We showed previously that Tyk2−/− natural killer cells lack the ability to lyse leukemic cells. As a consequence, the animals are leukemia prone. Here, we show that the impaired tumor surveillance extends to T cells. Challenging Tyk2−/− mice with EL4 thymoma significantly decreased disease latency. The crucial role of Tyk2 for CTL function was further characterized using the ovalbumin-expressing EG7 cells. Tyk2−/− OT-1 mice developed EG7-induced tumors significantly faster compared with wild-type (wt) controls. In vivo assays confirmed the defect in CD8+ cytotoxicity on Tyk2 deficiency and clearly linked it to type I IFN signaling. An impaired CTL activity was only observed in IFNAR1−/− animals but not on IFNγ or IL12p35 deficiency. Accordingly, EG7-induced tumors grew faster in IFNAR1−/− and Tyk2−/− but not in IFNγ−/− or IL12p35−/− mice. Adoptive transfer experiments defined a key role of Tyk2 in CTL-mediated tumor surveillance. In contrast to wt OT-1 cells, Tyk2−/− OT-1 T cells were incapable of controlling EG7-induced tumor growth. [Cancer Res 2009;69(1):203–11]
Signal transduction via the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway regulates cell proliferation, differentiation, and survival of hematopoietic cells (1–3). Aberrant activation of Jak/Stat signaling was observed in multiple tumors and leukemia, and great effort has been made in the past years to elucidate and define the role of the Jak/Stat pathway in cancer formation and tumor immune surveillance (4–9).
Tyrosine kinase 2 (Tyk2), one of the Jaks, is activated on binding of type I IFN, interleukin (IL) 6, IL10, IL12, and IL13 to the respective receptors (10). Mice deficient for Tyk2 (Tyk2−/− mice) are viable and fertile but were described to have defects in type I IFN, IL12, and also type II IFN (IFNγ) signaling (11, 12). Tyk2−/− mice show an increased susceptibility to various pathogens (11, 13–15) but are resistant against lipopolysaccharide (LPS)–induced endotoxin shock (16). In addition, Tyk2 is a key regulator for Th1/Th2 balance (17). Despite the broad interest for Tyk2 in infection, only sparse information is available on the role of Tyk2 for cancer progression. We have previously shown that Tyk2−/− mice are prone to develop B-cell leukemia in an Abelson-induced leukemia model. In this study, Tyk2−/− mice were found to have impaired tumor surveillance as a result of decreased natural killer (NK)-cell function (18). Search for cancer-associated genetic alterations has led to the identification of different germ-line mutations and sequence variants of Tyk2 in various tumors (19–21). Tomasson and colleagues describe a potential association of Tyk2 germ-line sequence changes to acute myelogenous leukemia pathogenesis. Elevated Tyk2 expression levels detected in breast cancer cell lines prompted the authors to propose Tyk2 as a potential biomarker for breast cancer diagnosis (22). Moreover, loss of Tyk2 expression and signaling has been shown to inhibit the invasiveness of human prostate cancer cells (23) and of leukemic B cells in a murine model system of human Burkitt's lymphoma (24). Complete loss of Tyk2 in humans results in enhanced allergic and impaired antimicrobial responses as recently described by Minegishi and colleagues (25).
NK cells and CTLs are regarded as the major mediators of the natural host response against developing tumors. It is nowadays widely accepted that the immune system cannot only distinguish self from nonself but is additionally able to identify and subsequently eliminate cancerous self. This process is referred to as tumor immune surveillance and is one of three phases that make up the concept of immunoediting (26). The elimination and immunoediting processes involve different cell types, most importantly αβ T and NK cells and cytokines such as IFNs and IL12.
Because IFN and IL12 signaling is compromised in Tyk2−/− mice, we postulated that not only the NK-cell–mediated antitumor response but also tumor surveillance by CTLs is impaired in these mice. Indeed, our study proves that Tyk2 is a central player in tumor surveillance mediated by cytotoxic T cells. Tyk2−/− CD8+ T cells exhibited a severe defect in the antigen-specific cytotoxicity in vitro and in vivo. We postulate a link between this functional defect and the compromised type I IFN signaling in Tyk2−/− animals, whereas type II IFN and IL12 seem to be of minor relevance.
Materials and Methods
Wild-type (wt), Rag2−/− (27), Tyk2−/− (11), IFNγ−/− (28), IFNAR1−/− (29), IL12p35−/− (30), and OT-1 (31) mice were on C57BL/6 background and were maintained under specific pathogen-free conditions at the Biomedical Research Institute, Medical University of Vienna (Vienna, Austria). Experiments were carried out with gender- and age-matched 6- to 12-wk-old mice. All experimental procedures were carried out according to Austrian Law (Tierversuchsgesetz BGBl Nr. 501/1989).
Culture of Tumor Cell Lines
EL4 and EG7 cells were maintained in DMEM (Sigma-Aldrich) containing 10% heat-inactivated FCS (PAA Laboratories), 100 units/mL penicillin-streptomycin (Life Technologies), 2 mmol/L l-glutamine (Life Technologies), nonessential amino acids (PAA Laboratories), and 5 μmol/L β-mercaptoethanol (Sigma-Aldrich).
Generation and Cultivation of Bone Marrow–Derived Dendritic Cells
Bone marrow was isolated from femora and tibiae of 6- to 8-wk-old mice. The immature dendritic cell (DC) population was enriched by panning over human IgG (Sigma-Aldrich)–coated plates (Optilux, Falcon, Becton Dickinson Labware). Cells were cultivated in complete DMEM supplemented with 10% (v/v) granulocyte macrophage colony-stimulating factor (GM-CSF) supernatant derived from the GM-CSF–producing hybridoma line X6310. Culture medium was exchanged every other day. On day 6, immature bone marrow–derived DCs (BMDC) were activated with LPS (100 ng/mL; Escherichia coli O111:B4; Calbiochem) or CpG-ODN 1668 (1 μmol/L; MWG-Biotech AG) and loaded with ovalbumin (OVA; 10 μg/mL, grade V; Sigma-Aldrich), OVA257-264 (SIINFEKL, 10 μg/mL, H2-Kb, derived from chicken OVA; Bachem), or left untreated. After 24 h, cells were harvested and used for subsequent experiments. The supernatants were collected for ELISA.
Measurement of Cytokine Production
Cytokine production of BMDCs was determined by measuring IL12p70 (Quantikine Mouse IL-12p70 Immunoassay, R&D Systems) and IL10 (OptEIA Mouse IL10 ELISA set, BD Biosciences) in cell culture supernatants of 24-h stimulated BMDCs.
CD8+ T Lymphocyte Isolation
Naive CD8+ cells were isolated from spleens of wt OT-1 and Tyk2−/− OT-1 mice using the CD8a+ Cell Isolation kit (Miltenyi Biotech). Purity of the enriched CD8a+ T cells was evaluated by fluorescence-activated cell sorting (FACS). Isolated cells were usually >90% CD8+ Vα2+.
Antigen-Specific T-Cell Proliferation and Activation
Tyk2−/− OT-1 and wt OT-1 splenocytes were stained with 2.5 μmol/L 6-carboxyfluoresein diacetate succinimidyl ester (CFSE; Molecular Probes). CD8+ cells (106) were then cocultivated with mature peptide-loaded BMDCs (T-cell to BMDC ratio = 10: 1). Cell samples were taken on 3 subsequent days and analyzed by FACS. Remaining T cells were harvested for RNA isolation.
Flow Cytometric Analysis (FACS)
The following antibodies were used: FITC-anti-Vα2, allophycocyanin (APC)-anti-CD8a, APC-anti-CD11c, R-phycoerythrin (R-PE)-anti-CD11b, FITC-anti-CD40, biotin-anti-CD80, FITC-anti-MHCII, biotin-anti-CD86, FITC-anti-CD107a, streptavidin-PerCP, R-PE-anti-CD44, and FITC-anti-CD69 (all BD Pharmingen). Samples were analyzed using a FACScan or FACScanto II (Becton Dickinson).
RNA Isolation and Semiquantitative Reverse Transcription-PCR
RNA was isolated with Trizol (TRI Reagent, Sigma-Aldrich) and subsequently DNase I treated (DNase I recombinant, Roche Diagnostics). The reverse transcription reaction was performed with the GeneAmp RNA PCR Core kit (Roche Diagnostics). Primer sequences are available on request. The cDNA concentration was normalized to the amount of β-actin.
Spleens and livers were fixed with 3.7% formaldehyde for 24 h, washed in PBS, and embedded in paraffin. Sections (5 μm) were stained with H&E using standard protocols. Confocal images (magnification, ×100) were taken using a Zeiss Axio Imager.Z1 microscope.
Systemic EL4 tumor model. Tyk2−/− and wt recipient mice received 5 × 103 EL4 cells in 200 μL sterile PBS via tail vein injection. Mice were monitored for disease onset. Sick mice were sacrificed and examined. Livers were isolated, weighed, and fixed in 3.7% formaldehyde and analyzed.
S.c. EG7 tumor formation. EG7 cells (106) in 100 μL PBS were injected s.c. into the shaved right flanks of recipient mice. Mice were checked every other day for the development of tumors. After 10 d, mice were sacrificed and tumors and spleens were isolated and weighed. Tumors and spleens were homogenized and subjected to FACS analysis.
CD8+ T-Cell Depletion
CD8+ T cells were depleted by i.p. injections of 100 μg 53-6.72 anti–CD8+-specific antibody (BioXCell) twice a week, starting 2 d before tumor cell injection.
Adoptive Cell Transfer
CD8+ T cells were isolated by MACS from wt OT-1 and Tyk2−/− OT-1 spleens. Two days after s.c. EG7 tumor cell injection, tumor-bearing mice received 106 wt OT-1 or Tyk2−/− OT-1 CD8+ cells in 100 μL PBS i.v. Tumor width (L) and length (l) were measured with a caliper and tumor size was calculated using the following formula: l × L × (l + L)/2. The experiment was terminated after 20 d. Tumors, lymph nodes, and spleens were excised, one part fixed in 3.7% formaldehyde, and the other part homogenized and subjected to FACS analysis.
In vitro Cytotoxicity Assay
A standard 51Cr-release assay was performed to measure the in vitro cytotoxicity of wt versus Tyk2−/− OT-1 T cells as described (32) with slight modifications. Splenocytes from wt and Tyk2−/− OT-1 mice were cocultured with irradiated (30 Gy), peptide-loaded (10 μg/mL, 1 h at 37°C) naive splenocytes in the presence of 100 units/mL IL2 (Fresenius Kabi) for 5 d. Effectors were harvested and analyzed for %CD8+Vα2+ cells by FACS and adjusted. Targets (104 per well) were incubated with effectors for 5 h at 37°C. Percent specific lysis was determined with following formula: [CTL-induced release (cpm) − spontaneous release (cpm)]/[maximum release (cpm) − spontaneous release (cpm)] × 100.
In vivo CTL Assay
Recipient mice were immunized via footpad injection (50 μL/footpad) with SIINFEKL (0.1 mg/mouse) alone, SIINFEKL in combination with adjuvant (IC31 or 50 μg CpG-ODN 1668), or PBS. Seven days later, immunized mice received differentially (0.025, 0.25, and 2.5 μmol/L) CFSE-labeled syngeneic target cells. The CFSElo population was unpulsed, the CFSEmi population was pulsed with an irrelevant peptide (mTRP-2181-188, VYDFFVWL; H2-Kb, derived from murine tyrosine-related protein-2; Bachem), and the CFSEhi population was pulsed with the relevant peptide (SIINFEKL, 10 μg/mL). Target cells were mixed at a 1:1:1 ratio and 3 × 107 cells were injected via tail vein into immunized mice. Eighteen to 24 h after the adoptive transfer, mice were sacrificed and peripheral blood, popliteal lymph nodes, and spleens were isolated and single-cell suspensions were analyzed by FACS. Specific killing was calculated as follows: % specific killing = [1 − (% CFSEhi / % CFSElo)] × 100. Results are expressed as the mean percentage of cell lysis observed for each experimental group ± SD.
Differences between mean values were calculated using an unpaired Student's t test, a Mann-Whitney U test, or a one-way ANOVA test, which was corrected by Dunnett's multiple comparison correction, as applicable. Differences in Kaplan-Meier plots were analyzed for statistical significance using the log-rank test. P values of <0.05 were considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Tyk2 deficiency facilitates systemic EL4-induced disease progression. We have previously shown that, due to a NK-cell defect, Tyk2−/− mice are highly susceptible to Abelson-induced B lymphoid leukemia formation (18). In the current study, we questioned whether Tyk2 is also required for CD8+ T-cell–mediated tumor surveillance. Therefore, we challenged Tyk2−/− recipient mice with the thymoma cell line EL4. The transplantation of EL4 cells and of their OVA-transfected derivatives, EG7 cells, is a frequently used tumor model for CD8+ T-cell–mediated cytotoxicity (33–35). I.v. injection of EL4 cells into mice induces a rapidly progressing leukemia/lymphoma. Tyk2−/− recipient mice injected with EL4 cells developed first signs of the disease significantly earlier than wt recipients (Fig. 1A,i). However, in spite of the significant difference in disease progression, the disease pattern was comparable: wt and Tyk2−/− animals had dramatically enlarged livers with macroscopic tumor nodules as illustrated in Fig. 1A,ii. The importance of cytotoxic T cells for tumor surveillance of EL4 cells was confirmed using CD8+ T-cell–depleted animals (Fig. 1B). Whereas CD8+ T-cell depletion exerted only a minor effect in Tyk2−/− animals, a significantly accelerated disease progression was observed in wt CD8+ T-cell–depleted mice when compared with control mice (P < 0.05). This finding supports our hypothesis of a key role for Tyk2 in CD8+-mediated tumor surveillance resulting in the significantly shortened disease latency in Tyk2−/− animals.
Tyk2 is dispensable for the maturation of BMDCs in vitro. An efficient antitumor CD8+ CTL response is initiated by activating signals provided by antigen-presenting cells (APC). In case of naive T cells, DCs serve as APCs, which interact via MHC class I and costimulatory receptors with the T cell. The diminished tumor surveillance in Tyk2−/− mice might therefore result from impaired DC function. To analyze the interaction between DCs and T cells after a defined stimulus, we intercrossed Tyk2−/− mice with transgenic OT-1 animals. OT-1 mice are transgenic for a T-cell receptor that recognizes the immunodominant OVA257-264 peptide SIINFEKL presented by H2-Kb.
BMDCs were prepared and maturation was induced by exposure to LPS or CpG-ODN. This procedure induces the up-regulation of CD40, CD80, CD86, and MHC II that are characteristic markers for mature BMDCs. No differences were observed between wt and Tyk2−/− BMDCs irrespective of the stimulus used (Fig. 2A; data not shown). Mature antigen-presenting DCs can activate an antigen-specific T-cell clone and induce T-cell proliferation. As depicted in Fig. 2B, LPS-stimulated or CpG-ODN–stimulated and SIINFEKL-loaded wt and Tyk2−/− BMDCs induced proliferation of purified OT-1 transgenic CD8+ cells equally well (Fig. 2B,, top). Hence, BMDCs do not require Tyk2 to induce the expansion of an antigen-specific T-cell population. To rule out that antigen processing is impaired, BMDCs were loaded with undigested OVA. Tyk2−/− and wt BMDCs loaded with OVA could equally well induce antigen-specific T-cell proliferation (data not shown). Mature BMDCs also secrete cytokines (IL10 and IL12) and thereby shift the balance to either the Th1 or Th2 side of the immune response. Again, no significant alterations in LPS-stimulated or CpG-ODN–stimulated cytokine secretion were detectable between wt and Tyk2−/− BMDCs (Fig. 2C and D).
Tyk2−/− OT-1 CD8+ T cells display an impaired in vitro cytotoxicity despite regular antigen-specific expansion. Because our experiments excluded severe defects in BMDC function, we next focused on CD8+ T-effector cells themselves. CD8+ T cells were purified from Tyk2−/− OT-1 and wt OT-1 animals and activated by coculture with LPS-stimulated or CpG-ODN–stimulated and peptide-loaded BMDCs. In this experimental setting, CD8+ cells expanded equally well irrespective of the genotype of the BMDCs or T cells (Fig. 2B). Accordingly, the expression of the T-cell activation markers CD44 and CD69 was induced to the same extent (Fig. 3A).
Activated CD8+ CTLs use two different mechanisms to kill their target cells: the granule-dependent (perforin/granzyme) or granule-independent (death receptor–induced cell death; e.g., Fas-FasL) pathway. As depicted in Fig. 3B, Tyk2−/− CD8+ cells expressed comparable levels of FasL, granzyme B, and perforin mRNA. This indicates that Tyk2−/− CD8+ T cells are equally well equipped to use either killing pathway. The exocytotic process itself (degranulation) can be quantified by analyzing surface expression of CD107a, also known as lysosome-associated membrane protein 1. After degranulation, CD107a appears on the cellular surface. As illustrated in Fig. 3C, CD107a surface expression was comparable on wt OT-1 and Tyk2−/− OT-1 T cells on activation by mature peptide-loaded BMDCs.
However, when OT-1 T cells were coincubated with EG7 cells and in vitro cytotoxicity was analyzed, we realized a significant difference: whereas wt OT-1 effector cells efficiently killed EG7 cells, Tyk2−/− OT-1 T cells displayed a highly significant reduction in specific lysis at all E:T ratios measured (Fig. 3D).
Enhanced tumor formation in Tyk2−/− OT-1 animals is linked to impaired CTL killing. Simplified in vitro settings may not sufficiently mimic the complex in vivo situation. Therefore, we challenged Tyk2−/− OT-1 T-cell function in vivo by s.c. injection of the EG7 cell line. Again, Tyk2−/− OT-1 animals were significantly less capable to suppress the proliferation of the injected EG7 cells (Fig. 4A). To unequivocally link this important defect to the T-cell compartment, we adoptively transferred either wt OT-1 or Tyk2−/− OT-1 CD8+ T cells into Tyk2−/− mice 2 days after EG7 tumor inoculation. As depicted in Fig. 4B (left), wt OT-1 cells significantly repressed EG7 tumor growth. In contrast, the injection of Tyk2−/− OT-1 cells failed to inhibit tumor progression (Fig. 4B,, middle). Tumors under the control of Tyk2−/− OT-1 CD8+ cells had a similar growth pattern when compared with PBS-injected controls (Fig. 4B,, right). This observation was confirmed by the comparison of tumor weights 17 days after the initial injection (Fig. 4C). The quantification of OT-1 cells (CD8+ Vα2+) in the spleens of the recipient animals revealed higher numbers of OT-1 T cells in wt OT-1–treated mice compared with control mice that, however, did not meet the criteria of statistical significance (wt OT-1: 0.98 ± 0.21%, Tyk2−/− OT-1: 0.47 ± 0.13%, PBS: 0.50 ± 0.15%). In a similar experiment but this time using CD8+ T cells from previously tumor cell–immunized wt and Tyk2−/− mice, comparable observations were made. Tumor development was delayed on adoptive transfer of wt CD8+ T cells but not on adoptive transfer of Tyk2−/− CD8+ T cells (data not shown). These experiments underscore and confirm the important function of Tyk2 in CTL-mediated tumor cell rejection.
The antigen-specific CTL function is compromised in the absence of Tyk2/IFNAR1 signaling. Thus far, our observations revealed a severe defect in CTL-mediated tumor surveillance in Tyk2−/− mice. This defect is attributed to an impaired T-cell function and is recapitulated in Tyk2−/− OT-1 animals. Previous experiments have shown that Tyk2 is required for IFN and IL12 signaling, which is also discussed to contribute to CTL-mediated cytotoxicity. To test whether one of these factors is indeed essential for CTL-mediated effector function, we performed in vivo CTL assays with wt, Tyk2−/−, IFNγ−/−, IFNAR1−/−, and IL12p35−/− mice. Wt mice that had been immunized with peptide and adjuvant showed a major cytotoxic effect (Fig. 5A,, arrow). In contrast, in Tyk2−/− mice, the antigen-specific cytotoxicity was significantly reduced (Fig. 5A, and Bi). This was accompanied by reduced numbers of IFNγ-secreting Tyk2−/− splenocytes (data not shown). Under the same assay conditions, antigen-specific cytotoxicity in IFNγ−/− and IL12p35−/− mice was unaltered (Fig. 5B,ii and Biii). However, the CTL response was severely and significantly impaired in IFNAR1−/− mice (Fig. 5B iv).
S.c. EG7 tumor growth is accelerated in Tyk2−/− and IFNAR1−/− mice. Our findings indicated that the severe reduction in CD8+ CTL function in Tyk2−/− animals is unrelated to an impaired IL12 and IFNγ signaling but rather associated with the inability to react to type I IFN. To test whether these findings are relevant for CTL-mediated tumor formation, 106 EG7 cells were injected s.c. into wt and Tyk2−/− animals. Tumor formation was analyzed 10 days later. As expected, tumor growth was significantly increased in Tyk2−/− animals. Depletion of CD8+ cells in wt mice significantly enhanced tumor growth but did not result in altered tumor formation in Tyk2−/− mice, confirming the CTL-mediated tumor surveillance for s.c. EG7 tumor rejection. No significant differences were observed when wt and IFNγ−/− mice were compared (Fig. 6B). We next compared EG7 tumor growth in IL12p35−/− and IFNAR1−/− mice. Interestingly and in accordance with our previous findings, EG7 cell–induced tumors were significantly bigger in IFNAR1−/− mice, whereas IL12p35 deficiency was irrelevant for the outcome of this experiment (Fig. 6C). Hence, these experiments define type I IFN as key component in this process.
A role of Tyk2 in NK-cell–mediated tumor surveillance against hematopoietic malignancies has recently been established (18). In our current study, we extend these findings and show that Tyk2 is also a critical component of CD8+-mediated tumor surveillance downstream of type I IFN signaling.
Tumors develop significantly faster in Tyk2−/− animals. The i.v. injection of the thymoma cell line EL4 in Tyk2−/− mice confirmed this result and again proves that Tyk2 deficiency renders animals highly susceptible for the development of malignant disease. The enhanced susceptibility for EL4-induced tumor formation has to be accredited, at least for the most part, to defects of CTL-mediated cytotoxicity. The depletion of CD8+ T cells significantly accelerated tumor growth in wt animals but had almost no effect in Tyk2−/− mice. The significant contribution of Tyk2 for CTL-mediated cytotoxicity was further supported by several other findings: Tyk2−/− mice immunized with antigen and adjuvant show a severe defect in CTL-mediated cytotoxicity and reduced IFNγ production. Moreover, we showed the detrimental effect of this defect on tumor surveillance in vivo: Tyk2−/− OT-1 mice develop significantly larger EG7 cell–initiated tumors than wt OT-1 control mice.
Tyk2 has been implicated in IFN and IL12 signaling, both of which are considered important for CD8+ T-cell effector functions (36, 37). Interestingly, IL12 and IFNγ were dispensable for target cell killing in the in vivo CTL assay. In line with this, IL12 or IFNγ deficiency did not confer a growth advantage on EG7-induced tumors, confirming the negligible role of these cytokines in CTL-mediated tumor surveillance in this experimental system. However, in IFNAR1−/− mice, the CTL-mediated cytotoxicity was severely impaired. IFNAR1−/− and Tyk2−/− animals displayed similar phenotypes indicating that both type I IFN and Tyk2 are key components of CTL-mediated cytotoxicity. Recent observations made in vaccination studies further support our hypothesis that type I IFN is of utmost importance for the generation of tumor-specific CTLs (38).
Although initially IFNγ was considered the key IFN involved in tumor surveillance, the importance of type I IFN has soon been recognized. Type I IFN exerts immune modulatory effects on several cell types, including NK cells, DCs, and effector cells of the adaptive immune response. These activities can, in part, be attributed to an increased expression of MHC I and costimulatory molecules, thus an enhancement of antigen presentation and T-cell activation. In addition, type I IFN was described to be critical for the antitumor function of NK cells as well as T lymphocytes (26, 39).
Our work now emphasizes that beside these well-known effects, type I IFNs are important components of CTL effector function in antitumor immunity and that these effects are mediated via Tyk2. One might speculate that type I IFN-dependent Tyk2 activation is a limiting factor for CTL-dependent cytotoxicity, whereas Tyk2 activation downstream of IFNγ and IL12 contributes to immune surveillance of tumors exerted by the innate immune system.
One way by which type I IFN signaling might impair CTL activation is via DC-mediated antigen presentation. We and others have failed to show any defects in Tyk2−/− DC maturation and their ability to stimulate T-cell proliferation in vitro (15). Nevertheless, we cannot yet fully exclude a contribution of Tyk2 for DC functions in vivo. This issue will ultimately be settled by the use of conditionally gene targeted animals.
Based on our experiments, we propose that type I IFN and Tyk2 critically contribute to the functional maturation of CTLs. The adoptive transfer of Tyk2−/− OT-1 CD8+ T cells did not suppress tumor growth in vivo. In contrast, the adoptive transfer of wt OT-1 CD8+ cells efficiently suppressed tumor growth. In this experimental setting, the adoptively transferred CTLs themselves determine the experimental outcome because all other experimental conditions and the immune environment are superimposable. Even under the premises that DC activation is of minor importance within the OT-1 system, the defect in the CTLs still remains a matter of fact. Thus, the adoptive transfer experiments point at an intrinsic defect within CD8+ T cells on Tyk2 deficiency. In view of our findings, it seems unlikely that the proliferation of CD8+ cells is impaired on Tyk2 deficiency. Tyk2−/− CD8+ cells proliferated with identical kinetics in vitro.
These findings strengthen the concept that Tyk2 inhibition is a powerful therapeutic option for patients suffering from diseases linked to the hyperactivation of the immune system. Tyk2 inhibition is supposed to efficiently suppress NK- and CTL-mediated killing and this renders Tyk2 inhibitors highly suitable for the treatment of autoimmune disorders or for patients undergoing transplantation. Similarly, Jak3 has been discussed as novel drug target for the treatment of rheumatoid arthritis, psoriasis, and transplant rejection (40). Clinical studies are ongoing to prove the usefulness of the Jak3 inhibitors (NCT00483756, NCT00263328, NCT00413699, and NCT00550446).9
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant support: Austrian Academy of Sciences at the Institute of Pharmacology DOC-FFORTE fellowship (O. Simma), Austrian National Bank project no. 9875 and FWF grant P19534-B13 (D. Stoiber), Austrian Federal Ministry of Science and Research grant BM_WFa GZ200.112/1-VI/1/2004 and FWF grant SFB F2808 (M. Müller), and FWF grants P15033 and SFB F2810 (V. Sexl).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Udo Losert and the staff of the Biomedical Research Institute for taking good care of our mice; Marina Karaghiosoff, Birgit Strobl, and Richard Moriggl for helpful discussions and support; and the Department of Neuro-Immunology, Center for Brain Research, Medical University of Vienna for help with histopathology.