Contrôle de la réponse immunitaire par l’indoleamine 2,3-dioxygénase : étude de la régulation d’une molécule immuno-suppressive dans les cellules cancéreuses et les lymphocytes B chez l’humain

Le système immunitaire se doit d’être étroitement régulé afin d’éviter que des réponses immunologiques inappropriées ou de trop forte intensité ne surviennent. Ainsi, différents mécanismes permettent de maintenir une tolérance périphérique, mais aussi d’atténuer la réponse lorsque celle-ci n’est plus nécessaire. De tels mécanismes sont cependant aussi exploités par les tumeurs, qui peuvent ainsi échapper à une attaque par le système immunitaire et donc poursuivre leur progression. Ces mécanismes immunosuppresseurs nuisent non seulement à la réponse naturelle contre les cellules tumorales, mais font aussi obstacle aux tentatives de manipulation clinique de l’immunité visant à générer une réponse anti-tumorale par l’immunothérapie. L’un des mécanismes par lesquels les tumeurs s’évadent du système immunitaire est l’expression d’enzymes responsables du métabolisme des acides aminés dont l’une des principales est l’indoleamine 2,3-dioxygénase (IDO). Cette dernière dégrade le tryptophane et diminue ainsi sa disponibilité dans le microenvironnement tumoral, ce qui engendre des effets négatifs sur la prolifération, les fonctions et la survie des lymphocytes T qui y sont présents. Bien que la régulation de l’expression de cette enzyme ait été largement étudiée chez certaines cellules présentatrices d’antigènes, dont les macrophages et les cellules dendritiques, peu est encore connu sur sa régulation dans les cellules tumorales humaines. Nous avons posé l’hypothèse que différents facteurs produits par les cellules immunitaires infiltrant les tumeurs (TIIC) régulent l’expression de l’IDO dans les cellules tumorales. Nous avons effectivement démontré qu’une expression de l’IDO est induite chez les cellules tumorales humaines, suite à une interaction avec des TIIC. Cette induction indépendante du contact cellulaire résulte principalement de l’interféron-gamma (IFN-g) produit par les lymphocytes T activés, mais est régulée à la baisse par l’interleukine (IL)-13. De plus, la fludarabine utilisée comme agent chimiothérapeutique inhibe l’induction de l’IDO chez les cellules tumorales en réponse aux lymphocytes T activés. Cette observation pourrait avoir des conséquences importantes en clinique sachant qu’une forte proportion d’échantillons cliniques provenant de tumeurs humaines exprime l’IDO. Enfin, les lymphocytes B, qui sont retrouvés également dans certaines tumeurs et qui interagissent étroitement avec les lymphocytes T, sont aussi susceptibles à une induction transcriptionnelle et traductionnelle de l’IDO. Cette enzyme est cependant produite sous une forme inactive dans les lymphocytes B, ce qui rend peu probable l’utilisation de l’IDO par les lymphocytes B comme mécanisme pour freiner la réponse immunitaire. Nos travaux apportent des informations importantes quant à la régulation de l’expression de la molécule immunosuppressive IDO dans les cellules cancéreuses. Ils démontrent que l’expression de l’IDO est influencée par la nature des cytokines présentes dans le microenvironnement tumoral. De plus son expression est inhibée par la fludarabine, un agent utilisé pour le traitement de certains cancers. Ces données devraient être prises en considération dans la planification de futurs essais immunothérapeutiques, et pourraient avoir un impact sur les réponses cliniques anti-tumorales.The immune system is under tight control to avoid inappropriate and excessive immunological responses. Many mechanisms allow the maintenance of peripheral tolerance and mediate attenuation of the immune response after pathogen clearance. Such mechanisms are also exploited by tumors, thereby favoring their escape from assault by the immune system. These immunosuppressive mechanisms hamper host natural immune responses against tumor cells, but also represent an obstacle to the successful clinical manipulation of the immune system in attempts to generate an anti-tumor response through immunotherapy. One immune escape mechanism used by tumors is the production of enzymes responsible for amino acid metabolism, amongst which indoleamine 2,3-dioxygenase (IDO) is of major importance. IDO degrades tryptophan, thus leading to its depletion from intracellular pools and local microenvironments. This culminates in multi-pronged negative effects on T lymphocytes neighboring IDO-expressing cells, notably on proliferation, function and survival. The regulation of IDO expression has been largely studied in antigen-presenting cells such as macrophages and dendritic cells, but its regulation in human tumor cells must still be characterized. We hypothesized that different factors produced by tumor-infiltrating immune cells (TIIC) regulate IDO expression in tumor cells. Accordingly, we have demonstrated that IDO expression is induced in human tumor cells upon interaction with TIIC. This induction is cell contact-independent, and results mainly from interferon-gamma (IFN-g) produced by activated T lymphocytes, while being antagonised by interleukin (IL)-13. Moreover, the chemotherapeutic agent fludarabine inhibits activated T lymphocyte-dependent IDO induction in tumor cells. This observation could have major clinical consequences, considering the large proportion of human cancer clinical samples expressing IDO. Finally, B lymphocytes, which interact closely with T lymphocytes and are found infiltrating human tumors, are also susceptible to transcriptional and translational IDO induction. This enzyme is however produced in an inactive form, suggesting that B lymphocytes do not exploit this mechanism to impede the immune response. In conclusion, our work brings crucial information on the regulation of the immunosuppressive molecule IDO in human tumor cells. We demonstrate that IDO expression is dependent on the nature of cytokines present in the tumor microenvironment. Furthermore, its expression is inhibited by fludarabine, a compound used to treat some types of cancer. These data should be taken into consideration in planning future immunotherapy trials and could impact anti-tumor clinical responses

Androgen-Regulated Expression of Arginase 1, Arginase 2 and Interleukin-8 in Human Prostate Cancer

BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens. CONCLUSION/SIGNIFICANCE: Our data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8

Fludarabine Downregulates Indoleamine 2,3-Dioxygenase in Tumors via a Proteasome-Mediated Degradation Mechanism

<div><p>Indoleamine 2,3-dioxygenase (IDO) is found in multiple malignancies and exerts immunosuppressive effects that are central in protecting tumors from host T lymphocyte rejection. IDO is an enzyme involved in the catabolism of tryptophan resulting in inhibition of T lymphocyte function. While inhibition of IDO enzymatic activity results in tumor rejection, it is still unknown how we can directly target IDO expression within tumors using drugs. We have chosen to interfere with IDO expression by targeting the key-signaling event signal transducer and activator of transcription 1 (STAT1). We evaluated the efficacy of fludarabine, previously described to inhibit STAT1 phosphorylation. Interestingly, fludarabine was efficient in suppressing protein expression and consequently IDO activity in two different cell lines derived from breast cancer and melanoma when IDO was activated with interferon-gamma (IFN-γ) or supernatants prepared from activated T lymphocytes. However, fludarabine had no inhibitory effect on STAT1 phosphorylation. Other IFN-γ-responsive genes were only marginally inhibited by fludarabine. The level of IDO transcript was unaffected by this inhibitor, suggesting the involvement of post-transcriptional control. Strikingly, we have found that the inhibition of proteasome partially protected IDO from fludarabine-induced degradation, indicating that fludarabine induces IDO degradation through a proteasome-dependent pathway. Currently used in the clinic to treat some malignancies, fludarabine has the potential for use in the treatment of human tumors through induction of IDO degradation and consequently, for the promotion of T cell-mediated anti-tumor response.</p></div

Fludarabine inhibits IDO protein independently of STAT1 phosphorylation on Y710 and S727.

<p><b>A-</b> PBMC were pre-treated with the indicated concentrations of fludarabine or DMSO (vehicle) for 24 h. Cells were washed and activated for 30 min (pSTAT1) or 24 h (total STAT1 and β-actin) with 50 U/ml of IFN-γ. <b>B-</b> MDA-231 were pre-treated with 100 µM fludarabine before activation with 50 U/ml of IFN-γ, anti-CD3 (OKT3) or IgG2a-activated TIL supernatants (Sup.). <b>C-</b> 624.38mel were pre-treated with 50 µM fludarabine, and cultured with anti-CD3 (OKT3) or IgG2a-activated CD4⁺ T lymphocyte supernatants. <b>B-C</b> Cells were harvested after 30 min (pSTAT1, STAT1 and β-actin) or 24 h (IDO and β-actin). <b>A-C</b> Proteins were extracted for immunoblot analysis. Results are representative of three independent experiments.</p

MHC I and PD-L1 expression levels remain unchanged following fludarabine treatment.

<p>MDA-231 and 624.38mel were pre-treated with the indicated concentrations of fludarabine or DMSO prior to IFN-γ activation with 50 U/ml for 24 h. Cells were harvested for flow cytometry analysis. MFI was assessed on viable populations for <b>A-</b> PD-L1 and <b>B-</b> HLA-ABC. Error bars represent standard deviation from one experiment. Results are representative of three independent experiments.</p

Fludarabine inhibits IDO via a proteasome-mediated degradation pathway.

<p><b>A-</b> MDA-231 cells were first activated with IFN-γ (50 U/ml) to induce IDO expression for 24 h. Cells were then washed and treated with 100 µM of fludarabine for 3-24 h. Proteins were extracted for Immunoblot analysis. Immunoblots are representative of three independent experiments <b>B-</b> To evaluate the role of the proteasome, MDA-231 cells were pre-treated with 100 µM of fludarabine for 24 h. Cells were washed and treated with indicated concentrations of bortezomib before IFN-γ (50 U/ml) activation. Proteins were extracted after 24 h for immunoblot analysis. D: DMSO control (bortezomib vehicle). Bortezomib concentrations are expressed in nM. Immunoblots are representative of four independent experiments <b>C-</b> Cells were pretreated with indicated concentration of fludarabine with or without 50 U/ml of IFN-γ and proteasomal activity was assessed using Proteasome-Glo kit. Results are represented as % of activity of untreated cells. Results combine data from 6 independent experiments and 3 independent experiments with IFN-γ. Black * p<0.01 t-test compared to untreated; grey * p<0.01 t-test compared to untreated with IFN-γ. <b>D-E</b> 50 U/ml of IFN-γ were used to activate the cells to express IDO. 24 h later, cells were washed and incubated with 100 µM of cycloheximide with or without 100 µM fludarabine. IDO protein stability was assessed at indicated time by immunoblot. Results are representative of three independent experiments. <b>E</b>- Densitometry of IDO/actin by immunoblot in <b>D</b>.</p

STAT1 is involved in IDO expression in response to T lymphocyte-derived factors.

<p>MDA-231 were transfected with siRNA against STAT1 or scrambled siRNA before activation with IFN-γ or supernatants of cultured TIL for 30 minutes (pSTAT1) or 24 h. Protein extracts were prepared for STAT1 (phosphorylated and total), IDO and β-actin immunoblot analysis. Results are representative of three independent experiments.</p

Fludarabine inhibits IDO activity independently of the mRNA level.

<p><b>A-</b> MDA-231 and 624.38 pre-treated with 100 µM of fludarabine or DMSO prior to IFN-γ activation with 50 U/ml for 24 h. RNA was extracted from activated cells. cDNA was prepared and IDO expression was evaluated by quantitative real-time RT-PCR and normalized to 18S rRNA. Error bars represent standard deviation. Results are representative of three independent experiments <b>B-</b> MDA-231 and 624.38 were pre-treated with 100 µM of fludarabine or DMSO prior to IFN-γ activation with 50 U/ml for 24 h. Cells were resuspended in HBSS with tryptophan with or without 1-MT and incubated for 4 h. Kynurenine was quantified by HPLC. Errors bars represent standard deviation of triplicates of one experiment. * p<0.05 t-test compared to IFN-© without inhibitor. Results are representative of two independent experiments.</p