Signal pathways which promote invasion and metastasis: critical and distinct contributions of extracellular signal-regulated kinase and Ral-specific guanine exchange factor pathways

Approximately 50% of metastatic tumors contain Ras mutations. Ras proteins can activate at least three downstream signaling cascades mediated by the Raf-MEK-extracellular signal-regulated kinase family, phosphatidylinositol-3 (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (RalGEFs). Here we investigated the contribution of RalGEF and ERK activation to the development of experimental metastasis in vivo and associated invasive properties in vitro. Each pathway contributes distinct properties to the metastatic phenotype. Following lateral tail vein injection, 3T3 cells transformed by constitutively active Raf or MEK produced lung metastasis that displayed circumscribed, noninfiltrating borders. In contrast, 3T3 cells transformed by Ras(12V,37G), a Ras effector mutant that activates RalGEF but not Raf or P13 kinase, formed aggressive, infiltrative metastasis. Dominant negative RalB inhibited Ras(12V,37G)-activated invasion and metastasis, demonstrating the necessity of the RalGEF pathway for a fully transformed phenotype. Moreover, 3T3 cells constitutively expressing a membrane-associated form of RalGEF (RalGDS-CAAX) formed invasive tumors as well, demonstrating that activation of a RalGEF pathway is sufficient to initiate the invasive phenotype. Despite the fact that Ras(12V,37G) expression does not elevate ERK activity, inhibition of this kinase by a conditionally expressed ERK phosphatase demonstrated that ERK activity was necessary for Ras(12V,37G)-transformed cells to express matrix-degrading activity in vitro and tissue invasiveness in vivo. Therefore, these experiments have revealed a hitherto-unknown but essential interaction of the RalGEF and ERK pathways to produce a malignant phenotype. The generality of the role of the RalGEF pathway in metastasis is supported by the finding that Ras(12V,37G) increased the invasiveness of epithelial cells as well as fibroblasts.     

Ras Signals to the Cell Cycle Machinery via Multiple Pathways To Induce Anchorage-Independent Growth

Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.     

Profound enhancement of the IL-12/IL-18 pathway of IFN-gamma secretion in human CD8+ memory T cell subsets via IL-15

Human memory CD8(+) T cell subsets, termed central memory and effector memory T cells, can be identified by expression of CD45RA, CD62 ligand (CD62L), and CCR7. Accordingly, functional differences have been described for each subset, reflecting unique roles in immunological memory. The common gamma-chain cytokines IL-15 and IL-7 have been shown to induce proliferation and differentiation of human CD8(+) T cell subsets, as well as increased effector functions (i.e., cytokines, cytotoxicity). In this study, we observed that addition of IL-15 or IL-7 to cultures of human CD8(+) T cells profoundly enhanced the IL-12-IL-18 pathway of IFN-gamma production. Importantly, IL-15 and IL-7 lowered the threshold concentrations of IL-12 and IL-18 required for induction of IFN-gamma by 100-fold. Comparison of IL-15 and IL-7 demonstrated that IL-15 was superior in its ability to enhance IL-12-IL-18-induced IFN-gamma, without evidence of a synergistic effect between IL-15 and IL-7. We also observed that IL-15- and IL-7-mediated enhancement of IL-12-IL-18-induced IFN-gamma production was a functional property of effector memory CD8(+) T cells. Despite a lack of association between cell division and acquisition of IL-12-IL-18-induced IFN-gamma, down-regulation of CD62L expression correlated well with increased IL-12-IL-18-induced IFN-gamma. Purified central memory T cells stimulated with IL-15 and IL-7 down-regulated CD62L and acquired potent IL-12-IL-18-induced IFN-gamma similar to effector memory T cells. Thus, in addition to its known role in development of T cell memory, IL-15 may amplify memory CD8(+) T cell effector functions by increasing sensitivity to proinflammatory cytokine stimulation.     

Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase

Ras proteins activate diverse effector molecules. Depending on the cellular context, Ras activation may have different biological consequences: induction of cell proliferation, senescence, survival, or death. Augmentation and selective activation of particular effector molecules may underlie various Ras actions. In fact, Ras effector-loop mutants interacting with distinctive effectors provide evidence for such selectivity. Interactions of active Ras with escort proteins, such as galectin-1, could also direct Ras selectivity. Here we show that in comparison with Ras transfectants, H-Ras/galectin-1 or K-Ras4B/galectin-1 co-transfectants exhibit enhanced and prolonged epidermal growth factor (EGF)-stimulated increases in Ras-GTP, Raf-1 activity, and active extracellular signal-regulated kinase. Galectin-1 antisense RNA inhibited these EGF responses. Conversely, Ras and galectin-1 co-transfection inhibited the EGF-stimulated increase in phosphoinositide 3-kinase (PI3K) activity. Galectin-1 transfection also inhibited Ras(G12V)-induced PI3K but not Raf-1 activity. Galectin-1 co-immunoprecipitated with Ras(G12V) or with Ras(G12V/T35S) that activate Raf-1 but not with Ras(G12V/Y40C) that activates PI3K. Thus, galectin-1 binds active Ras and diverts its signal to Raf-1 at the expense of PI3K. This demonstrates a novel mechanism controlling the duration and selectivity of the Ras signal. Ras gains selectivity when it is associated with galectin-1, mimicking the selectivity of Ras(T35S), which activates Raf-1 but not PI3K.     

Dual role of the IL-12/IFN-gamma axis in the development of autoimmune myocarditis: induction by IL-12 and protection by IFN-gamma.

IL-12 and IFN-gamma positively regulate each other and type 1 inflammatory responses, which are believed to cause tissue damage in autoimmune diseases. We investigated the role of the IL-12/IFN-gamma (Th1) axis in the development of autoimmune myocarditis. IL-12p40-deficient mice on a susceptible background resisted myocarditis. In the absence of IL-12, autospecific CD4(+) T cells proliferated poorly and showed increased Th2 cytokine responses. However, IFN-gamma-deficient mice developed fatal autoimmune disease, and blockade of IL-4R signaling did not confer susceptibility to myocarditis in IL-12p40-deficient mice, demonstrating that IL-12 triggers autoimmunity by a mechanism independent of the effector cytokines IFN-gamma and IL-4. In conclusion, our results suggest that the IL-12/IFN-gamma axis is a double-edged sword for the development of autoimmune myocarditis. Although IL-12 mediates disease by induction/expansion of Th1-type cells, IFN-gamma production from these cells limits disease progression.     

An IFN-gamma-dependent pathway controls stimulation of memory phenotype CD8+ T cell turnover in vivo by IL-12, IL-18, and IFN-gamma

Unlike naive T cells, memory phenotype (CD44(high)) T cells exhibit a high background rate of turnover in vivo. Previous studies showed that the turnover of memory phenotype CD8(+) (but not CD4(+)) cells in vivo can be considerably enhanced by products of infectious agents such as LPS. Such stimulation is TCR independent and hinges on the release of type I IFNs (IFN-I) which leads to the production of an effector cytokine, probably IL-15. In this study, we describe a second pathway of CD44(high) CD8(+) stimulation in vivo. This pathway is IFN-gamma rather than IFN-I dependent and is mediated by at least three cytokines, IL-12, IL-18, and IFN-gamma. As for IFN-I, these three cytokines are nonstimulatory for purified T cells and under in vivo conditions probably act via production of IL-15.     

T cells undergo rapid ON/OFF but not ON/OFF/ON cycling of cytokine production in response to antigen

Inflammatory cytokines such as IFN-gamma and TNF produced by Ag-stimulated CD4(+) and CD8(+) T cells are important in defense against microbial infection. However, production of these cytokines must be tightly regulated to prevent immunopathology. Previous studies, conducted with BALB/c mice, have suggested that 1) CD8(+) T cells maintain IFN-gamma production but transiently produce TNF in the continued presence of Ag and 2) lymphocytic choriomeningitis virus-specific and in vitro-propagated effector CD8(+) T cells could rapidly cycle IFN-gamma production ON/OFF/ON in response to Ag exposure, removal, and re-exposure. In contrast with CD8(+) T cells, our results show that Listeria monocytogenes-specific CD4(+) T cells from C57BL/6 mice rapidly initiate (ON cycling) and maintain production of both IFN-gamma and TNF in the continued presence of Ag. Upon Ag removal, production of both cytokines rapidly ceases (OFF cycling). However, if the initial stimulation was maximal, Ag-specific CD4(+) T cells were unable to reinitiate cytokine production after a second Ag exposure. Furthermore, L. monocytogenes-specific CD8(+) T cells in the same mice and lymphocytic choriomeningitis virus-specific CD8(+) T cells in BALB/c mice also underwent ON/OFF cycling, but if the initial Ag stimulus was maximal, they could not produce IFN-gamma after Ag re-exposure. As the initial Ag dose was reduced, the number of cells producing cytokine in response to the second Ag exposure exhibited a corresponding increase. However, T cells that were marked for IFN-gamma secretion during the first stimulation did not contribute cytokine production during the second stimulation. Thus, T cells are not able to undergo rapid ON/OFF/ON cytokine cycling in vitro in response to Ag.     

Strong TCR signaling, TLR ligands, and cytokine redundancies ensure robust development of type 1 effector T cells

T cell effector function is a central mechanism of adaptive immunity, and accordingly, protection of the host against pathogens. One of the primary effector molecules produced by T cells in response to such pathogens is the cytokine, IFN-gamma. Although the signaling pathways associated with the production of IFN-gamma are well established, disparate in vivo and in vitro results indicate that distinct pathways may become more prominent dependent upon the nature of the infection, inflammatory milieu and tissue localization. We have examined the roles and requirements of the major IFN-gamma-inducing pathways in vivo and in vitro, specifically: strength of TCR signal; paracrine release of IL-12, IL-23, and IL-18; and autocrine production of IFN-gamma. Our data show a dynamic interaction between these activation pathways, which allows the host a degree of flexibility and redundancy in the induction of IFN-gamma. Upon strong signaling through the TCR, IL-12, IL-18, and IL-23 play negligible roles in the induction of IFN-gamma, whereas autocrine IFN-gamma is an important component in sustaining its own secretion. However, the absence of any one of these factors during a weaker TCR signal, results in strikingly impaired T cell IFN-gamma production. Of note, TLR-activated dendritic cells (DCs) were capable of overcoming the absence of a strong TCR signal, IL-12, IL-23, or IL-18 revealing an important additional mechanism for ensuring a robust IFN-gamma response. Our findings clarify the hierarchical requirements of the major IFN-gamma inducing pathways and highlight the important role TLR ligand-activated DCs have to preserve them.     

Epigenetic remodeling of the IL-2 and IFN-gamma loci in memory CD8 T cells is influenced by CD4 T cells

Memory T cells (T(M)) are able to rapidly exert effector functions, including immediate effector cytokine production upon re-encounter with Ag, which is critical for protective immunity. Furthermore, this poised state is maintained as T(M) undergo homeostatic proliferation over time. We examined the molecular basis underlying this enhanced functional capacity in CD8 T(M) by comparing them to defective CD8 T(M) generated in the absence of CD4 T cells. Unhelped CD8 T(M) are defective in many functions, including the immediate expression of cytokines, such as IL-2 and IFN-gamma. Our data show that this defect in IL-2 and IFN-gamma production is independent of clonal selection, functional avidity maturation, and the integrity of proximal TCR signaling, but rather involves epigenetic modification of these cytokine genes. Activated Ag-specific CD8 T cells exhibit rapid DNA demethylation at the IL-2 and IFN-gamma loci and substantial histone acetylation at the IFN-gamma promoter and enhancer regions. These epigenetic modifications occur early after infection at the effector stage and are maintained through memory development. However, activated unhelped CD8 T cells, which fail to develop into functional memory and are incapable of rapid cytokine production, exhibit increased DNA methylation at the IL-2 promoter and fail to acetylate histones at the IFN-gamma locus. Thus, CD4 T cell help influences epigenetic modification during CD8 T(M) differentiation and these epigenetic changes provide a molecular basis for the enhanced responsiveness and the maintenance of a "ready-to-respond" state in CD8 T(M).     

Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN-gamma production by dendritic cells

Mouse splenic dendritic cells (DCs) produce IFN-gamma in response to IL-12. In the present study, we analyzed effects of Th1 and Th2 cytokines on IFN-gamma production by DCs. IL-18 produced by DCs and macrophages acts in an autocrine manner and augments IL-12-induced IFN-gamma production by DCs as also observed in T and NK cells. Surprisingly, IL-4, a Th2 cytokine, also acts synergistically with IL-12 on IFN-gamma production by DCs. In addition, IL-4 markedly enhances IFN-gamma production when DCs are stimulated through CD40 or MHC class II. These results indicate that both Th1 and Th2 cytokines act on DCs during T cell-DC interaction upon Ag presentation. p38 mitogen-activated protein kinase is constitutively activated in mature DCs and is required for IFN-gamma production by DCs. IL-18 but not IL-4 or IL-12 further activates the p38 mitogen-activated protein kinase activity, suggesting that IL-4 and IL-18 enhance IFN-gamma production through distinct intracellular signal transduction pathways in DCs.     

Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts

Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65(Pak)). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho.     

Bacillus Calmette-Guérin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles

The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA-CCR7-CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.     

IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-gamma and nitric oxide synthetase 2

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-gamma is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-gamma) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12-/- mice were far more susceptible than IFN-gamma-/- mice, and primary CTL responses against VV were absent in IL-12-/- mice but remained intact in IFN-gamma-/- mice. Both CD4+ and CD8+ T cells from IL-12-/- mice were unimpaired in IFN-gamma production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4-/- mice and, even more strikingly, in IL-10-/- mice, which both produced elevated levels of the proinflammatory cytokines IL-1alpha and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-gamma and show that IL-4 and IL-10 modulate host protective responses to VV.     

IL-21 promotes differentiation of naive CD8 T cells to a unique effector phenotype

IL-21, the most recently described member of the common gamma-chain cytokine family, is produced by activated CD4 T cells, whereas CD8 T cells express the IL-21 receptor. To investigate a possible role for IL-21 in the priming of naive CD8 T cells, we examined responses of highly purified naive OT-I CD8 T cells to artificial APCs displaying Ag and B7-1 on their surface. We found that IL-21 enhanced OT-I clonal expansion and supported development of cytotoxic effector function. High levels of IL-2 did not support development of effector functions, but IL-2 was required for optimal responses in the presence of IL-21. IL-12 and IFN-alpha have previously been shown to support naive CD8 T cell differentiation and acquisition of effector functions through a STAT4-dependent mechanism. Here, we show that IL-21 does not require STAT4 to stimulate development of cytolytic activity. Furthermore, IL-21 fails to induce IFN-gamma or IL-4 production and can partially block IL-12 induction of IFN-gamma production. CD8 T cells that differentiate in response to IL-21 have a distinct surface marker expression pattern and are characterized as CD44(high), PD-1(low), CD25(low), CD134(low), and CD137(low). Thus, IL-21 can provide a signal required by naive CD8 T cells to differentiate in response to Ag and costimulation, and the resulting effector cells represent a unique effector phenotype with highly effective cytolytic activity, but deficient capacity to secrete IFN-gamma.     

Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes

The TNF-like cytokine TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 and IL-12/IL-18-stimulated peripheral blood (PB) T cells. However, only a small subset of PB T cells respond to TL1A stimulation with IFN-gamma production. PB CCR9+ T cells represent a small subset of circulating T cells with mucosal T cell characteristics and a Th1/Tr1 cytokine profile. In the current study, we show that TL1A enhanced IFN-gamma production by TCR- or CD2/CD28-stimulated CCR9(+)CD4+ PB T cells. However, TL1A had the most pronounced effect on augmenting IFN-gamma production by IL-12/IL-18-primed CCR9(+)CD4+ PB T cells. TL1A enhanced both the percentage and the mean fluorescence intensity of IFN-gamma in CCR9(+)CD4+ T cells as assessed by intracellular cytokine staining. IL-12 plus IL-18 up-regulated DR3 expression in CCR9(+)CD4+ T cells but had negligible effect on CCR9(-)CD4+ T cells. CCR9(+)CD4+ T cells isolated from the small intestine showed a 37- to 105-fold enhancement of IFN-gamma production when TL1A was added to the IL-12/IL18 cytokine combination. Cell membrane-expressed TL1A was preferentially expressed in CCR9(+)CD4+ PB T cells, and a blocking anti-TL1A mAb inhibited IFN-gamma production by cytokine-primed CCR9(+)CD4+ T cells by approximately 50%. Our data show that the TL1A/DR3 pathway plays a dominant role in the ultimate level of cytokine-induced IFN-gamma production by CCR9+ mucosal and gut-homing PB T cells and could play an important role in Th1-mediated intestinal diseases, such as Crohn's disease, where increased expression of IL-12, IL-18, TL1A, and DR3 converge in the inflamed intestinal mucosa.     

Peripheral immature CD2-/low T cell development from type 2 to type 1 cytokine production

Immature myeloid and NK cells exist, and undergo cytokine-induced differentiation, in the periphery. In this study, we show that also immature CD2(-/low) T cells exist in peripheral blood. These cells produce the type 2 cytokines IL-13, IL-4, and IL-5, but not IFN-gamma or IL-10, and, upon culture with IL-12- and TCR-mediated stimuli, differentiate to IL-13(+)IFN-gamma(+) cells producing high IL-2 levels, and finally IL-13(-)IFN-gamma(+) cells. The monokine combination IL-12, IL-18, and IFN-alpha substitutes for TCR-mediated stimulation to induce the same differentiation process in both immature CD2(-/low) and primary mature CD2(+) IL-13(+) T cells. IFN-alpha is needed to maintain high level IL-2 production, which is confined to type 2 cytokine-producing cells and lost in the IFN-gamma(+) ones. Upon TCR-mediated stimulation, IFN-gamma(+) cells are then induced to produce IL-10 as they undergo apoptosis. These data indicate that peripheral type 2 cytokine(+) T cells are immature cells that can differentiate to effector IFN-gamma(+) cells following a linear monokine-regulated pathway identical with that previously described for NK cells. They define the cellular bases to support that cell-mediated immune responses are regulated not only via Ag-induced activation of mature effector cells, but also via bystander monokine-induced maturation of immature T cells.     

Signal 3 availability limits the CD8 T cell response to a solid tumor

CD8 T cells need a third signal, along with Ag and costimulation, for effective survival and development of effector functions, and this can be provided by IL-12 or type I IFN. Adoptively transferred OT-I T cells, specific for H-2K(b) and OVA, encounter Ag in the draining lymph nodes of mice with the OVA-expressing E.G7 tumor growing at a s.c. site. The OT-I cells respond by undergoing limited clonal expansion and development of effector functions (granzyme B expression and IFN-gamma production), and they migrate to the tumor where they persist but fail to control tumor growth. In contrast, OT-I T cells deficient for both the IL-12 and type I IFN receptors expand only transiently and rapidly disappear. These results suggested that some signal 3 cytokine is available, but that it is insufficient to support a CTL response that can control tumor growth. Consistent with this, administration of IL-12 at day 10 of tumor growth resulted in a large and sustained expansion of wild-type OT-I cells with enhanced effector functions, and tumor growth was controlled. This did not occur when the OT-I cells lacked the IL-12 and type I IFN receptors, demonstrating that the therapeutic effect of IL-12 results from direct delivery of signal 3 to the CD8 T cells responding to tumor Ag in the signal 3-deficient environment of the tumor.     

Increased nonobese diabetic Th1:Th2 (IFN-gamma:IL-4) ratio is CD4+ T cell intrinsic and independent of APC genetic background

Autoreactive CD4(+) T cells play a major role in the pathogenesis of autoimmune diabetes in nonobese diabetic (NOD) mice. We recently showed that the non-MHC genetic background controlled enhanced entry into the IFN-gamma pathway by NOD vs B6.G7 T cells. In this study, we demonstrate that increased IFN-gamma, decreased IL-4, and decreased IL-10 production in NOD T cells is CD4 T cell intrinsic. NOD CD4(+) T cells purified and stimulated with anti-CD3/anti-CD28 Abs generated greater IFN-gamma, less IL-4, and less IL-10 than B6.G7 CD4(+) T cells. The same results were obtained in purified NOD.H2(b) vs B6 CD4(+) T cells, demonstrating that the non-MHC NOD genetic background controlled the cytokine phenotype. Moreover, the increased IFN-gamma:IL-4 cytokine ratio was independent of the genetic background of APCs, since NOD CD4(+) T cells generated increased IFN-gamma and decreased IL-4 compared with B6.G7 CD4(+) T cells, regardless of whether they were stimulated with NOD or B6.G7 APCs. Cell cycle analysis showed that the cytokine differences were not due to cycle/proliferative differences between NOD and B6.G7, since stimulated CD4(+) T cells from both strains showed quantitatively identical entry into subsequent cell divisions (shown by CFSE staining), although NOD cells showed greater numbers of IFN-gamma-positive cells with each subsequent cell division. Moreover, 7-aminoactinomycin D and 5-bromo-2'-deoxyuridine analysis showed indistinguishable entry into G(0)/G(1), S, and G(2)/M phases of the cell cycle for both NOD and B6.G7 CD4(+) cells, with both strains generating IFN-gamma predominantly in the S phase. Therefore, the NOD cytokine effector phenotype is CD4(+) T cell intrinsic, genetically controlled, and independent of cell cycle machinery.     

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.