high IL7R, SELL, CCR7, CXCR4) with preservation of replicative potential. Furthermore, co-cultured TCR8+CD4+ T cells expressed more co-stimulatory and less activation/ exhaustion markers. In addition, co-cultured TCR8+CD4+ T cells heavily relied on oxidative phosphorylation and had higher mitochondrial activity compared to co-cultured TCR+ or TCR8+ CD8+ T cells. In stress co-cultures with multiple rounds of tumor challenge, TCR8+CD4+ T cells outperformed TCR+CD8+ T cells (number of killings TCR8+CD4+ vs TCR+CD8+: 3.3±0.5 vs 1.3±1.1, p=0.01, n=7), but were comparable to TCR8+CD8+ T cells (TCR8+CD4+ vs TCR8+CD8+: 3.3±0.5 vs 2±1.4,p=NS, n=7). TCR8+CD4+ T cells expanded significantly better than TCR+CD8+ T cells (p=0.002) and TCR8+CD8+ T cells (p=0.015) and produced TH1 type cytokines. In the xenograft mouse model, we observed significant BV173 leukemia control in mice treated with TCR+CD8+ T cells compared to controls (NT), and further enhancement in mice treated with TCR8+CD8+ T cells (NT vs TCR: p=0.0002, NT vs TCR8: p&lt;0.0001, TCR vs TCR8: p=0.01, n=5). TCR8+CD4+ T cells also significantly delayed leukemia progression compared to TCR+CD4+ or NT T cells (p=0.001, n=5).
Conclusion:Transgenic TCR8 expression has previously been proposed as a strategy to enhance TCR-pMHC recognition. Here we identify profound transcriptional changes involving multiple pathways that are important for sustained anti-tumor function upon adoptive T cell transfer in vivo, such as cytotoxicity, co-stimulation, cell cycle and metabolism. Our results point towards previously unrecognized mechanisms by which TCR8 transgenes mediate their beneficial effect in both CD4+ and CD8+ T cells.

Disclosures
Brenner: T Scan: Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Equity Ownership; Allovir: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Equity Ownership; Memgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees. Arber:Cell Medica: Patents & Royalties.