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Transgenic CD8?? co-receptor rescues endogenous TCR function in TCR-transgenic virus-specific T cells.

ABSTRACT: BACKGROUND:Genetically engineered virus-specific T cells (VSTs) are a platform for adoptive cell therapy after allogeneic hematopoietic stem cell transplantation. However, redirection to a tumor-associated antigen by the introduction of a transgenic T-cell receptor (TCR) reduces anti-viral activity, thereby impeding the possibility of preventing or treating two distinct complications-malignant relapse and viral infection-with a single cell therapy product. Availability of CD8?? co-receptor molecules can significantly impact class I restricted T-cell activation, and thus, we interrogated whether transgenic CD8?? improves anti-viral activity mediated by native VSTs with or without a co-expressed transgenic TCR (TCR8). METHODS:Our existing clinical VST manufacturing platform was adapted and validated to engineer TCR+ or TCR8+ VSTs targeting cytomegalovirus and Epstein-Barr virus. Simultaneous anti-viral and anti-tumor function of engineered VSTs was assessed in vitro and in vivo. We used pentamer staining, interferon (IFN)-? enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), cytotoxicity assays, co-cultures, and cytokine secretion assays for the in vitro characterization. The in vivo anti-tumor function was assessed in a leukemia xenograft mouse model. RESULTS:Both transgenic CD8?? alone and TCR8 had significant impact on the anti-viral function of engineered VSTs, and TCR8+ VSTs had comparable anti-viral activity as non-engineered VSTs as determined by IFN-? ELISpot, ICS and cytotoxicity assays. TCR8-engineered VSTs had improved anti-tumor function and greater effector cytokine production in vitro, as well as enhanced anti-tumor function against leukemia xenografts in mice. CONCLUSION:Incorporation of transgenic CD8?? into vectors for TCR-targetable antigens preserves anti-viral activity of TCR transgenic VSTs while simultaneously supporting tumor-directed activity mediated by a transgenic TCR. Our approach may provide clinical benefit in preventing and treating viral infections and malignant relapse post-transplant.

SUBMITTER: Bajwa G

PROVIDER: S-EPMC7640589 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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

Bajwa Gagan G Lanz Inès I Cardenas Mara M Brenner Malcolm K MK Arber Caroline C

Journal for immunotherapy of cancer 20201101 2

<h4>Background</h4>Genetically engineered virus-specific T cells (VSTs) are a platform for adoptive cell therapy after allogeneic hematopoietic stem cell transplantation. However, redirection to a tumor-associated antigen by the introduction of a transgenic T-cell receptor (TCR) reduces anti-viral activity, thereby impeding the possibility of preventing or treating two distinct complications-malignant relapse and viral infection-with a single cell therapy product. Availability of CD8αβ co-recept ...[more]

PMID: 33148692

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Project description:The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously established TCR discovery platform using 2D TCR affinity and sequence test would allow for selection of TCRs specific to any given antigen with the desirable attributes of high TCR affinity, CD8 co-receptor independence and functional superiority. Utilizing TCRs with less CD8 contribution could be beneficial for adoptive cell transfer immunotherapies using naturally occurring or genetically engineered T cells against viral or cancer-associated antigens.

Project description:Purpose: To identify potential underlying molecular programs associated with the observed dysfunctional TCRTg101 phenotype, RNA sequencing (seq) was performed on TCRTg101 isolated from livers of leukemia-bearing mice at several time points following their adoptive transfer (days 6-7, 13-14, and 167-18), andas well as on TCRTg101 isolated from control (leukemia-free) analysis Methods: TCRTg101 in livers and spleens of leukemia-bearing mice were isolated at various time points following adoptive transfer (day 0, day 6-7, day 13-14, day 16-18) by FACS and were re-suspended in Trizol (Life Technologies). TCRTg101 RNA was isolated via chloroform extraction. Low input RNA sequencing was performed in the University of Chicago Genomic. Genes with fewer than 10 reads in at least 6 samples were filtered out, resulting in a dataset of 11,164 genes. Differential gene expression analysis was performed on raw aligned read counts using DESeq2 (Love et al., 2014), with batch effects accounted for in the design formula. Genes were considered to be differentially expressed if they had an adjusted p-value < 0.05 using a Benjamini-Hochberg test (FDR). Counts per gene were regular log (rlog)-transformed, and batch effects were removed using the removeBatchEffect function from limma (Ritchie et al., 2015) for principal component analysis (PCA), sample clustering based on Euclidean distance, and heatmaps depicting z-scores of gene expression. rlog-transformed, batch-corrected counts were also used for weighted gene correlation network analysis (WGCNA) (Langfelder and Horvath, 2008) while additionally filtering out the 50% of genes with the lowest variance to reduce noise, resulting in a set of 5,582 genes. Adjacency was determined using a signed analysis and a soft thresholding power of 14, which was determined by scale-free fit index (Zhang and Horvath, 2005). 12 clusters of genes were initially identified, 2 of which contained a combined 4,269 genes (76.5% of the dataset), and roughly corresponded to genes whose expression increased or decreased over the experimental time course. Of the other 10 clusters, 2 were identified as containing genes which were transiently upregulated or downregulated. These two clusters contained 448 genes, combined. The remaining 865 genes were assigned to eight different clusters, which appeared to be the result of high variance within sample groups, either due to low overall gene expression or low outlier values. No conclusions were drawn regarding these clusters due to uncertainty in the data. Core Facility on the Illumina HiSeq 2500 platform in two batches. Reads were mapped onto the University of California Santa Cruz mouse genome using kallisto (Bray et al., 2016) Results: In total, 4,075 genes were found to be significantly differentially expressed across all pairwise comparisons Conclusion:Dysfunctional TCRTg101 acquire a transcriptional program canonically associated with T cell exhaustion

Dataset Information

Transgenic CD8?? co-receptor rescues endogenous TCR function in TCR-transgenic virus-specific T cells.

Publications

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

Similar Datasets

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