Absence Of APC Research Articles

Abstract A014: Identifying and overcoming resistance to RAS targeted therapy in colorectal cancer

Abstract KRAS is the most frequently mutated oncogene in human cancer and a potent oncogenic driver. In colorectal cancer (CRC), pathogenic KRAS mutations are observed in up to 50% of cases, yet there is little understanding of how distinct mutations dictate tumor biology including initiation, progression and therapy response. The recent approval of targeted KRASG12C inhibitors brings promise to transform the therapeutic landscape for many patients who lack effective, personalized treatment. Clinically acquired resistance is already observed, highlighting the need to identify mechanisms of resistance and approaches to overcome them. The aim of this study is to understand how distinct Kras alterations drive tumorigenesis and identify reasons underlying the failure of KRAS inhibitors in CRC. To explore the clinical significance of distinct KRAS mutations in tumor development and therapy response, we used Cre- conditional (Lox- stop- Lox) Kras mutant mouse strains crossed to the colon specific promoter Fabp1-Cre and Apc(flox/flox) mice. In the absence of Apc, we demonstrated that distinct Kras mutations G12C and G13D drive unique phenotypes in the colon which demonstrates that subtle mutational changes can dramatically alter the physiological response in vivo, and make distinct KRAS alleles an attractive target for targeted therapeutics. In tumor bearing models of CRC, we observed that KrasG12D drives large numbers of smaller tumors, while KrasG13D drives smaller numbers of larger tumors. To investigate the differences observed in tumorigenesis, we are studying the potential role of Kras to prime the colon for APC loss and the consequences of Kras alterations on the stem cell dynamics in driving tumorigenesis. Further, we employed our unique LSL-Kras mutant mouse strains and colon derived organoids to assess the therapeutic response of G12C and pan Kras inhibitors alone or in combination with upstream target drugs including inhibitors targeting EGFR signaling (EGFR, SHP2, SOS1 and MEK) and the Wnt signaling pathway. Together, this work has identified that distinct mutations in KRAS creates colon cancer specific vulnerabilities which dictates tumor biology. The unique in vivo and ex vivo organoid models provide an ideal platform to identify the underlying cause of failure of G12C inhibitors and developing effective approaches to target RAS in CRC. Citation Format: Marie J. Parsons, Maria Zafra, Sukanya Goswami, Maria Teresa Calvo Fernandez, John E. Wilkinson, Lukas E. Dow. Identifying and overcoming resistance to RAS targeted therapy in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A014.

Ten Color Multiparameter Flow Cytometry in Bone Marrow and Apheresis Products for Assessment and Outcome Prediction in Multiple Myeloma Patients.

ObjectiveIn clinical trials (CTs), the assessment of minimal residual disease (MRD) has proven to have prognostic value for multiple myeloma (MM) patients. Multiparameter flow cytometry (MFC) and next-generation sequencing are currently used in CTs as effective tools for outcome prediction. We have previously described 6- and 8-color MFC panels with and without kappa/lambda, which were equally reliable in detecting aberrant plasma cells (aPC) in myeloma bone marrow (BM) specimens. This follow-up study a) established a highly sensitive single-tube 10-color MFC panel for MRD detection in myeloma samples carrying different disease burden (monoclonal gammopathy of unknown significance (MGUS), smoldering multiple myeloma (SMM), MM), b) evaluated additional, rarely used markers included in this panel, and c) assessed MRD levels and the predictive value in apheresis vs. BM samples of MM patients undergoing autologous stem cell transplantation (ASCT).Methods + ResultsThe 10-color MFC was performed in BM and apheresis samples of 128 MM and pre-MM (MGUS/SMM) patients. The markers CD28, CD200, CD19, and CD117 underwent closer examination. The analysis revealed distinct differences in these antigens between MM, MGUS/SMM, and patients under treatment. In apheresis samples, the 10-color panel determined MRD negativity in 44% of patients. Absence of aPC in apheresis corresponded with disease burden, cytogenetics, and response to induction. It also determined MRD negativity in BM samples after ASCT and was associated with improved progression-free survival.ConclusionThese results highlight the significance of the evaluation of both BM and apheresis samples with a novel highly sensitive 10-color MFC panel.

Validation and standardization of the ETP-based activated protein C resistance test for the clinical investigation of steroid contraceptives in women: an unmet clinical and regulatory need.

Background Regulatory bodies recommend the use of an assay based on the assessment of the endogenous thrombin potential (ETP) for the investigation of the activated protein C resistance (APCr) in the development of steroid contraceptives in women. However, the assays described in the literature are home-made and not standardized regarding the method, the reagents, the reference plasma and the quality controls. In the absence of any commercially available method, we aimed at validating the ETP-based APCr assay. Methods The validation was performed according to regulatory standards. The method targets a 90% inhibition of the ETP in healthy donors in the presence of APC compared to the same condition in the absence of APC. As a large-scale production of a pool of plasma from well-selected healthy donors is impossible, algorithms were applied to a commercial reference plasma to correlate with the selected pool. Results Repeatability and intermediate precision passed the acceptance criteria. The assay demonstrated a curvilinear dose response to protein S and APC concentrations (R2 > 0.99). Analysis of plasma samples from 47 healthy individuals (22 women not taking combined hormonal contraceptives [CHC], and 25 men not Factor V Leiden carriers) confirmed the validity of the test, with a mean inhibition percentage of 90%. Investigations in 15 women taking different contraceptives and in two subjects with Factor V Leiden confirmed the good sensitivity and performance of the assay. Conclusions This validation provides the pharmaceutical industry, the regulatory bodies and physicians with a reproducible, sensitive and validated gold-standard ETP-based APCr assay.

Identifying anti-EGFR (EGFRi) response subgroups using evidence of ctDNA selective pressure.

3587 Background: Metastatic colorectal cancers (mCRC) that respond to EGFRi display a robust circulating tumor DNA (ctDNA) signature that reflects selective pressure and clonal evolution. Conversely, non-responding tumors do not exhibit this signature. On this basis, we developed a novel method that defines EGFRi sensitivity with improved biological confidence with fewer patients (pts), and does not rely on clinical trial outcomes where responses may be confounded by concurrent chemotherapy. We used this method to further elucidate the association of several features that have been previously reported to be associated with EGFRi resistance, namely tumor sidedness, BRAF, PIK3CA, or ERBB2 ( HER2) MTs, and the absence of APC/ TP53MT. Methods: We analyzed 112 pts with baseline tissue based RASWT mCRC who had progressed following EGFRi, and with plasma samples available for ctDNA sequencing using a blood based NGS assay. Using our previously validated EGFRi exposure signature, we identified pts with evidence of selective pressure. Results: Post EGFRi ctDNA found 37% and 33% of pts with left sided and transverse tumors displayed evidence of selective pressure, respectively. 0 pts with right sided tumors displayed evidence of selective pressure; p= 0.01. Similarly, BRAFV600EMT displayed no evidence of selective pressure vs 30% of WT pts; in contrast, selective pressure was evident in pts with PIK3CAMT, ERBB2MT and pts with absence of APC/TP53MT (42% vs 28%, 67% vs 28%, 24% vs 43%, respectively for MT vs WT, p= NS for all). BRAF, PIK3CA, ERBB2, and APC/TP53 MT were present in 4/117, 12/108, 3/118 and 30/91 pts, respectively. ctDNA shedding was similar for all subgroups, as was time from previous EGFRi, indicating that these factors were not confounders. Conclusions: Consistent with prior large randomized studies, no pts with right sided tumors or BRAFMT had evidence of biologic benefit as assessed by presence of selective pressure. In contrast a number of pts with transverse tumors, ERBB2MT, PIK3CAMT or absence of APC/TP53MT had evidence of EGFR selective pressure, confirming that these are not absolute predictors of EGFR resistance and suggesting a subset of these pts were deriving benefit from EGFR inhibition. This biology based approach has the potential to more efficiently evaluate biomarkers of targeted therapy in the future without reliance on large randomized datasets.

Regulation of Notch activation in CD4+ T cells

Abstract Notch signaling provides an important cue in the mammalian developmental process. It is a key player in T cell development and function. Mature T cells express Notch receptors 1, 2 and 3. Data from our lab and others indicate that T cell receptor (TCR) signaling activates Notch. Conventional ligand dependent Notch signaling involves, interaction of the Notch receptor with ligands such as Delta like ligand (DLL) 1,3, 4 or Jagged (JAG) 1, 2. However, in T cells it is unclear whether TCR induced activation of Notch is dependent upon engagement of Notch receptor with ligand. In-vitro stimulation of purified CD4+T cells, using antibodies against CD3 and costimulatory molecule CD28, results in activation of Notch. This occurs in absence of APC or any apparent ligand presenting cell. Thus, the mechanism that drives Notch activation during TCR stimulation is unclear. When naïve CD4+ T cells were cultured with bone marrow derived dendritic cells pulsed with antigen, we observed activation of Notch in the apparent absence of ligand on APC or activated T cells. However, when we examined ligand expression on naïve CD4+ T cell, activated by anti-CD3/CD28, we found resting T cells express constitutively low levels of DLL1 which increases with activation and is maintained through activated state. Additionally, DLL4 and JAG1 are expressed only after T cell activation and maintained only for 24hrs after activation. This upregulation is completely dependent upon signaling through CD28. However, whether the expression of ligand is sufficient to activate Notch remains unclear. Our preliminary data thus suggests that TCR mediated signaling in the absence of ligand may be is sufficient to activate Notch.

Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK Cells.

Lactobacilli are probiotic commensal bacteria and potent modulators of immunity. When present in the gut or supplemented as probiotics, they beneficially modulate ex vivo immune responsiveness. Further, factors derived from several lactobacilli strains act immune regulatory in vitro. In contrast, Staphylococcus aureus (S. aureus) is known to induce excessive T cell activation. In this study, we aimed to investigate S. aureus-induced activation of human mucosal-associated invariant T cells (MAIT cells), γδ T cells, NK cells, as well as of conventional CD4+ and CD8+ T cells in vitro. Further, we investigated if lactobacilli-derived factors could modulate their activation. PBMC were cultured with S. aureus 161:2 cell-free supernatants (CFS), staphylococcal enterotoxin A or CD3/CD28-beads alone, or in combination with Lactobacillus rhamnosus GG-CFS or Lactobacillus reuteri DSM 17938-CFS and activation of T and NK cells was evaluated. S. aureus-CFS induced IFN-γ and CD107a expression as well as proliferation. Costimulation with lactobacilli-CFS dampened lymphocyte-activation in all cell types analyzed. Preincubation with lactobacilli-CFS was enough to reduce subsequent activation, and the absence of APC or APC-derived IL-10 did not prevent lactobacilli-mediated dampening. Finally, lactate selectively dampened activation of unconventional T cells and NK cells. In summary, we show that molecules present in the lactobacilli-CFS are able to directly dampen in vitro activation of conventional and unconventional T cells and of NK cells. This study provides novel insights on the immune-modulatory nature of probiotic lactobacilli and suggests a role for lactobacilli in the modulation of induced T and NK cell activation.

Direct TLR-2 Costimulation Unmasks the Proinflammatory Potential of Neonatal CD4+ T Cells.

Neonatal CD4(+) T cells have traditionally been viewed as deficient in their capacity to produce Th1 cytokines in response to polyclonal or Ag-specific stimuli. Thus, defining unique aspects of CD4(+) T cell activation and development into Th1 effector cells in neonates is essential to the successful development of novel vaccines and immunotherapies to protect infants from intracellular pathogens. Using highly purified naive CD4(+) T cells derived from cord and adult peripheral blood, we compared the impact of anti-CD3 stimulation plus costimulation through TLR-2 performed in the absence of APC on CD4(+) T cell cytokine production, proliferation, and expression of activation markers. In both age groups, TLR-2 costimulation elicited activation of naive CD4(+) T cells, characterized by robust production of IL-2 as well as key Th1-type cytokines IFN-γ and TNF-α. TLR-2 costimulation also dramatically reduced naive T cell production of the immunosuppressive cytokine IL-10. We observed that neonatal naive CD4(+) T cells are uniquely sensitive to TLR-2-mediated costimulation, which enabled them to produce equivalent amounts of IFN-γ and more IL-2 when compared with adult responses. Thus, neonatal CD4(+) T cells have a distinctive propensity to use TLR-2-mediated costimulation for development into proinflammatory Th1 effectors, and interventions that target CD4(+) T cell TLR-2-mediated responses may be exploited to enhance neonatal adaptive immunity.

Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing

Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.

T cell-suppression during acute Toxoplasma gondii infection does not involve APC and is a consequence of Treg-mediated IL-2 deprivation and death of proliferating T cells (P1279)

Abstract Foxp3+ Treg (Treg) mediate the suppression of T-cell proliferation observed during the acute Toxoplasma gondii infection in mice. This suppression in T-cell proliferation is related to an IL-2 mechanism. We studied the suppression mechanisms of Treg on T cells in a model of toxoplasmosis using transgenic Foxp3EGFP mice. We analyzed if APC were involved during Treg-suppression of T-cell. Removal of APC from spleen cells from infected mice did not modify suppression and further elimination of Treg led to a complete restored proliferation, demonstrating that APC are not involved in Treg-mediated suppression. Low IL-2 levels were detected in supernatants of APC-free T cells from infected mice. IL-2 levels and T-cell proliferation were restored when Treg and T cells were separated by transwells, indicating that Treg require contact to induce suppression. Although Treg did not inhibit il-2 mRNA levels in T cells, they were able to reduce proliferation of CTLL-2 cells in the IL-2 bioassay, strongly suggesting that Treg act by IL-2 deprivation. Kinetic analysis of T cell proliferation showed that T cells from infected mice die after a few rounds of division in vitro, but addition of rIL-2 or removal of Treg abolished this effect. We conclude that suppression of T-cell proliferation during acute T. gondii infection is mediated by Treg in the absence of APC by IL-2 deprivation, requiring cell contact between T cells and Treg, thus leading to death of proliferating T cells.

Intermediate Filaments: Not Just for Structure Anymore

A recent paper has identified the tumor suppressor APC as a linker protein between intermediate filaments and microtubules. In the absence of APC, intermediate filaments collapse and the cells are no longer polarized and fail tomigrate.

Importance of TLR2 in the direct response of T lymphocytes to Schistosoma mansoni antigens

The immunomodulatory effect of Schistosoma mansoni antigens has often been attributed to interaction with PRR expressed on APC. Our previous work has shown that S. mansoni-soluble egg antigen (SEA) can induce, together with a Th2 response, TGF-β-dependent Foxp3 expression in naïve CD4(+) T cells from NOD mice. We found that SEA can directly upregulate the expression of surface-bound TGF-β in purified CD4(+) T cells in the absence of accessory cell interactions. In this study, we show that the C-type lectin receptors DEC-205 and galectin-3 were involved in the direct interaction between S. mansoni antigens and CD4(+) T cells. SEA was able to enhance CD4(+) T-cell secretion of bioactive TGF-β in response to TLR2 ligand stimulation, in the absence of APC. We also show that TLR2 expressed on CD4(+) T cells was important for the Foxp3 expression induced by SEA.

Abstract C150: SOX9 modulates Wnt/beta-catenin singal in cancer cells by regulating TCF4 and LRP6 expression

Abstract Introduction: Cancers frequently exploit developmental genes/pathways to sustain their uncontrolled growth or invasion. SOX9, an important developmental transcription factor is one of the signature genes of the basal-like breast cancers. SOX9 over-expression has been demonstrated in various cancers and is associated with a poor prognosis. In prostate cancer, SOX9 supports tumor proliferation, angiogenesis and local invasion in xenografts. The role of SOX9 as a biomarker and molecular mechanisms of its biological functions were further explored in this study. Methods: Immunohistochmical analysis of SOX9 protein expression was performed on a tissue microarray of 129 breast cancer samples. Gene profiles of the breast cancer samples were examined by ANOVA analysis to identify genes that are expressed at significantly higher level among SOX9-high protein expressing samples. SOX9 regulated genes were validated by their corresponding changes in expression levels to SOX9 silencing in cancer cell lines. The effect of SOX9 silencing on cancer progression was examined in the MCF10A DCIS.com xenografts. Results: SOX9 protein expression was highly represented in the basal-like breast cancers and was associated with poor tumor grades. Among the genes correlated with higher SOX9 expression were two major components of the canonical Wnt/ -catenin signal pathway, ie TCF4 and LRP6. These genes were up- or down-regulated in various cancer cell lines in response to SOX9 overexpression or silencing. More importantly, the Wnt3a induced -catenin activity was significantly reduced when SOX9 protein was specifically down-regulated. SOX9 down-regulation in MCF10A DCIS xenografts lead to a squamous differentiation and a block in the progression from DCIS to invasive carcinoma. Conclusion: This study identified a novel function of SOX9 in modulating Wnt/β-catenin signaling by regulating TCF4 and LRP6 expression. SOX9 enhanced Wnt/β-catenin activity in the absence of APC or β-catenin mutations may play an important role in determining tumor subtype, maintaining tumor stem cells or supporting tumor growth or progression. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C150.

Engagement of TLR2 Does not Reverse the Suppressor Function of Mouse Regulatory T Cells, but Promotes Their Survival

TLRs are a class of conserved pattern recognition receptors that are used by cells of the innate immune system. Recent studies have demonstrated the expression of TLRs on both human and mouse T cells raising the possibility that TLRs play a direct role in adaptive immunity. TLR2 is activated primarily by bacterial wall components including peptidoglycan and lipoproteins. Several studies have shown that mouse regulatory T (Treg) cells express TLR2 and claimed that engagement of TLR2 by synthetic ligands reversed their suppressive function. In contrary, enhancement of Treg function was observed following engagement of TLR2 on human Treg. We have reexamined the expression and function of TLR2 on mouse Treg purified from Foxp3-GFP knock-in mice. TLR2 ligation by TLR2 agonist, the synthetic bacterial lipoprotein Pam3CSK4, enhanced the proliferative responses of both conventional T cells and Treg in response to TLR stimulation in the absence of APC. Treatment of Foxp3+ Treg with Pam3CSK4 did not alter their suppressive function in vitro or in vivo and did not reduce their level of Foxp3 expression. An additional effect of TLR2 stimulation of Treg was induction of Bcl-x(L) resulting in enhanced survival in vitro. Treatment of mice with the TLR2 agonist enhanced the Ag-driven proliferation of Treg in vivo, but did not abolish their ability to suppress the development of experimental autoimmune encephalomyelitis. Development of methods to selectively stimulate TLR2 on Treg may lead to a novel approaches for the treatment of autoimmune diseases.

IL‐10 interferes directly with TCR‐induced IFN‐γ but not IL‐17 production in memory T cells

IL-10 is a potent immunoregulatory and anti-inflammatory cytokine. However, therapeutic trials in chronic inflammation have been largely disappointing. It is well established that IL-10 can inhibit Th1 and Th2 cytokine production via indirect effects on APC. Less data are available about the influence of IL-10 on IL-17 production, a cytokine which has been recently linked to chronic inflammation. Furthermore, there are only few reports about a direct effect of IL-10 on T cells. We demonstrate here that IL-10 can directly interfere with TCR-induced IFN-gamma production in freshly isolated memory T cells in the absence of APC. This effect was independent of the previously described effects of IL-10 on T cells, namely inhibition of IL-2 production and inhibition of CD28 signaling. In contrast, IL-10 did not affect anti-CD3/anti-CD28-induced IL-17 production from memory T cells even in the presence of APC. This might have implications for the interpretation of therapeutic trials in patients with chronic inflammation where Th17 cells contribute to pathogenesis.

APC ain't always necessary for axons

Like many neuroscientists, Rusan et al. considered that the mechanism controlling axonal positioning and outgrowth might be the same in all neurons. As they now show, however, one neuron's must-have axon-promoting protein is virtually dispensable in other neurons. Figure 1 A central brain neuron grows a perfectly good axon even in the absence of APC. In a young neuron, multiple mini neurites protrude until one is chosen to become the axon. A microtubule-associated protein called APC was thought to be required for axon outgrowth, as dominant-negative APC expression in cultured neurons reduced axon growth dramatically. Other reports had suggested that APC delivers a polarity protein called Par3, necessary for axon growth, to the chosen neurite. Despite the strong evidence for APC's axon-promoting role, definitive knockout experiments in neurons were limited to one study, which showed that medulla neurons behaved much as expected—no APC, no axon extension. Now, the same lab have found that neurons from the central brain, mushroom body, and lobular plug will happily grow axons without APC. Cells from these parts of the brain were not entirely normal: precursor neuroblasts had slightly longer cell cycles and some had bent spindles. Though, neither problem prevented mitosis. The need for APC in some neurons but not others seems to reflect a requirement for Wnt signaling. As well as its cytoskeletal role, APC is a downstream regulator of Wnt, and while medulla neurons need Wnt for axon growth, the other types of neurons did not. It will be of interest to check whether hippocampal neurons, where the dominant-negative experiments were carried out, are also Wnt responsive. Rusan, N.M., et al. 2008. J. Cell Biol. .10.1083/jcb.200807079 [PubMed] [Cross Ref]

Activated protein C inhibits chemotaxis and interleukin-6 release by human neutrophils without affecting other neutrophil functions

BackgroundActivated protein C (APC) therapy reduces mortality in high-risk patients with severe sepsis. The effects of APC on inflammatory responses have also been reported. Neutrophils are key cells involved in early host defence mechanisms in sepsis. We hypothesized that APC may have effects on neutrophil function. MethodsNeutrophils were isolated from 10 healthy volunteers and incubated in the presence of lipopolysaccharide (LPS) with and without a range of therapeutically relevant concentrations of recombinant human APC. Respiratory burst activity was determined using flow-activated cell sorting (FACS) analysis. Apoptosis was determined using Annexin-V staining and FACS analysis. Cytokine bead array was used to simultaneously measure three key cytokines in culture supernatants: interleukin (IL)-1β, −6, and −8. For chemotaxis, neutrophil migration through a 5 μm membrane was measured in response to formyl–methyl–leucine–phenylalanine (FMLP) or IL-8 in the presence and absence of APC. ResultsExposure to LPS resulted in significant increases in respiratory burst activity, IL-1β, −6, and −8 expression (all P<0.0001) and decreased the number of apoptotic cells (P<0.0001). The APC exposure resulted in a significant release of IL-6 (P=0.04) without affecting other cytokines. Respiratory burst and apoptosis were also unaffected by APC. Neutrophil chemotaxis in response to either FMLP or IL-8 was reduced by APC (P=0.005 and 0.007, respectively). ConclusionsThis pilot study showed that APC treatment of human neutrophils results in a decreased IL-6 expression and chemotaxis, without affecting other cytokines, apoptosis, or respiratory burst activity.

Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17

Recent evidence from several groups indicates that IL-17-producing Th17 cells, rather than, as once was thought, IFN-gamma-producing Th1 cells, can represent the key effector cells in the induction/development of several autoimmune and allergic disorders. Although Th17 cells exhibit certain phenotypic and developmental differences from Th1 cells, the extent of the differences between these two T cell subsets is still not fully understood. We found that the expression profile of cell surface molecules on Th17 cells has more similarities to that of Th1 cells than Th2 cells. However, although certain Th1-lineage markers [i.e., IL-18 receptor alpha, CXCR3, and T cell Ig domain, mucin-like domain-3 (TIM-3)], but not Th2-lineage markers (i.e., T1/ST2, TIM-1, and TIM-2), were expressed on Th17 cells, the intensity of expression was different between Th17 and Th1 cells. Moreover, the expression of CTLA-1, ICOS, programmed death ligand 1, CD153, Fas, and TNF-related activation-induced cytokine was greater on Th17 cells than on Th1 cells. We found that IL-23 or IL-17 can suppress Th1 cell differentiation in the presence of exogenous IL-12 in vitro. We also confirmed that IL-12 or IFN-gamma can negatively regulate Th17 cell differentiation. However, these cytokines could not modulate such effects on T cell differentiation in the absence of APC.

Cyclin D1 Is Not an Immediate Target of β-Catenin following Apc Loss in the Intestine

Cyclin D1 is postulated to be a target of the canonical Wnt pathway and critical for intestinal adenoma development. We show here that, unlike cyclin D1 reporter assays, endogenous cyclin D1 levels are not affected following antagonism of the Wnt pathway in vitro, nor is cyclin D1 immediately up-regulated following conditional loss of Apc in vivo. Cyclin D1 levels do, however, increase in a delayed manner in a small subset of cells, suggesting such up-regulation occurs as a secondary event. We also analyzed the immediate consequences of Apc loss in a cyclin D1(-/-) background and failed to find any cyclin D1-dependent phenotypes. However, we did observe elevated cyclin D1 expression in lesions developing 20 days after Apc loss. In these circumstances, all adenomas (but not smaller lesions) showed cyclin D1 up-regulation. Finally in a smaller study, we analyzed whether cyclin D1 deficiency affected adenoma formation 20 days following induced loss of Apc. Unlike AhCre(+) Apc(fl/fl) mice (which all developed adenomas), doubly mutant AhCre(+) Apc(fl/fl) cyclin D1(-/-) mice only developed small lesions. Taken together, this argues that cyclin D1 up-regulation in intestinal neoplasia is important for tumor progression rather than initiation.

TGF-β1 Uses Distinct Mechanisms to Inhibit IFN-γ Expression in CD4+ T Cells at Priming and at Recall: Differential Involvement of Stat4 and T-bet

TGF-beta1 plays a critical role in restraining pathogenic Th1 autoimmune responses in vivo, but the mechanisms that mediate TGF-beta1's suppressive effects on CD4(+) T cell expression of IFN-gamma expression remain incompletely understood. To evaluate mechanisms by which TGF-beta1 inhibits IFN-gamma expression in CD4(+) T cells, we primed naive wild-type murine BALB/c CD4(+) T cells in vitro under Th1 development conditions in the presence or the absence of added TGF-beta1. We found that the presence of TGF-beta1 during priming of CD4(+) T cells suppressed both IFN-gamma expression during priming as well as the development of Th1 effector cells expressing IFN-gamma at a recall stimulation. TGF-beta1 inhibited the development of IFN-gamma-expressing cells in a dose-dependent fashion and in the absence of APC, indicating that TGF-beta1 can inhibit Th1 development by acting directly on the CD4(+) T cell. During priming, TGF-beta1 strongly inhibited the expression of both T-bet (T box expressed in T cells) and Stat4. We evaluated the importance of these two molecules in the suppression of IFN-gamma expression at the two phases of Th1 responses. Enforced expression of T-bet by retrovirus prevented TGF-beta1's inhibition of Th1 development, but did not prevent TGF-beta1's inhibition of IFN-gamma expression at priming. Conversely, enforced expression of Stat4 partly prevented TGF-beta1's inhibition of IFN-gamma expression during priming, but did not prevent TGF-beta1's inhibition of Th1 development. These data show that TGF-beta1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4(+) T cells at priming and at recall.

Direct processing and presentation of antigen from malaria sporozoites by professional antigen‐presenting cells in the induction of CD8+ T‐cell responses

Irradiated malaria sporozoites induce better protection than viable untreated sporozoites. We observed early differences between irradiated and viable untreated sporozoites in priming responses in vivo to a protective CD8 T-cell epitope, pb9, of the circumsporozoite protein of Plasmodium berghei. Sporozoites were processed for MHC class I presentation by dendritic cells (DC) to prime pb9-specific IFN-gamma-producing CD8 T cells. DC pulsed with untreated and irradiated sporozoites were similarly capable of priming central memory T-cell responses, detectable by the IFN-gamma cultured ELISPOT assay. However, irradiation significantly enhanced sporozoites' ability to prime effector T-cell responses detectable by the IFN-gammaex vivo ELISPOT assay. Irradiation also enhanced the ability of splenic APC to process and present sporozoites in order to re-stimulate pb9-specific polyclonal and clonal T-cell responses. Sporozoites did not stimulate T cells in the absence of APC. Over-irradiation decreased the sporozoites' T-cell stimulating capacity in vitro at high parasite doses, which may indicate that an optimal irradiation dose is necessary to induce protective immunity by sporozoite inoculation. The induction of sporozoite-specific CD8 T-cell responses without the need for liver stage infection identifies a potentially important mechanism in the development of pre-erythrocytic immunity.