Interplays between mouse mammary tumor virus and the cellular and humoral immune response.

Mouse mammary tumor virus has developed strategies to exploit the immune response. It requires vigorous immune stimulation to achieve efficient infection. The infected antigen-presenting cells present a viral superantigen on the cell surface which stimulates strong CD4-mediated T-cell help but CD8 T-cell responses are undetectable. Despite the high frequency of superantigen-reactive T cells, the superantigen-induced immune response is comparable to classical antigen responses in terms of T-cell priming, T-cell-B-cell collaboration as well as follicular and extra-follicular B-cell differentiation. Induction of systemic anergy is observed, similar to classical antigen responses where antigen is administered systemically but does not influence the role of the superantigen-reactive T cells in the maintenance of the chronic germinal center reaction. So far we have been unable to detect a cytotoxic T-cell response to mouse mammary tumor virus peptide antigens or to the superantigen. This might yet represent another step in the viral infection strategy.

Transplantation tolerance: Clinical potential of regulatory T cells.

The major challenge in transplantation medicine remains long-term allograft acceptance, with preserved allograft function under minimal chronic immunosuppression. To safely achieve the goal of sustained donor-specific T and B cell non-responsiveness, research efforts are now focusing on therapies based on cell subsets with regulatory properties. In particular the transfusion of human regulatory T cells (Treg) is currently being evaluated in phase I/II clinical trials for the treatment of graft versus host disease following hematopoietic stem cell transplantation, and is also under consideration for solid organ transplantation. The purpose of this review is to recapitulate current knowledge on naturally occurring as well as induced human Treg, with emphasis on their specific phenotype, suppressive function and how these cells can be manipulated in vitro and/or in vivo for therapeutic purposes in transplantation medicine. We highlight the potential but also possible limitations of Treg-based strategies to promote long-term allograft survival. It is evident that the bench-to-beside translation of these protocols still requires further understanding of Treg biology. Nevertheless, current data already suggest that Treg therapy alone will not be sufficient and needs to be combined with other immunomodulatory approaches in order to induce allograft tolerance.

Molecular mechanisms of follicular associated epithelium differentiation

Les muqueuses respiratoires, genitales et digestives sont continuellement exposées aux antigènes de l?alimentation, à la flore intestinale et aux pathogènes. Cela implique une activité immunologique intense et finement régulée dans ces tissus. On admet que la modulation de ces réponses immunitaires muqueuses s?effectue dans des organes sentinels spécifiques appelés o-MALT (organized mucosal associated lymphoid tissues). Ces processus de modulation et la biologie de ces sites immuno-inducteurs sont peu connus. Ceci est pourtant d?une grande relevance si l?on veut faire un design rationnel de drogues et de vaccins muqueux. Dans l?intestin grèle, ces organes sont composés de follicules multiples et sont appelés plaques de Peyer. Ils sont constitués de follicules enrichis en cellules B comprenant ou non un centre germinatif, de regions interfolliculaires comprenant des cellules T, et d?une région en d ome riche en cellules dendritiques, cellules B naives et cellules T CD4+, surmontée par un epithelium specialisé, le FAE (epithelium associé aux follicules). Le FAE contient des cellules M spécialisées dans le transport de macromolécules et micro-organismes de la lumière intestinale au tissu lymphoide sous-jacent. Ce transport des antigènes est une condition obligatoire pour induire une réponse immunitaire. Les cellules du FAE, outre les cellules M, expriment un programme de différenciation distinct de celui des cellules associées aux villosités. Ceci est characterisé par une baisse des fonctions digestives et de défenses, et l?expression constitutive des chimiokines: CCL20 et CCL25. Le but de l?étude présentée ici est de rechercher les facteurs cellulaires et/ou moléculaire responsables de cette différenciation. Certaines études ont démontré l?importance du contact entre le compartiment mésenchymateux et l?épithelium pour la morphogenèse de ce dernier. En particulier, les molécules de la matrice extracellulaire peuvent activer des gènes clefs qui, à leur tour, vont controler l?adhésion et la differenciation cellulaire. Dans l?intestin, les cellules mésenchymateuses différencient en myofibroblastes qui participent à l?élaboration de la matrice extracellulaire. Dans cette étude, nous avons décrit les différences d?expression de molécules de la matrices sous le FAE et les villosités. Nous avons également montré une absence de myofibroblastes sous le FAE. Suite à plusieurs évidences expérimentales, certains ont proposé une influence des composés présents dans la lumière sur la différenciation et/ou la maturation des plaques de Peyer. La chimiokine CCL20, capable de recruter des cellules initiatrices de la réponse immunitaire, constitue notre seul marqueur positif de FAE. Nous avons pu montrer que la flagelline, un composé du flagelle bactérien, était capable d?induire l?expression de CCL20 in vitro et in vivo. Cet effet n?est pas limité aux cellules du FAE mais est observé sur l?ensemble de l?épithelium intestinal. Molecular mechanisms of FAE differenciation. La signalement induit par la lymphotoxine ß est critique pour l?organogenèse des plaques de Peyer, car des souris déficientes pour cette molécules ou son récepteur n?ont ni plaque de Peyer, ni la plupart des ganglions lymphatiques. Nous avons obtenus plusieurs évidences que la lymphotoxine ß était impliquée dans la régulation du gène CCL20 in vitro et in vivo.<br/><br/>Mucosal surfaces of the respiratory, genital and digestive systems are exposed to food antigens, normal bacterial flora and oral pathogens. This justifies an intense and tuned immunological activity in mucosal tissues. The modulation of immune responses in the mucosa is thought to occur in specific sentinel sites, the organized mucosa associated lymphoid tissues (o-MALT). This immune modulation and the biology of these immune-inductive sites are poorly understood but highly important and relevant in the case of drugs and vaccines design. In the small intestine, these organs (gut associated lymphoid tissue : GALT) consists of single or multiple lymphoid follicles, the so-called Peyer?s patches (PP), with typical B cell-enriched follicles and germinal centers, inter-follicular T cell areas, and a dome region enriched in dendritic cells, naive B cells, and CD4+ T cells under a specialized follicle associated epithelium (FAE). To trigger protective immunity, antigens have to cross the mucosal epithelial barrier. This is achieved by the specialized epithelial M cells of the FAE that are able to take up and transport macromolecules and microorganisms from the environment into the underlying organized lymphoid tissue. The ontogeny of M cells remains controversial: some data are in favor of a distinct cell lineage, while others provide evidence for the conversion of differentiated enterocytes into M cells. In this study we mapped the proliferative, M cells and apoptotic compartments along the FAE. Enterocytes acquire transient M cell features as they leave the crypt and regain enterocyte properties as they move towards the apoptotic compartment at the apex of the FAE, favouring the hypothesis of a plastic phenotype. The follicle-associated epithelium (FAE) is found exclusively over lymphoid follicles in mucosal tissues, including Peyer?s patches. The enterocytes over Peyer?s patches express a distinct phenotype when compared to the villi enterocytes, characterized by the down regulation of digestive and defense functions and the constitutive expression of chemokines, i.e. CCL20 and CCL25. The purpose of this study was to investigate and identify the potential cells and/or molecules instructing FAE differentiation. Contact between the epithelial and the mesenchymal cell compartment is required for gut morphogenesis. Extracellular matrix molecules (ECM) can activate key regulatory genes which in turn control cell adhesion and differentiation. In the gut, mesenchymal cells differentiate into myofibroblats that participate to the elaboration of ECM. We have described a differential expression of extracellular matrix components under the FAE, correlating with the absence of subepithelial myofibroblats. Molecular mechanisms of FAE differenciation. Different studies proposed an influence of the luminal compartment in the differentiation and/or the maturation of PP. CCL20, a chemokine able to recruit cells that initiate adaptive immunity constitutes our first positive FAE molecular marker. We have shown that CCL20 gene expression is inducible in vitro and in vivo in intestinal epithelium by flagellin, a component of bacterial flagella. This effect was not restricted to the FAE. Lymphotoxin ß (LTß) signaling is critical for PPs organogenesis as LT deficient mice as well as LTß-receptor-/- mice lack PPs and most of the lymph nodes (LN). The continuous signaling via LTßR-expressing cells appears necessary for the maintenance throughout the life of PP architecture. We obtained in vitro and in vivo evidence that LTß signalling is involved in CCL20 gene expression.

KLF2 mutation is the most frequent somatic change in splenic marginal zone lymphoma and identifies a subset with distinct genotype.

To characterise the genetics of splenic marginal zone lymphoma (SMZL), we performed whole exome sequencing of 16 cases and identified novel recurrent inactivating mutations in Kruppel-like factor 2 (KLF2), a gene whose deficiency was previously shown to cause splenic marginal zone hyperplasia in mice. KLF2 mutation was found in 40 (42%) of 96 SMZLs, but rarely in other B-cell lymphomas. The majority of KLF2 mutations were frameshift indels or nonsense changes, with missense mutations clustered in the C-terminal zinc finger domains. Functional assays showed that these mutations inactivated the ability of KLF2 to suppress NF-κB activation by TLR, BCR, BAFFR and TNFR signalling. Further extensive investigations revealed common and distinct genetic changes between SMZL with and without KLF2 mutation. IGHV1-2 rearrangement and 7q deletion were primarily seen in SMZL with KLF2 mutation, while MYD88 and TP53 mutations were nearly exclusively found in those without KLF2 mutation. NOTCH2, TRAF3, TNFAIP3 and CARD11 mutations were observed in SMZL both with and without KLF2 mutation. Taken together, KLF2 mutation is the most common genetic change in SMZL and identifies a subset with a distinct genotype characterised by multi-genetic changes. These different genetic changes may deregulate various signalling pathways and generate cooperative oncogenic properties, thereby contributing to lymphomagenesis.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties.

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

IL-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs.

The series of events leading to tertiary lymphoid organ (TLO) formation in mucosal organs following tissue damage remain unclear. Using a virus-induced model of autoantibody formation in the salivary glands of adult mice, we demonstrate that IL-22 provides a mechanistic link between mucosal infection, B-cell recruitment, and humoral autoimmunity. IL-22 receptor engagement is necessary and sufficient to promote differential expression of chemokine (C-X-C motif) ligand 12 and chemokine (C-X-C motif) ligand 13 in epithelial and fibroblastic stromal cells that, in turn, is pivotal for B-cell recruitment and organization of the TLOs. Accordingly, genetic and therapeutic blockade of IL-22 impairs and reverses TLO formation and autoantibody production. Our work highlights a critical role for IL-22 in TLO-induced pathology and provides a rationale for the use of IL-22-blocking agents in B-cell-mediated autoimmune conditions.

Cerebriforms cells in cerebrum: an unusual finding

Background. Mycosis Fungoides (MF) is the most common cutaneous T-cell lymphoma, and large cell trasformation (tMF) is an adverse prognostic event. Extra-cutaneous dissemination can occur in the course of the disease, but dissemination to the central nervous system (CNS) is uncommon. Moreover, CNS lymphomas are overall rare and most often of B-cell phenotype. We report a case of CNS large T-cell lymphoma presenting as multiple cerebral lesions in a patient with a history of MF. Methods. We report a case of a 33-year-old woman, known since the age of 16 for erythematous plaques thought to be atopic dermatitis, who developed, end 2012, multiple nodular skin lesions and peripheral adenopathies. Two skin lesions were biopsied simultaneously, and diagnosed as MF and tMF. A lymph node biopsy showed dermatopathic changes without lymphoma (Stage IIB). She received local treatment (UVB, PUVA and radiation therapy) and interferon therapy, and experienced almost complete remission. In 2015 neurological symptoms lead to evidence multiple cerebral lesions, suspicious for lymphoma, evaluated by stereotaxic biopsies. We compared histopathological and molecular features of these with previous skin specimens. After negative bone marrow staging biopsy, she was recently started on chemotherapy (MATRIX). Short follow-up shows rapidly worsening clinical conditions. Results. One of the initial skin biopsies showed atypical lymphoid cells with epidermotropism, Pautrier abcesses and CD4+ CD30- phenotype; the other revealed diffuse dermal infiltration by predominantly large cerebriform tumor cells with high proliferative fraction, and CD2−CD3 −CD4+/−CD7−CD30+ALK- EMA- non-cytotoxic immunophenotype. Altogether, these results led us to diagnose MF and tMF, respectively. The brain was infiltrated by large atypical lymphoid cells with cerebriform nuclei, somewhat anaplastic features and perivascular distribution. By immunohistochemistry, tumor cells were highly proliferative, with a CD2−CD3+CD5−CD7+CD30+ activated cytotoxic immunophenotype. A diagnosis of CD30+ cytotoxic peripheral T-cell lymphoma was retained. TRG and TRB clonality analyses revealed clonal rearrangements in skin and CNS biopsies, with identical patterns in both skin specimens but only minimally overlapping profiles when compared to the CNS sample. Der Pathologe 6 ? 2015 | 633 Conclusions. The reported case illustrates an uncommon finding of a CNS T-cell lymphoma in a patient with previous MF, questioning the clonal relationship between the two diseases and challenging the adequate classification of this CNS lymphoma as either a progression or a de novo lymphoma. Despite differences in immunophenotype and clonality patterns, this CNS lymphoma could possibly represent an aggressive divergent evolution of a primary cutaneous T-cell lymphoma. Additional sequencing is ongoing to try to solve the question.

Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

MYC-IG rearrangements are negative predictors of survival in DLBCL patients treated with immunochemotherapy: a GELA/LYSA study.

Diffuse large B-cell lymphoma (DLBCL) with MYC rearrangement (MYC-R) carries an unfavorable outcome. We explored the prognostic value of the MYC translocation partner gene in a series of MYC-R de novo DLBCL patients enrolled in first-line prospective clinical trials (Groupe d'Etudes des Lymphomes de l'Adulte/Lymphoma Study Association) and treated with rituximab-anthracycline-based chemotherapy. A total of 774 DLBCL cases characterized for cell of origin by the Hans classifier were analyzed using fluorescence in situ hybridization with BCL2, BCL6, MYC, immunoglobulin (IG)K, and IGL break-apart and IGH/MYC, IGK/MYC, and IGL/MYC fusion probes. MYC-R was observed in 51/574 (8.9%) evaluable DLBCL cases. MYC-R cases were predominantly of the germinal center B-cell-like subtype 37/51 (74%) with no distinctive morphologic and phenotypic features. Nineteen cases were MYC single-hit and 32 cases were MYC double-hit (MYC plus BCL2 and/or BCL6) DLBCL. MYC translocation partner was an IG gene in 24 cases (MYC-IG) and a non-IG gene (MYC-non-IG) in 26 of 50 evaluable cases. Noteworthy, MYC-IG patients had shorter overall survival (OS) (P = .0002) compared with MYC-negative patients, whereas no survival difference was observed between MYC-non-IG and MYC-negative patients. In multivariate analyses, MYC-IG predicted poor progression-free survival (P = .0051) and OS (P = .0006) independently from the International Prognostic Index and the Hans classifier. In conclusion, we show in this prospective randomized trial that the adverse prognostic impact of MYC-R is correlated to the MYC-IG translocation partner gene in DLBCL patients treated with immunochemotherapy. These results may have an important impact on the clinical management of DLBCL patients with MYC-R who should be routinely characterized according to MYC partner gene. These trials are individually registered at www.clinicaltrials.gov as #NCT00144807, #NCT01087424, #NCT00169143, #NCT00144755, #NCT00140660, #NCT00140595, and #NCT00135499.

The paracaspase MALT1: biological function and potential for therapeutic inhibition.

The paracaspase MALT1 has a central role in the activation of lymphocytes and other immune cells including myeloid cells, mast cells and NK cells. MALT1 activity is required not only for the immune response, but also for the development of natural Treg cells that keep the immune response in check. Exaggerated MALT1 activity has been associated with the development of lymphoid malignancies, and recently developed MALT1 inhibitors show promising anti-tumor effects in xenograft models of diffuse large B cell lymphoma. In this review, we provide an overview of the present understanding of MALT1's function, and discuss possibilities for its therapeutic targeting based on recently developed inhibitors and animal models.

B and T lymphocyte attenuator regulates CD8+ T cell-intrinsic homeostasis and memory cell generation.

B and T lymphocyte attenuator (BTLA) is a negative regulator of T cell activation, but its function in vivo is not well characterized. Here we show that mice deficient in full-length BTLA or its ligand, herpesvirus entry mediator, had increased number of memory CD8(+) T cells. The memory CD8(+) T cell phenotype resulted from a T cell-intrinsic perturbation of the CD8(+) T cell pool. Naive BTLA-deficient CD8(+) T cells were more efficient than wild-type cells at generating memory in a competitive antigen-specific system. This effect was independent of the initial expansion of the responding antigen-specific T cell population. In addition, BTLA negatively regulated antigen-independent homeostatic expansion of CD4(+) and CD8(+) T cells. These results emphasize two central functions of BTLA in limiting T cell activity in vivo.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

Leishmania major-specific B cells are necessary for Th2 cell development and susceptibility to L. major LV39 in BALB/c mice.

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.

Clara cell protein and surfactant protein B in garbage collectors and in wastewater workers exposed to bioaerosols

OBJECTIVES: Inhalation of bioaerosols has been hypothesised to cause "toxic pneumonitis" that should increase lung epithelial permeability at the bronchioloalveolar level. Serum Clara cell protein (CC16) and serum surfactant protein B (SPB) have been proposed as sensitive markers of lung epithelial injury. This study was aimed at looking for increased lung epithelial permeability by determining CC16 and SPB in workers exposed to bioaerosols from wastewater or garbage. METHODS: Subjects (778 wastewater, garbage and control workers; participation 61%) underwent a medical examination, lung function tests [American Thoracic Society (ATS) criteria], and determination of CC16 and SPB. Symptoms of endotoxin exposure and several potential confounders (age, gender, smoking, kidney function, obesity) were looked for. Results were examined with multiple linear or logistic regression. RESULTS: Exposure to bioaerosols increased CC16 concentration in the wastewater workers. No effect of exposure on SPB was found. No clue to work-related respiratory diseases was found. CONCLUSIONS: The increase in CC16 in serum supports the hypothesis that bioaerosols cause subclinical "toxic pneumonitis", even at low exposure. [Authors]

Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.