Characterization of the aryl hydrocarbon receptor as a potential candidate to improve cancer T cell therapies

Abstract

The efficacy of T-cell-based cancer therapies can be limited by the tumor microenvironment which can lead to T cell dysfunction. Multiple studies, particularly in murine models, have demonstrated the capacity of the aryl hydrocarbon receptor (AHR) to negatively regulate antitumor T cell functions. AHR is a cytoplasmic receptor and transcription factor that was originally identified as a xenobiotic sensor, but has since been shown to play a significant role in the gene regulation of various immune cells, including T cells. Given the insights from murine studies, AHR emerges as a promising candidate to invalidate for optimizing T cell-based cancer therapies. However, the controversial role of AHR in human T cells underscores the need for a more comprehensive characterization of AHR expressing T cells. This study aims to investigate the regulatory mechanisms of AHR in human T cell biology to better understand its impact on reducing antitumor immune responses. Here, we knocked-out AHR in human T cells using CRISPR-Cas9 technology to characterize AHR's function in an in vitro chronic stimulation model. Engineered T cells exhibited enhanced effector- and memory-like profiles and expressed reduced amount of CD39 and TIGIT. AHR knockout enhanced human CAR-T cells' functionality and persistence upon tumor chronic stimulation. Collectively, these results highlight the role of AHR in human CAR-T cells efficiency.

Keywords: AHR; CAR-T cell therapy; CRISPR-Cas9; T cell dysfunction.

Fig. 1

AHR is induced upon T cell activation and is maintained over time upon chronic stimulation. a Follow-up of AHR protein expression over time in activated T cells. (n = 2 to 7 according to timepoints) b Subcellular localization of AHR after treatment with 50 µM of kynurenine. (n = 8) c Methodology of in vitro chronic stimulation assay consisting of a chronic stimulation with αCD3/CD28 dynabeads. d Follow-up of protein AHR expression prior to T cell activation and after short-term restimulation (T cells restimulated once) or chronic stimulation (T cells restimulated four times). (n = 4 (non-restimulated), n = 28 (short-term restimulation), n = 5 (chronic stimulation))

Fig. 2

In vitro chronic stimulation induces a T cell dysfunctional state. a, b Follow-up of the proliferation (a) and viability (b) of T cells chronically stimulated and treated with 50 µM of kynurenine. (n = 9) c Principal Component Analysis (PCA) plot illustrating the distribution of T cells stimulated once or four times in the first two principal components (PC1 and PC2). (n = 3) d Volcano plot displaying the differential expression of genes between T cells stimulated once or four times. (n = 3) e–g CD39, PD-1, TIM-3 and TIGIT expression after short-term restimulation (T cells restimulated once) or chronic stimulation (T cells stimulated four times). (n = 9 (e), n = 12 (f, g)) h Knockout validation of AHR in activated T cells. (n = 10 (non-stimulated), n = 28 (short-term restimulation), n = 5 (chronic stimulation)) i, j Follow up of the proliferation (i) and viability (j) of AHR knocked-out T cells treated with 50 µM of kynurenine. (n = 6)

Fig. 2

In vitro chronic stimulation induces a T cell dysfunctional state. a, b Follow-up of the proliferation (a) and viability (b) of T cells chronically stimulated and treated with 50 µM of kynurenine. (n = 9) c Principal Component Analysis (PCA) plot illustrating the distribution of T cells stimulated once or four times in the first two principal components (PC1 and PC2). (n = 3) d Volcano plot displaying the differential expression of genes between T cells stimulated once or four times. (n = 3) e–g CD39, PD-1, TIM-3 and TIGIT expression after short-term restimulation (T cells restimulated once) or chronic stimulation (T cells stimulated four times). (n = 9 (e), n = 12 (f, g)) h Knockout validation of AHR in activated T cells. (n = 10 (non-stimulated), n = 28 (short-term restimulation), n = 5 (chronic stimulation)) i, j Follow up of the proliferation (i) and viability (j) of AHR knocked-out T cells treated with 50 µM of kynurenine. (n = 6)

Fig. 3

AHR knockout promotes effector- and memory-like profiles upon T cell stimulation. a Methodology of AHR knocked-out T cells’ RNA-sequencing. b Heatmap showing differential expression of genes regulated by AHR, analyzed using DESeq2. (n = 3) c Pathway enrichment analysis using GSEA (Gene Set Enrichment Analysis) based on GO (Gene Ontology) Biological Process. The size of each dot reflects the number of genes contributing to the enrichment of each pathway (count), while the color gradient represents the adjusted p-value (p.adj), with darker colors indicating higher statistical significance. (n = 3) d Heatmap showing differential expression of immune effector and memory genes. (n = 3) e RT-qPCR validation of RNA-sequencing data. (n = 5) f–h Cytokine analysis of AHR knocked-out T cells treated with 50 µM of kynurenine. (n = 11)

Fig. 4

AHR knockout prevents increased expression of CD39 upon chronic T cell stimulation. a Heatmap showing differential expression of inhibitory receptors (n = 3). b, c CD39 expression (b) after short-term restimulation (T cells restimulated once) or chronic stimulation (T cells restimulated four times) in AHR knocked-out T cells treated with 50 µM of kynurenine and CD39 mean of fluorescence (c) after chronic stimulation (T cells restimulated four times) in CD39.⁺ T cells treated with 50 µM of kynurenine. (n = 13 (b), n = 11 (c)) d, e TIGIT expression (d) after short-term restimulation or chronic stimulation in AHR knocked-out T cells treated with 50 µM of kynurenine and TIGIT mean of fluorescence (e) after chronic stimulation (T cells restimulated four times) treated with 50 µM of kynurenine. (n = 10 (d), n = 6 (e))

Fig. 5

AHR knocked-out CAR-T cells are functional. a Methodology. b, c Knockout validation (b) and transduction efficiency (detected with a percentage of the truncated protein CD19 as a reporter) (c) of AHR knocked-out CAR-T cells. (n = 14) d Cytotoxic assay of AHR knocked-out CAR-T cells after co-culture at different effector:target ratios for 24 h. (n = 8 (ratio 1:1), n = 3 (ratio 1:10)) e CD107a degranulation assay of AHR knocked-out CAR-T cells after co-culture at an effector:target ratio of 1:1 for 5 h treated with 50 µM of kynurenine. (n = 11) f CD39 expression in AHR knocked-out CAR-T cells after co-culture at an effector:target ratio of 1:1 for 24 h treated with 50 µM of kynurenine. (n = 14)

Fig. 6

AHR knockout CAR-T cells persist longer than wild type CAR-T cells upon chronic stimulation. a Methodology of in vitro chronic stimulation assay for a hematologic cancer cell line. b Follow-up of the percentage of CAR-T remaining after chronic stimulation. (n = 8) c CD4 and CD8 ratio upon chronic stimulation. (n = 8) d, e CD39 and TIGIT expression over time after chronic stimulation in AHR knocked-out CAR-T cells. (n = 3)