Cytotoxic T cells induce proliferation of chronic myeloid leukemia stem cells by secreting interferon-γ

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia arising from the oncogenic break point cluster region/Abelson murine leukemia viral oncogene homolog 1 translocation in hematopoietic stem cells (HSCs), resulting in a leukemia stem cell (LSC). Curing CML depends on the eradication of LSCs. Unfortunately, LSCs are resistant to current treatment strategies. The host’s immune system is thought to contribute to disease control, and several immunotherapy strategies are under investigation. However, the interaction of the immune system with LSCs is poorly defined. In the present study, we use a murine CML model to show that LSCs express major histocompatibility complex (MHC) and co-stimulatory molecules and are recognized and killed by leukemia-specific CD8+ effector CTLs in vitro. In contrast, therapeutic infusions of effector CTLs into CML mice in vivo failed to eradicate LSCs but, paradoxically, increased LSC numbers. LSC proliferation and differentiation was induced by CTL-secreted IFN-γ. Effector CTLs were only able to eliminate LSCs in a situation with minimal leukemia load where CTL-secreted IFN-γ levels were low. In addition, IFN-γ increased proliferation and colony formation of CD34+ stem/progenitor cells from CML patients in vitro. Our study reveals a novel mechanism by which the immune system contributes to leukemia progression and may be important to improve T cell–based immunotherapy against leukemia.

The apoptotic and proliferation rate of tumour budding cells in colorectal cancer outlines a heterogeneous population of cells with various impacts on clinical outcome

AIMS In colorectal cancer (CRC), tumour buds represent an aggressive cell type at the invasive front with apparently low proliferation. The aim of this study was to determine proliferation and apoptotic rates of buds in comparison to tumour centre, front and mucosa. METHODS AND RESULTS Whole tissue sections from 188 CRC patients underwent immunohistochemistry for Ki67. Ten high-power fields (HPFs) were evaluated in mucosa, tumour centre, tumour front and tumour buds (total = 40 HPFs/case). Caspase-3 and M30 immunohistochemistry were performed on a multipunch tissue microarray from the same cohort. Ki67, caspase-3 and M30 immunoreactivity were correlated with outcome. The average percentage of cells showing Ki67 positivity was 5.2% in mucosa, and was not significantly different between the centre and front of the tumour (38.2% and 34.9%; P < 0.0001); 0.3% of buds showed Ki67 positivity (P < 0.0001). Caspase-3 expression was similar in mucosa, tumour centre and tumour front, but lower in tumour buds (<0.1%; P < 0.0001). M30 staining in buds was decreased (0.01%; P < 0.0001) in comparison to other areas. Ki67 positivity in buds was detrimental to survival in univariate (P = 0.0352) and multivariate (P = 0.0355) analysis. Caspase-3-positive tumours showed better outcome than negative tumours (P = 0.0262); but tumours with caspase-3-positive buds showed a worse outcome than those with caspase-3-negative buds (P = 0.0235). CONCLUSIONS Ki67, caspase-3 and M30 staining is absent in most tumour buds, suggesting decreased proliferation and apoptosis. However, the fact that Ki67 and caspase-3 immunoreactivity was associated with unfavourable prognosis points to a heterogeneous population of tumour buds.

Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma

Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma Timothy Christopher Graham, B.S. Supervisory Professor: Patrick Zweidler-McKay, MD/PhD Neuroblastoma is the most common extra-cranial solid tumor in children, arising from neural crest precursor cells. The neurotrophin receptors (TrkA/B/C) have been implicated as important prognostic markers, linking the biology of the tumor to patient outcome. High expression of TrkA and TrkC receptors have been linked to favorable biological features and high patient survival, while TrkB is expressed in unfavorable, aggressive tumors. Several studies suggest that high levels and activation of TrkB by its ligand brain-derived neurotrophic factor (BDNF) stimulates tumor cell survival, proliferation, and chemoresistance. However, little is known about the molecular mechanisms that regulate proliferation. The TrkB signaling pathway in neuroblastoma cells has been difficult to evaluate due to the loss of TrkB expression when the cells are used in vitro. Here we determined the role of proximal signaling pathways downstream of TrkB on neuroblastoma proliferation. By analyzing a panel of neuroblastoma cell lines, we found that the SMS-KCN cells express detectable levels of protein and mRNA levels of TrkB as analyzed by western, RT-PCR, and surface expression by flow cytometry. By the addition of exogenous human recombinant BDNF, we showed that activation of TrkB is important in the proliferation of the cells and can be repressed by inhibiting TrkB kinase function. By BDNF stimulation and use of specific kinase inhibitors, the common pathways involving PLCg, PI3K/AKT, and MAPK were initially investigated in addition to PI3K/MTOR and FYN pathways. We demonstrate for the first time that Fyn plays a critical role in TrkB mediated proliferation in neuroblastoma. Constitutively active and over-expressed Fyn reduced neuroblastoma proliferation, as measured by PCNA expression. Knockdown of Fyn by shRNA was shown to cooperate with activated TrkB for an enhanced proliferative response. Although TrkB activation has been implicated in the proliferation of neuroblastoma cells, little is known about its effects on cell cycle regulation. Protein levels of pRB, CDK2, CDK4, CDC25A, cyclin D1, and cyclin E were analyzed following BDNF stimulation. We found that BDNF mediated activation of TrkB induces multiple common proximal signaling pathways including the anti-proliferative Fyn pathway and drives cell cycle machinery to enhance the proliferation of neuroblastoma cells.

Enhanced estrogen-induced proliferation in obese rat endometrium.

OBJECTIVE: We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese vs lean animals. STUDY DESIGN: Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated proproliferative and antiproliferative genes after short-term treatment with estradiol. RESULTS: No significant morphologic/histologic difference was seen between the obese rats and the lean rats. Estrogen-induced proproliferative genes cyclin A and c-Myc messenger RNA expression were significantly higher in the endometrium of obese rats compared with those of the lean control. Expression of the antiproliferative gene p27Kip1 was suppressed by estrogen treatment in both obese and lean rats; however, the decrease was more pronounced in obese rats. Estrogen more strongly induced the antiproliferative genes retinaldehyde dehydrogenases 2 and secreted frizzled-related protein 4 in lean rats but had little or no effect in obese rats. CONCLUSION: Enhancement of estrogen-induced endometrial proproliferative gene expression and suppression of antiproliferative gene expression was seen in the endometrium of obese vs lean animals.

Suppression of peripheral blood mononuclear cell proliferation by a periodontopathic bacteria {\it Actinobacillus actinomycetemcomitans\/}

Actinobacillus actinomycetemcomitans (Aa) is a gram-negative coccobacillus implicated as a major pathogen in juvenile periodontitis. The immunosuppressive activity of a sonic extract (designated 100SN) derived from Aa was investigated. 100SN suppressed spontaneous proliferation as well as proliferative response to the mitogens, PHA and PWM, of human peripheral blood mononuclear cells (PBMC). 100SN-induced suppression of PHA-stimulated proliferation was heat-sensitive, inactivated by pronase and trypsin, dose-dependent and non-cytotoxic. There were no significant changes in the CD4$\sp+$ or CD8$\sp+$ subsets of PBMC after 7-day incubation with 100SN. There was a trend toward increased levels of the CD4$\sp+$CD45R$\sp{\rm hi}$CDw29$\sp{\rm lo}$ (naive cells, associated with suppressor-inducer activity) and CD4$\sp+$CDw29$\sp{\rm hi}$CD45R$\sp{\rm lo}$ (memory cells, associated with helper-inducer activity) subsets. The target of 100SN appeared to be the non-adherent cells and suppression by 100SN could not be reversed by indomethacin (IDM), the cyclo-oxygenase inhibitor of prostaglandin (PG) synthesis. The mechanism of 100SN-induced suppression was studied in terms of inhibition involving IL-2-regulated T cell proliferation and the results point to the possibility that suppression occurred subsequent to IL-2 receptor binding.^ The suppressive activity observed could occur through multiple mechanisms including cell-cell; contact or release of soluble factors. Supernatants derived from 7-day cultures of PBMC and 100SN (designated CSN-A) were able to suppress proliferative response of PBMC to PHA without affecting cell viability. Analysis of CSN-A showed that it contained PGE2 and soluble IL-2 receptors. Suppression by CSN-A could be partially overcome by either IDM or exogenous IL-2. Significant suppression was also maintained when both IDM and exogenous IL-2 were added at the same time. These findings suggest that PGE2 and soluble IL-2 receptors contribute to the suppression observed but other suppressive cytokine(s) may be involved. Collectively, the data indicate that a factor derived from oral bacteria associated with juvenile periodontitis have profound effects on cellular immune responses, and that these effects may be partially mediated by secondary factors produced by the host in response to the bacteria. ^

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^

Mechanism of dendritic epidermal T cell-mediated tolerance induction and inhibition of proliferation

Dendritic epidermal T cells (DETC) comprise a unique population of T cells that reside in mouse epidermis and whose function remains unclear. Most DETC express a $\gamma\delta$ TCR, although some, including our DETC line, AU16, express an $\alpha\beta$ TCR. Additionally, AU16 cells express CD3, Thy-1, CD45, CD28, B7, and AsGM-1. Previous studies in our laboratory demonstrated that hapten-conjugated AU16 could induce specific immunologic tolerance in vivo and inhibit T cell proliferation in vitro. Both these activities are antigen-specific, and the induction of tolerance is non-MHC-restricted. In addition, AU16 cells are cytotoxic to a number of tumor cell lines in vitro. These studies suggested a role for these cells in immune surveillance. The purpose of my studies was to test the hypothesis that these functions of DETC (tolerance induction, inhibition of T cell proliferation, and tumor cell killing) were mediated by a cytotoxic mechanism. My specific aims were (1) to determine whether AU16 could prevent or delay tumor growth in vivo; and (2) to determine the mechanism whereby AU16 induce tolerance, using an in vitro proliferation assay. I first showed that AU16 cells killed a variety of skin tumor cell lines in vitro. I then demonstrated that they prevented melanoma growth in C3H mice when both cell types were mixed immediately prior to intradermal (i.d.) injection. Studies using the in vitro proliferation assay confirmed that DETC inhibit proliferation of T cells stimulated by hapten-bearing, antigen-presenting cells (FITC-APC). To determine which cell was the target, $\gamma$-irradiated, hapten-conjugated AU16 were added to the proliferation assay on d 4. They profoundly inhibited the proliferation of naive T cells to $\gamma$-irradiated, FITC-APC, as measured by ($\sp3$H) TdR uptake. This result strongly suggested that the T cell was the target of the AU16 activity because no APC were present by d 4 of the in vitro culture. In contrast, the addition of FITC-conjugated splenic T cells (SP-T) or lymph node T cells (LN-T) was less inhibitory. Preincubation of the T cells with FITC-AU16 cells for 24 h, followed by removal of the AU16 cells, completely inhibited the ability of the T cells to proliferate in response to FITC-APC, further supporting the conclusion that the T cell was the target of the AU16. Finally, AU16 cells were capable of killing a variety of activated T cells and T cell lines, arguing that the mechanism of proliferation inhibition, and possibly tolerance induction is one of cytotoxicity. Importantly, $\gamma\delta$ TCR$\sp+$ DETC behaved, both in vivo and in vitro like AU16, whereas other T cells did not. Therefore, these results are consistent with the hypothesis that AU16 cells are true DETC and that they induce tolerance by killing T cells that are antigen-activated in vivo. ^

Mechanisms of liposomal all-{\it trans\/} retinoic acid and vitamin D3 induced inhibition of cell proliferation and stimulation of apoptosis in aggressive B-cell non-Hodgkin's lymphoma

The Non-Hodgkin's Lymphoma (NHLs) are neoplasms of the immune system. Currently, less than 1% of the etiology of the 22,000 newly diagnosed lymphoma cases in the U.S.A. every year is known. This disease has a significant prevalence and high mortality rate. Cell growth in lymphomas has been shown to be an important parameter in aggressive NHL when establishing prognosis, as well as an integral part in the pathophysiology of the disease process. While many aggressive B cell NHLs respond initially to chemotherapeutic regimens such as CHOP-bleo (adriamycin, vincristine and bleomycin) etc., relapse is common, and the patient is then often refractory to further salvage treatment regimens.^ To assess their potential to inhibit aggressive B cell NHLs and induce apoptosis (also referred to as programmed cell death (PCD)), it was proposed to utilize the following biological agents-liposomal all-trans retinoic acid (L-ATRA) which is a derivative of Vitamin A in liposomes and Vitamin D3. Preliminary evidence indicates that L-ATRA may inhibit cell growth in these cells and may induce PCD as well. Detailed studies were performed to understand the above phenomena by L-ATRA and Vitamin D3 in recently established NHL-B cell lines and primary cell cultures. The gene regulation involved in the case of L-ATRA was also delineated. ^

Effects of galectin-1 and galectin-3 expression on prostate cancer cell adhesion, differentiation, proliferation, and tumorigenicity

The vertebrate $\beta$-galactoside-binding lectins galectin-1 and galectin-3 have been proposed to function in diverse cellular processes such as adhesion, proliferation, differentiation, and tumorigenesis. Experiments were initiated to further study the functional properties of these molecules. A prostate cancer cell line, LNCaP, was identified which expressed neither galectin. This line was stably transfected with cDNA for either galectin-1 or galectin-3. The resultant clones were used to study effects on critical cell processes. LNCaP cells expressing galectin-1 on the surface were found to bind more rapidly than control lines to the human extracellular matrix proteins laminin and fibronectin, although overall binding was not increased. To analyze effects on differentiation, LNCaP cells were studied which had either been transfected with galectin-1 or which had been induced to express endogenous galectin-1 by treatment with the differentiation agent sodium butyrate. In both cases, cells displayed a slower rate of growth and increased rate of apoptosis. A transient decrease in expression of prostate specific antigen was seen in the butyrate treated cells but not in the transfected cells. To investigate the role of galectins in the process of malignant transformation and progression, immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections of human prostate tissue, the premalignant lesion prostatic intraepithelial neoplasia, primary adenocarcinoma of the prostate, and foci of metastatic prostate cancer. Galectin-1 expression was relatively constant throughout in contrast to galectin-3 which demonstrated significantly less expression in primary and metastatic tumors. LNCaP cells transfected with galectin-3 cDNA displayed lower proliferation rates, increased spontaneous apoptosis, and G1 growth phase arrest compared to controls. Four of six galectin-3 lines tested were less tumorigenic in nude mice than controls. The following conclusions are drawn regarding the role of galectin-1 and galectin-3 expression in the context of prostate cancer: (1) galectin-1 may participate in the early stages of cancer cell adhesion to extracellular matrix proteins; (2) galectin-1 expression results in a differentiated phenotype and may contribute to differentiation induction by butyrate; (3) galectin-3 expression correlates inversely with prostate cell tumorigenesis and prostate cancer metastasis. ^

Suppression of cell proliferation and programmed cell death by dexamethasone during postnatal lung development

Prematurely born babies are often treated with glucocorticoids. We studied the consequences of an early postnatal and short dexamethasone treatment (0.1-0.01 microg/g, days 1-4) on lung development in rats, focusing on its influence on peaks of cell proliferation around day 4 and of programmed cell death at days 19-21. By morphological criteria, we observed a dexamethasone-induced premature maturation of the septa (day 4), followed by a transient septal immatureness and delayed alveolarization leading to complete rescue of the structural changes. The numbers of proliferating (anti-Ki67) and dying cells (TdT-mediated dUTP nick end labeling) were determined and compared with controls. In dexamethasone-treated animals, both the peak of cell proliferation and the peak of programmed cell death were reduced to baseline, whereas the expression of tissue transglutaminase (transglutaminase-C), another marker for postnatal lung maturation, was not significantly altered. We hypothesize that a short neonatal course of dexamethasone leads to severe but transient structural changes of the lung parenchyma and influences the balance between cell proliferation and cell death even in later stages of lung maturation.

Differential effects of interferon-gamma and tumor necrosis factor-alpha on Toxoplasma gondii proliferation in organotypic rat brain slice cultures.

Organotypic slice culture explants of rat cortical tissue infected with Toxoplasma gondii tachyzoites were applied as an in vitro model to investigate host-pathogen interactions in cerebral toxoplasmosis. The kinetics of parasite proliferation and the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in infected organotypic cultures were monitored by light microscopy, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (PCR) assay. As assessed by the loss of the structural integrity of the glial fibrillary acidic protein-intermediate filament network, tachyzoites infected and proliferated mainly within astrocytes, whereas neurons and microglia remained largely unaffected. Toxoplasma gondii proliferation was severely inhibited by IFN-y. However, this inhibition was not linked to tachyzoite-to-bradyzoite stage conversion. In contrast, TNF-alpha treatment resulted in a dramatically enhanced proliferation rate of the parasite. The cellular integrity in IFN-gamma-treated organotypic slice cultures was severely impaired compared with untreated and TNF-alpha-treated cultures. Thus, on infection of organotypic neuronal cultures, IFN-gamma and TNF-alpha exhibit largely detrimental effects, which could contribute to either inhibition or acceleration of parasite proliferation during cerebral toxoplasmosis.

The FUS protein is required for cell proliferation

FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed and highly conserved RNA binding protein, has been linked to a variety of cellular processes from mRNA processing to DNA repair. However, the precise function of FUS is not well understood. Recently, mutations in the FUS gene have been identified in familial and sporadic patients of Amyotrophic Lateral Sclerosis, a fatal neurodegenerative disorder characterized by dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function situation (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we have generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS that efficiently depletes the protein. In order to characterize this cell line, we have characterized the poly(A) fraction by RNA deep sequencing. Preliminary results show that FUS depletion affects both mRNA expression and alternative splicing. Upon FUS depletion 330 genes are downregulated and 81 are upregulated. We also found that 395 splicing isoforms were downregulated, while 426 were upregulated. Currently, we are focusing our attention on the pathways which are mostly affected by FUS depletion. In addition, we are currently characterizing how FUS depletion affects cell proliferation and survival. We find that the lack of FUS impairs cell proliferation but does not induce apoptosis. Finally, since MEFs and B-lymphocytes derived from FUS knockdown mice display major sensitivity to ionizing radiation and chromosomal aberrations [1,2], we are exploring the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). Taken together, these studies may contribute to our knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.