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TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells

ABSTRACT: In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. Results: We show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

ORGANISM(S): Homo sapiens

PROVIDER: GSE256035 | GEO | 2024/02/27

REPOSITORIES: GEO

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Project description:The foodborne pathogen Listeria monocytogenes experiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. While osmotic stress induced changes in expression have been investigated for specific genes, identifying and understanding genome-wide temporal changes in the transcriptome due to salt stress will provide insights into how this pathogen adapts to and survives this stress, leading to development of new intervention strategies. To determine the short-term and long-term responses to salt stress, we exposed exponential phase cells of L. monocytogenes H7858 to 6% NaCl in Brain Heart Infusion (BHI) broth at 7°C and 37°C and extracted RNA after 2.5%, 5%, 10%, and 20% of lag phase and during exponential growth in BHI + 6% NaCl, and evaluated temporal changes in transcript levels with microarrays. The temperature dependent short-term response to salt stress included a significant increase in transcript levels of the alternative sigma factor σB, with maximum expression at 2.5-5% of lag phase at 37°C, and at 20% of lag phase at 7°C. Transcript levels of genes known to be regulated by σB, including inlAB, opuCA, glpFK, and gadBC, were significantly upregulated at the same relative time points as σB As indicated by significantly elevated transcript levels during exponential growth in BHI + 6% NaCl, genes encoding proteins involved in purine and pyrimidine synthesis and amino acid biosynthesis are part of the long-term response to salt stress at 37°C. Transcript levels of virulence genes plcA, mpl, actA, and plcB were also significantly upregulated during exponential growth under salt stress at 37°C. Genes encoding proteins involved in protein degradation and stability and cell membrane modifications are part of the long-term response to salt stress at 7°C. Overall, an initial alteration in the transcriptome occurs, decreasing transcript levels of genes encoding proteins involved in energy conversion and metabolism, while increasing transcript levels of those involved in the stress response controlled by σB, followed by a long-term response, including increased expression of genes encoding compatible solute transporters and general stress proteins, facilitating growth under salt stress. A reference design was used to analyze gene expression differences. Exponential phase cultures at 37C and at 7C were used as a reference for salt stressed samples at each temperature. Salt stressed samples from the same relative time point were compared across temperatures. 4 biological replicates were tested for each comparison.

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TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells

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