Table_1_Expression of TRX1 optimizes the antitumor functions of human CAR T cells and confers resistance to a pro-oxidative tumor microenvironment.xlsx

Use of chimeric antigen receptor (CAR) T cells to treat B cell lymphoma and leukemia has been remarkably successful. Unfortunately, the therapeutic efficacy of CAR T cells against solid tumors is very limited, with immunosuppression by the pro-oxidative tumor microenvironment (TME) a major contributing factor. High levels of reactive oxygen species are well-tolerated by tumor cells due to their elevated expression of antioxidant proteins; however, this is not the case for T cells, which consequently become hypo-responsive. The aim of this study was to improve CAR T cell efficacy in solid tumors by empowering the antioxidant capacity of CAR T cells against the pro-oxidative TME. To this end, HER2-specific human CAR T cells stably expressing two antioxidant systems: thioredoxin-1 (TRX1), and glutaredoxin-1 (GRX1) were generated and characterized. Thereafter, antitumor functions of CAR T cells were evaluated under control or pro-oxidative conditions. To provide insights into the role of antioxidant systems, gene expression profiles as well as global protein oxidation were analyzed. Our results highlight that TRX1 is pivotal for T cell redox homeostasis. TRX1 expression allows CAR T cells to retain their cytolytic immune synapse formation, cytokine release, proliferation, and tumor cell-killing properties under pro-oxidative conditions. Evaluation of differentially expressed genes and the first comprehensive redoxosome analysis of T cells by mass spectrometry further clarified the underlying mechanisms. Taken together, enhancement of the key antioxidant TRX1 in human T cells opens possibilities to increase the efficacy of CAR T cell treatment against solid tumors.</p

DataSheet_1_Expression of TRX1 optimizes the antitumor functions of human CAR T cells and confers resistance to a pro-oxidative tumor microenvironment.pdf

Use of chimeric antigen receptor (CAR) T cells to treat B cell lymphoma and leukemia has been remarkably successful. Unfortunately, the therapeutic efficacy of CAR T cells against solid tumors is very limited, with immunosuppression by the pro-oxidative tumor microenvironment (TME) a major contributing factor. High levels of reactive oxygen species are well-tolerated by tumor cells due to their elevated expression of antioxidant proteins; however, this is not the case for T cells, which consequently become hypo-responsive. The aim of this study was to improve CAR T cell efficacy in solid tumors by empowering the antioxidant capacity of CAR T cells against the pro-oxidative TME. To this end, HER2-specific human CAR T cells stably expressing two antioxidant systems: thioredoxin-1 (TRX1), and glutaredoxin-1 (GRX1) were generated and characterized. Thereafter, antitumor functions of CAR T cells were evaluated under control or pro-oxidative conditions. To provide insights into the role of antioxidant systems, gene expression profiles as well as global protein oxidation were analyzed. Our results highlight that TRX1 is pivotal for T cell redox homeostasis. TRX1 expression allows CAR T cells to retain their cytolytic immune synapse formation, cytokine release, proliferation, and tumor cell-killing properties under pro-oxidative conditions. Evaluation of differentially expressed genes and the first comprehensive redoxosome analysis of T cells by mass spectrometry further clarified the underlying mechanisms. Taken together, enhancement of the key antioxidant TRX1 in human T cells opens possibilities to increase the efficacy of CAR T cell treatment against solid tumors.</p

HPV38 E6 and E7 expression is not required for the viability of cancer cells in K14 HPV38 E6/E7 Tg mice.

<p>(A) Electroporated lesions were kept under control and the diameter was recorded weekly. On the day of injection, the lesion diameter varied between 1.2 mm and 2.5 mm for the lesions injected with the Luc plasmid, and between 1.3 mm and 2.6 mm for the lesions injected with the Cre-Luc plasmid. To standardize the measurement, each lesion diameter was set to an arbitrary value of 1 on the day of injection, and the following measurements were adjusted accordingly. The difference in tumour growth between the lesions injected with the Luc plasmid and the lesions injected with the Cre-Luc plasmid was not significant according to an unpaired two-sample Student’s <i>t</i>-test (<i>p</i> = 0.3108, <i>t</i> = 1.052; <i>df</i> = 14). The test was run on data from the fourth week, because afterwards the number of living animals was substantially reduced. (B) Representative images of SCC sections from two different HPV38 E6/E7 Tg mice. Sections were taken from tumours initially electroporated with pS/MARt-Luc plasmid (Luc) or with pS/MARt-Luc-P2A-Cre plasmid (Cre-Luc). The morphological analysis revealed no substantial differences between the specimens; the tumours were all classified as invasive cSCC, with deep penetration into the dermis or into the muscular fibres, and clear and diffuse atypia. The loss of the viral mRNA in the tumours injected with the Cre-Luc plasmid was confirmed by <i>in situ</i> RNA hybridization using a complementary (antisense) riboprobe, while the staining with a sense probe confirmed the specificity of the signal.</p

Cancer-related genes recurrently mutated in cSCCs of K14 HPV38 E6/E7 Tg mice.

<p>(A) Circos presentation of mutations occurring in the same genes between the different mice. From the centre to the outside, the skin samples (white), the lesion samples (yellow), and the SCC samples (grey) are displayed for <i>n</i> = 3 mice each. Each track (three per colour) corresponds to one animal. Red dots represent C:G > T:A mutations, and black dots represent the other types of mutations. For Circos A, only the mutations that occur in genes present in the Cancer Gene Census list from the COSMIC database are displayed, with the number of recurrent mutations in these genes in parentheses. (B) For the epigenetic drivers/modifiers, only the mutations that occur in the epi-driver or the epi-modifier gene lists are displayed. Blue gene names correspond to genes that are only involved in epigenetic processes, and purple gene names correspond to genes that are involved in epigenetic processes and that are present in the Cancer Gene Census list. The total number of recurrent mutations occurring in each of these genes is also displayed in parentheses.</p

Schematic representation of well-known and hypothetical models of virus-associated carcinogenesis.

<p>Schematic representation of well-known and hypothetical models of virus-associated carcinogenesis.</p

ΔNp73α mRNA levels are high in the skin of HPV38 E6/E7 Tg mice, but are decreased in the UV-induced skin lesions harbouring p53 mutations.

<p>Total RNA was extracted from the skin of WT (<i>n</i> = 4) or K14 HPV38 E6/E7 Tg animals (<i>n</i> = 5) as well as histologically confirmed pre-malignant (pre-m) and SCC from three independent mice and harbouring mutated p53. ΔN73α levels were measured by quantitative RT-PCR. The data shown are the mean of two independent experiments. The differences in ΔN73α mRNA levels between WT and K14 HPV38 E6/E7 Tg animals were statistically significant: * <0.05.</p

Luciferase expression vectors can be efficiently electroporated into skin lesions of K14 HPV38 E6/E7 Tg mice.

<p>(A) Schematic diagram of the electroporation procedure of skin lesions of K14 HPV38 E6/E7 Tg mice. (B) Luciferase activity is detected in lesions electroporated with the control vector (Luc) as well as in lesions electroporated with the plasmid coding for the Cre recombinase and luciferase genes (CRE-Luc). Mice and tumour growth are closely monitored at regular intervals.</p

Several genes mutated in human skin lesions are also mutated in the UV-induced skin lesions of cSCCs of K14 HPV38 E6/E7 Tg mice.

<p>(A) Heatmap of significantly mutated genes, corresponding to genes recurrently mutated in at least two mouse SCC samples and reported in the Cancer Gene Census list from the COSMIC database (left panel) or having an impact on epigenetic regulation processes (right panel). The types of mutation represented by colours are chosen according to the most prevalent mutation type in each sample. The data for the human samples displayed in the first column are derived from a previous publication on cutaneous SCC. (B) Heatmap of mutations in genes in normal skin, pre-malignant lesions, and cSCC from different mice (M1–3) reported as significantly mutated in human cSCC.</p

HPV38 E6 and E7 induce an increased steady-state level of UV-induced mutations in mouse skin keratinocytes.

<p>(A) UV-induced cSCCs in K14 HPV38 E6/E7 Tg mice have a vast number of somatic mutations. SCCs display a very high mutational load, with each Tg animal (Tg1–3) harbouring almost 3 times the number of variants compared with pre-malignant lesions (Pre-m). All differences in number of DNA mutations among the tree types of specimens were statistically significant: * ≤0.05; ** ≤0.01; **** ≤0.0001. (B) cSCCs of K14 HPV38 E6/E7 Tg mice display the classic UV-induced mutation signature with a very high number of C:G > T:A mutations. This type of mutation represents the majority of the SNV type in SCC samples of the three Tg animals. (C) Mutation spectrum of pooled SCC samples from the three mice. This spectrum displays the high prevalence of C:G > T:A mutations, especially in the 5′-T_N-3′ and 5′-C_N-3′ context. The <i>y</i> axis represents the percentage of mutations, and the <i>x</i> axis the trinucleotide sequence context.</p