Two unique HLA-A*0201 restricted peptides derived from cyclin E as immunotherapeutic targets in leukemia.

Immunotherapy targeting leukemia-associated antigens has shown promising results. Because of the heterogeneity of leukemia, vaccines with a single peptide have elicited only a limited immune response. Targeting several peptides together elicited peptide-specific cytotoxic T lymphocytes (CTLs) in leukemia patients, and this was associated with clinical responses. Thus, the discovery of novel antigens is essential. In the current study, we investigated cyclin E as a novel target for immunotherapy. Cyclin E1 and cyclin E2 were found to be highly expressed in hematologic malignancies, according to reverse transcription polymerase chain reaction and western blot analysis. We identified two HLA-A*0201 binding nonameric peptides, CCNE1M from cyclin E1 and CCNE2L from cyclin E2, which both elicited the peptide-specific CTLs. The peptide-specific CTLs specifically kill leukemia cells. Furthermore, CCNE1M and CCNE2L CTLs were increased in leukemia patients who underwent allogeneic hematopoietic stem cell transplantation, and this was associated with desired clinical outcomes. Our findings suggest that cyclin E1 and cyclin E2 are potential targets for immunotherapy in leukemia.

Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis.

Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions.

[Gateway Reflex, a regulator of the inflammation feedback loop by regional neural activation].

Inflammation is observed in many diseases and disorders. We discovered a key machinery of inflammation, the inflammation amplifier, which is induced by the simultaneous activation of NFκB and STAT3 followed by the hyper-activation of NFκB in non-immune cells, including endothelial cells and fibroblasts. Since that discovery, we found the Gateway Reflex, which describes regional neural activations that enhance the inflammation amplifier to create a gateway for immune cells to bypass the blood-brain barrier. In addition, we have identified over 1,000 positive regulators and over 500 targets of the inflammation amplifier, which include a significant numbers of human disease-associated genes. In parallel, we performed a comprehensive analysis of human disease samples and found that the inflammation amplifier was activated during the development of chronic inflammation. Thus, we concluded that the inflammation amplifier is associated with various human diseases and disorders, including autoimmune diseases, metabolic syndromes, neurodegenerative diseases, and other inflammatory diseases. We are now attempting drug discovery for inflammatory diseases and disorders based on the inflammation amplifier and Gateway Reflex. In this review, we discuss the Gateway Reflex as an example for the neuro-immune interaction in vivo.

The thymus in immunity and in malignancy.

The thymus is an essential organ for the generation of the adaptive immune system. By now, the cellular selection events taking place in ongoing life before sexual maturity have been worked out even at the molecular level, and thus thymic lymphocyte development represents one of the best-studied systems in mammalian development. Because thymic lymphocyte development involves ample proliferation and generation of new cells, it is not astonishing that the thymus also represents an organ where malignancy can develop. In this Masters of Immunology primer, the development of lymphocytes and the role of intracellular Notch 1 and cyclins in lymphocytic malignancy are reviewed, offering new therapeutic possibilities.

Serum anti-CCNY autoantibody is an independent prognosis indicator for postoperative patients with early-stage nonsmall-cell lung carcinoma.

Cyclin Y (CCNY) is a novel cyclin and almost nothing is known about its role in human cancers. To investigate the clinical significance of serum anti-CCNY autoantibodies in nonsmall-cell lung carcinoma (NSCLC), the serum levels of CCNY protein in 264 patients with NSCLC, 103 patients with tuberculosis, and 89 healthy controls were analyzed by immunohistochemistry. The result shows that, compared with normal lung tissues, the NSCLC tissues contained higher levels of CCNY protein. The levels of anti-CCNY autoantibodies were higher in the sera of the patients with NSCLC than in the sera of the healthy controls (P < 0.001) or the patients with tuberculosis (P = 0.027). Moreover, in a Cox regression analysis, anti-CCNY autoantibody was an independent factor that predicted poor prognosis for postoperative patients with early-stage NSCLC (P = 0.026) as well as for those with distant metastasis (P = 0.012). Our data indicated that Anti-CCNY autoantibody may be useful as a latent tumor marker to facilitate diagnosis and may represent a novel prognostic indicator for patients with early stage NSCLC.

The cyclins: a family of widely expressed tumor antigens?

Continuous cell division is a hallmark of cancer and cell-cycle regulators therefore represent relevant target molecules for tumor therapy. Among these targets the cyclins are of particular interest as they are overexpressed in various tumor entities with little expression in normal tissue. Here we review evidence that these molecules are recognized by the immune system, summarize why cyclins A, B and D in particular appear to be interesting targets for active and passive immunotherapy, and discuss whether the entire family could be an interesting novel class of tumor antigens for cancer treatment and prevention.

p35, the non-cyclin activator of Cdk5, protects podocytes against apoptosis in vitro and in vivo.

Cyclin-dependent kinase-5 is widely expressed and predominantly regulated by the non-cyclin activator p35. Since we recently showed that expression of p35 in the kidney is restricted to podocytes, we examined here its function in mice in which p35 was genetically deleted. The mice did not exhibit kidney abnormalities during glomerular development or during adult life. Conditionally immortalized cultured podocytes, derived from these null mice, did not have any change in their morphology, differentiation, or proliferation. However, when these cultured podocytes were exposed to UV-C irradiation, serum depletion, puromycin aminonucleoside, or transforming growth factor-beta-1, they showed increased apoptosis compared to those from wild-type mice. Levels of Bcl-2 were decreased in these null podocytes but increased after transduction with human p35. Restoration of p35 or the ectopic expression of Bcl-2 reduced the susceptibility of p35-null podocytes to apoptosis. Experimental glomerulonephritis, characterized by podocyte apoptosis and subsequent crescent formation, was utilized to test these findings in vivo. Podocyte apoptosis was significantly increased in diseased p35-null compared with wild-type mice, accompanied by increased glomerulosclerosis and decreased renal function. Our study shows that p35 does not affect glomerulogenesis but controls podocyte survival following injury, in part, by regulating Bcl-2 expression.

Ras orchestrates exit from the cell cycle and light-chain recombination during early B cell development.

Signals through the pre-B cell antigen receptor (pre-BCR) and interleukin 7 receptor (IL-7R) coordinate pre-B cell population expansion with subsequent recombination of the locus encoding immunoglobulin kappa-chain (Igk). Although many 'downstream' effectors of each receptor are known, how they integrate to mediate development has remained unclear. Here we report that pre-BCR-mediated activation of the Ras-MEK-Erk signaling pathway silenced transcription of Ccnd3 (encoding cyclin D3) and coordinated exit from the cell cycle with induction of the transcription factor E2A and the initiation of Igk recombination. IL-7R-mediated activation of the transcription factor STAT5 opposed this pathway by promoting Ccnd3 expression and concomitantly inhibiting Igk transcription by binding to the Igk intronic enhancer and preventing E2A recruitment. Our data show how pre-BCR signaling poises pre-B cells to undergo differentiation after escape from IL-7R signaling.

Measurement of changes in Cdk2 and cyclin o-associated kinase activity in apoptosis.

Many cell cycle regulatory proteins have been shown to be able to regulate cell death. Activation of Cdk2 has been shown to be necessary for apoptosis of quiescent cells such as thymocytes, neurons, and endothelial cells. This activation is stimulus-specific because it occurs in glucocorticoid and DNA damage but not in CD95-induced apoptosis in thymocytes. Apoptotic Cdk2 activation in lymphoid cells is controlled by a recently identified protein, cyclin O, and its activity is modulated by p53 and members of the Bcl-2 protein family. In this chapter, we describe methods for measuring changes in Cdk2 activity during apoptosis. In addition, we also show the details of the generation of an antibody able to immunoprecipitate the cyclin O complexes from apoptotic cells in native conditions and its use to measure the kinase activity associated with this proapoptotic cyclin.

Foxo3-/- mice demonstrate reduced numbers of pre-B and recirculating B cells but normal splenic B cell sub-population distribution.

B cell antigen receptor (BCR) cross-linking promotes proliferation and survival of mature B cells. Phosphoinositide-3-kinase-mediated down-regulation of pro-apoptotic and anti-mitogenic genes such as the Foxo family of transcription factors is an important component of this process. Previously, we demonstrated that BCR signaling decreases expression of transcripts for Foxo1, Foxo3 and Foxo4. We now show that BCR-induced down-regulation of Foxo3 and Foxo4 mRNA expression occurs via distinct mechanisms from those established for Foxo1. While Foxo1, Foxo3 and Foxo4 bind the same DNA sequence, the differential control of their expression upon B cell activation suggests that they may have unique functions in the B lineage. To begin to address this issue, we evaluated B cell development and function in Foxo3-/- mice. No effect of Foxo3 deficiency was observed with respect to the following parameters in the splenic B cell compartment: sub-population distribution, proliferation, in vitro differentiation and expression of the Foxo target genes cyclin G2 and B cell translocation gene 1. However, Foxo3-/- mice demonstrated increased basal levels of IgG2a, IgG3 and IgA. A significant reduction in pre-B cell numbers was also observed in Foxo3-/- bone marrow. Finally, recirculating B cells in the bone marrow and peripheral blood were decreased in Foxo3-/- mice, perhaps due to lower than normal expression of receptor for sphingosine-1 phosphate, which mediates egress from lymphoid organs. Thus, Foxo3 makes a unique contribution to B cell development, B cell localization and control of Ig levels.

Saikosaponin a inhibits the proliferation and activation of T cells through cell cycle arrest and induction of apoptosis.

In the present study, we aimed at examining the immunosuppressive activity of saikosaponin a, a triterpene saponin derived from Bupleurum falcatum L. (Umbelliferae), and the underlying mechanisms. Saikosaponin a significantly inhibited the proliferation and activation of T cells activated by concanavalin A (Con A) in a concentration-dependent manner. Additionally, it potently suppressed Con A-stimulated IL-2, IFN-gamma and TNF-alpha production in mouse T cells. Saikosaponin a also caused G0/G1 arrest of activated T cells through down-regulating the protein levels of CDK6 and Cyclin D3 and up-regulating the protein level of p27(kip). Furthermore, the compound dose-dependently induced apoptosis of Con A-activated T cells rather than those non-activated, as determined by Annexin V/PI staining. Besides, it induced a remarkable collapse of mitochondrial membrane potential and caused significant release of cytochrome c from mitochondria to cytosol. In summary, these results suggest that the G0/G1 arrest as well as the induction of apoptosis via mitochondrial pathway are involved in the immunosuppressive activity of saikosaponin a against activated T cells. This may herald a novel approach for further studies of saikosaponin a as a candidate for the treatment of inflammatory and autoimmune diseases.

Using CD40-activated B cells to efficiently identify epitopes of tumor antigens.

The rapid development of genomics and proteomics has accelerated the discovery of antigens that play a role in host-tumor interaction and can be potentially targeted in tumor immunotherapy. Several independent approaches to characterize such antigens and identify the relevant epitopes have been developed. However, the detection, expansion, and characterization of antigen-specific T cells are essential steps common to all strategies. Efficient identification of epitopes, in particular in a preclinical setting, is often hampered by lack of significant numbers of antigen presenting cells at sufficient purity that readily expand low-frequency T-cell precursors. Using the cylins as a model family of self-tumor antigens, we show that CD40-activated primary human B cells can be used very efficiently to identify novel epitopes and characterize such tumor antigen-specific T cells.

Cell cycle arrest effects of large-dose FTY720 on lymphocytes in mouse skin transplantation models.

To probe into the mechanism of immunosuppression of FTY720, the authors study the cell cycle arrest effects of large-dose FTY720 on lymphocytes in mouse skin transplantation models and the expression of cell cycle-related factors in cell cycle regulation system in mouse skin allograft models using flow cytometry, Immunohistochemical staining, and reverse transcriptase-polymerase chain reaction (RT-PCR). FTY720 could prolong survival days of graft in mouse skin transplantation models obviously (p < 0.01). In thymus gland and lymph node, compared with the control group, cell percentage in G0-G1 increases and cell percentage in G2-M and S decreases in the treated group (p < 0.01). Expression of P16 increases and expression of cyclin D1 decreases in the treated group (p < 0.05). In thymus gland, it is shown by semiquantitative RT-PCR that the quantity of mRNA of P16 increases in the treated group (p < 0.01), and the quantity of mRNA of cyclin D1 decreases in the treated group (p < 0.01). FTY720 is a kind of effective immunosuppressant. FTY720 could induce cell cycle arrest in thymus gland and lymph node and change the expression of protein and the transcription of mRNA of cell cycle-related factor.

Deacetylase activity is required for STAT5-dependent GM-CSF functional activity in macrophages and differentiation to dendritic cells.

After interaction with its receptor, GM-CSF induces phosphorylation of the beta-chain in two distinct domains in macrophages. One induces activation of mitogen-activated protein kinases and the PI3K/Akt pathway, and the other induces JAK2-STAT5. In this study we describe how trichostatin A (TSA), which inhibits deacetylase activity, blocks JAK2-STAT5-dependent gene expression but not the expression of genes that depend on the signal transduction induced by the other domain of the receptor. TSA treatment inhibited the GM-CSF-dependent proliferation of macrophages by interfering with c-myc and cyclin D1 expression. However, M-CSF-dependent proliferation, which requires ERK1/2, was unaffected. Protection from apoptosis, which involves Akt phosphorylation and p21(waf-1) expression, was not modified by TSA. GM-CSF-dependent expression of MHC class II molecules was inhibited because CIITA was not induced. The generation of dendritic cells was also impaired by TSA treatment because of the inhibition of IRF4, IRF2, and RelB expression. TSA mediates its effects by preventing the recruitment of RNA polymerase II to the promoter of STAT5 target genes and by inhibiting their expression. However, this drug did not affect STAT5A or STAT5B phosphorylation or DNA binding. These results in GM-CSF-treated macrophages reveal a relationship between histone deacetylase complexes and STAT5 in the regulation of gene expression.

Vitamin A potentiates CpG-mediated memory B-cell proliferation and differentiation: involvement of early activation of p38MAPK.

Foreign CpG-DNA from viruses and bacteria can activate memory B cells through binding to toll-like receptor 9, and this pathway has been hypothesized to be involved in the continuous activation of memory B cells ensuring life-long humoral immunity. In this study, we demonstrate that retinoic acid (RA) is a potent coactivator of this pathway in human B cells. RA enhanced the CpG-mediated proliferation of CD27(+) memory B cells, and the proliferative response was accompanied by increased immunoglobulin (Ig) secretion indicative of plasma-cell formation. The RA-induced proliferation was preceded by enhanced expression of cyclin D3, and both the expression of cyclin D3 and the induced Ig secretion were found to be dependent on IL-10. Of importance, RA increased the CpG-induced phosphorylation of ERK1/2, p38MAPK, and IkappaB as early as 30 minutes after stimulation. By using specific inhibitors, all the RA-mediated events, including proliferation, cyclin D3 expression, IL-10 secretion, and Ig secretion, were shown to be dependent on p38MAPK. Hence, we propose that RA can strengthen humoral immunity by promoting CpG-mediated stimulation of CD27(+) B cells via activation of p38MAPK resulting in increased proliferation and differentiation to Ig-secreting plasma cells.

The immunohistochemical outline of p27kip1, cyclin B1 and cyclin D3 in pleomorphic adenomas.

AIM: Pleomorphic adenomas of salivary glands are benign lesions which may sometimes relapse even after complete surgical removal. This risk has led to the search for methods to provide predictive data on the biological behaviour of such neoplasia. The authors intend to evaluate the degree of cellular aggression of these tumours by finding prognostic data using the antigens involved in cellular proliferative activity. Therefore they have chosen for this study: p27kip1, cyclin B1 and Cyclin D3. METHODS: Seventeen mixed tumours, 2 of them relapsed, underwent the direct immunohystochemical PAP technique for the determination of antigens p27kip1, cyclins B1 and D3 of the tissue. RESULTS: The results obtained show that the verification of these markers may reveal a potential risk of biological deviation and that their expression is independent of the degree of cellularity in neoplasias. CONCLUSIONS: On the basis of the results, the conclusion is drawn that there is no relation between the expressivity of the mentioned antigens and histological characters of pleomorphic adenomas.

Defective cell cycle induction by IL-2 in naive T-cells antigen stimulated in the presence of refractory T-lymphocytes.

CD4+ T cells enter a transient refractory period after stimulation. Upon re-stimulation the refractory cells produce little IL-2 and show diminished proliferation. We previously demonstrated that refractory T cells can also, like anergic and CD4+CD25+ regulatory T cells, suppress in trans the proliferation of antigen-stimulated naive T cells. The suppressed T cells up-regulate high-affinity IL-2R but do not produce IL-2. This IL-2 deficit could potentially explain the proliferation failure, but does not appear to do so. Supplementation of refractory-naive co-cultures with exogenous IL-2 fails to alleviate both the proliferation suppression and IL-2 production defects. This does not result from a failure of IL-2 to stimulate its receptor. Proximal IL-2 signaling into suppressed T cells through STAT5 and Akt is intact. However, refractory cell-co-cultured T cells fail to up-regulate cyclins and c-myc and incompletely down-regulate p27kip1 in response to IL-2, and the downstream consequences of this signaling are therefore dissociated. IL-2 signaling is not fully disabled as IL-2 up-regulates the anti-apoptotic protein Bcl-xL to control levels. This up-regulation correlates with enhanced survival of refractory cell-co-cultured T cells placed in IL-2 when compared with cells cultured without IL-2. Thus, refractory T cells are able to suppress naive T-cell proliferative responses in part by blocking both IL-2 production and the mitogenic but not anti-apoptotic effects of IL-2. These results have implications for how activation-refractory T cells may influence nascent immune responses.

Cyclin D(1) and D(3) expression in vestibular schwannomas.

OBJECTIVES: The G1 regulators of the cell cycle, cyclin D(1) and D(3), have been implicated in the regulation of Schwann cell proliferation and differentiation. The purpose of this study is to evaluate cyclin D(1) and D(3) protein expression and the corresponding clinical characteristics of vestibular schwannomas. STUDY DESIGN AND METHODS: Tissue sections of 15 sporadic vestibular schwannomas were prepared. Immunohistochemical analysis of the vestibular schwannomas was performed with anticyclin D(1) and anticyclin D(3) antibodies. The immunoreactivity was evaluated in comparison with adjacent vestibular nerves. Tissue sections of breast carcinoma and prostate carcinoma were used as positive controls for cyclin D(1) and D(3) staining, respectively. Patient demographics, tumor characteristics, and cyclin D expression were reviewed, and statistical analysis was performed. RESULTS: While the breast carcinoma control expressed abundant cyclin D(1) protein, none of the 15 vestibular schwannomas showed detectable cyclin D(1) staining. In contrast, seven of 15 vestibular schwannomas stained positive for the cyclin D(3) protein. Cyclin D(3) staining was taken up in the nucleus of schwannoma tumor cells in greater proportion than Schwann cells of adjacent vestibular nerve. Although sample size was small, no significant difference in the average age of presentation, tumor size, and male to female ratios for the cyclin D(3)(+) or cyclin D(3)(-) groups was found. CONCLUSION: The Cyclin D(1) protein does not appear to play a prominent role in promoting cell cycle progression in vestibular schwannomas. In contrast, cyclin D(3) expression was seen in nearly half of the tumors examined, suggesting that it may have a growth-promoting role in some schwannomas. Further studies are needed to define its cellular mechanism.