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Conventional cytotoxic drugs preferentially eliminate differentiated cancer cells but spare relatively more resistant stem-like cancer cells capable to initiate recurrence. Due to cancer cell plasticity, the stem-like phenotype can be also acquired by cancer cells refractory to treatment with cytotoxic drugs. We investigated whether drugs inhibiting receptor tyrosine kinases could be used to target human colon cancer cells initiating cancer regrowth following conventional cytotoxic treatment. The moderately differentiated cell line HT-29 and poorly differentiated cell line HCT-116 were exposed to 5-fluorouracil (5-FU). Cells that resisted the exposure to 5-FU were subsequently treated with imatinib or sunitinib. Both drugs reduced clonogenicity of 5-FU-refractory cells under normoxic and hypoxic culture conditions. The expression of numerous stemness-related genes was upregulated in cancer cells following the exposure to 5-FU, and remained at a high level in 5-FU-refractory cells undergoing renewal under normoxia, but decreased spontaneously under hypoxia. Imatinib downregulated the expression of stemness-related genes in cells undergoing renewal under normoxia. A combination of imatinib with PRI-2191, an analogue of 1,25-dihydroxyvitamin D3, downregulated stemness-related genes in HCT-116/5-FU cells more efficiently than imatinib alone. A synthetic analogue of 1,25-dihydroxyvitamin D2 (PRI-1906) abolished the effect of imatinib on gene expression in HCT-116/5-FU cells undergoing renewal under normoxia. Sunitinib promoted shift of phenotype of HT-29/5-FU cells undergoing renewal toward stem-like one. It suggests that the phenotype shift toward stemness induced by sequential sunitinib treatment following 5-FU treatment could increase a risk of cancer recurrence. In contrast to sunitinib, imatinib could be used both to interfere with cancer regrowth after conventional chemotherapy and to downregulate the expression of stemness-related genes in residual colon cancer cells capable to initiate cancer recurrence. The findings suggest that imatinib could also be combined with vitamin D analogue PRI-2191 to prevent recurrence more efficiently than imatinib alone and to compensate for vitamin D deficiency resulting from imatinib treatment.

Growing tumors avoid recognition and destruction by the immune system. During continuous stimulation of tumor-infiltrating lymphocytes (TILs) by tumors, TILs become functionally exhausted, thus, they become unable to kill tumor cells and to produce certain cytokines and lose their ability to proliferate. This collectively results in the immune escape of cancer cells. Here, we show that breast cancer cells expressing PD-L1 can accelerate exhaustion of persistently activated human effector CD4+ T cells, manifesting in high PD-1 and PD-L1 expression level son T cell surfaces, decreased glucose metabolism genes, strong downregulation of SWI/SNF chromatin remodeling complex subunits, and p21 cell cycle inhibitor upregulation. This results in inhibition of T cell proliferation and reduction of T cell numbers. The RNAseq analysis on exhausted CD4+ T cells indicated strong overexpression of IDO1 and genes encoding pro-inflammatory cytokines and chemokines. Some interleukins were also detected in media from CD4+ T cells co-cultured with cancer cells. The PD-L1 overexpression was also observed in CD4+ T cells after co-cultivation with other cell lines overexpressing PD-L1, which suggested the existence of a general mechanism of CD4+ T cell exhaustion induced by cancer cells. The ChIP analysis on the PD-L1 promoter region indicated that the BRM recruitment in control CD4+ T cells was replaced by BRG1 and EZH2 in CD4+ T cells strongly exhausted by cancer cells. These findings suggest that epi-drugs such as EZH2 inhibitors may be used as immunomodulators in cancer treatment.

We have previously found that ex vivo expanded human CD4+CD25+Treg cells suppress proliferation of lymphoma B-cell lines. Here we demonstrate that the immunomodulatory drug lenalidomide potentiates suppression of lymphoma B-cell proliferation by freshly isolated CD4+CD25+Tregs, as well as suppression by Tregs expanded polyclonally in the presence of rapamycin from CD4+CD25+T cells or CD4+CD25+CD127loT cells. The regulation of lymphoma cell proliferation by Tregs pre-expanded with "third-party" allogeneic MoDCs in the presence of rapamycin was also potentiated by lenalidomide. Lenalidomide contributed to the suppression exerted by Tregs despite concomitant downregulation of Treg proliferation. Lenalidomide did not reduce the suppression of conventional T cells by expanded Tregs. The exposure of polyclonally expanded Tregs to lenalidomide did not significantly alter their phenotype. There was no uniform pattern of lenalidomide effect on Treg-mediated regulation of lymphoma B cells freshly isolated from patients. Freshly isolated lymphoma cells activated with multimeric CD40L and IL-4 to support their survival in vitro varied in their sensitivity to lenalidomide, and the regulatory effect of Tregs on such lymphoma cells ranged from suppression to help in individual patients. Lenalidomide potentiated or attenuated Treg effects on the survival of freshly isolated lymphoma cells. A combination of lenalidomide treatment with adoptive transfer of CD4+CD25+Tregs or CD4+CD25+CD127loTregs expanded ex vivo could be used to suppress proliferation of residual lymphoma in select patients with lymphoma responsive to the regulation by Tregs and sensitive to lenalidomide.

Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinases and subsequent inactivation of FOXO1 is a critical pro-survival component of tonic BCR signaling. In murine models, conditional deletion of FOXO1 protected quiescent peripheral B cells from apoptosis mediated by inducible loss of the BCR, demonstrating that PI3K-AKT-FOXO1 axis plays a central role in B-cell homeostasis. Disruption of the BCR signaling by SYK inhibitor leads also to the apoptosis of BCR-dependent DLBCLs, at least in part via a mechanism involving decreased activity of PI3K/AKT axis. Herein, we investigated the role of FOXO1 in toxicity of BCR pathway/SYK inhibition in human BCR-dependent lymphomas. Methods: BCR-dependent DLBCL cell lines (DHL4, DHL6, Ly1, Ly7) were incubated with SYK inhibitor R406 (4 µM) and AKT phosphorylation, FOXO1 activation and transcriptional activity were assessed by phospho-specific flow cytometry, Western Blot and qPCR. Toxicity of active FOXO1 was assessed by transducing DLBCL cells with a constitutively nuclear FOXO1 mutant. FOXO1 knock-down in DLBCL cell was achieved with shRNA. DREAM cleavage and HRK expression were assessed by Western Blot and qPCR, respectively, in the absence or presence of caspase inhibitors. DLBCL cell viability and apoptosis after incubation with SYK and/or AKT inhibitors R406 and MK2206, were assessed by MTS assay and Annexin V/PI staining. Drug interactions were quantitated by CompuSyn software. The expression of p-SYK and FOXO1 in DLBCL samples was assessed by immunohistochemistry (IHC) in 60 DLBCL patients. Results: Since FOXO1 is a major effector of tonic BCR signaling, we assessed the activity of FOXO1 in DLBCL cells after SYK inhibition. In all tested cell lines, AKT and FOXO1 phosphorylations decreased after incubation with SYK inhibitor. Diminished FOXO1 phosphorylation resulted in its nuclear relocalization and induction of FOXO1-dependent expression of p27, BIM, TRAIL and GADD45A. To assess whether the increased activity of FOXO1 is sufficient to induce apoptosis of DLBCL cells, we transduced DHL4 cells with a constitutively nuclear and transcriptionally active FOXO1-3A mutant. The mutant induced G1/S cell cycle arrest and triggered apoptosis, whereas wild-type FOXO1 did not change proliferation or cellular viability, demonstrating that FOXO1 activation is sufficient to induce apoptosis of DLBCL cells. Next, we assessed the toxicity of SYK inhibitor in cells lacking FOXO1. Cells with depleted FOXO1 exhibited 70% lower sensitivity to SYK inhibitor than control cells (p Conclusions: Taken together, our results demonstrate that targeting the BCR signaling at multiple levels is a rational therapeutic strategy. Proapoptotic activity of FOXO1 is an important effector of SYK and AKT inhibition in DLBCLs and its expression is required for SYK-and AKT inhibitor-induced toxicity. The underlying mechanism linking FOXO1 activation and cell death involves caspase-dependent cleavage of transcriptional repressor DREAM and subsequent induction of a proapoptotic BCL2 family member, HRK. Disclosures No relevant conflicts of interest to declare.

This study was aimed to determine whether hypocalcemic analogs of active forms of vitamins D modulate expression of genes related to stem-like phenotype in colon cancer cell lines HT-29 and HCT-116 undergoing renewal after the treatment with 5-fluorouracil (5-FU). Both lines express vitamin D receptor, but differ in differentiation stage and vitamin D sensitivity. Cells that resisted the 5-FU exposure were treated with synthetic analog of 1,25-dihydroxyvitamin D2 (PRI-1906) and analogs of 1,25-dihydroxyvitamin D3 (PRI-2191 and PRI-2205). Proliferative activity was more profoundly affected by vitamin D analogs in HT-29/5-FU than in HCT-116/5-FU cells. In HT-29/5-FU cells, analogs PRI-1906 and PRI-2191 downregulated the expression of genes related to survival, re-growth, and invasiveness during renewal, while PRI-2205 increased expression of genes related to differentiation only. In HCT-116/5-FU cells, PRI-2191 decreased the expression of stemness- and angiogenesis-related genes, whereas PRI-1906 augmented their expression. The effects in HCT-116/5-FU cells were observed at higher concentrations of the analogs than those used for HT-29/5-FU cells. Out of the series of analogs studied, PRI-2191 might be used to counteract the renewal of both moderately and poorly differentiated cancer cells following conventional treatment.

A b s t r a c t Opiate analgesics like morphine or fentanyl are the most widely used medicines for relieving severe acute or chronic pain, including cancer pain. Unfortunately, chronic pain treatment is associated with fast development of tolerance that creates the need to escalate the treatment doses. In addition, opiates may stimulate progression of cancer. There - fore, a new type of effective analgesic especially designed for chronic cancer pain treatment is needed. In this paper, a new opioid peptide analogue has been described as a new analgesic. The compound is characterized by very high agonist affinities to MOR and also high, but ten times lower affinity to DOR. Affinity to hNK1 as an antagonist is on the level of C-terminal hexapeptide fragment analogue of Substance P. The compound expressed reasonable antipro - liferative properties toward various cancer cells. Interestingly, the peptide did not interfere with the proliferation of fibro - blasts. Therefore, the compound should be considered as a new analgesic for treatment of cancer-related pains with adjuvant anticancer properties which may support cancer treatments.

This study aimed to evaluate the capacity of hypocalcemic analogues of 1α,25-dihydroxyvitamin D2 (1,25D2) and 1α,25-dihydroxyvitamin D3 (1,25D3) to inhibit regrowth and regulate the stemness-related gene expression in colon cancer cells undergoing renewal after exposure to 5-fluorouracil (5-FU). All of the tested analogues of 1,25D2 equally potently decreased the clonogenicity and the proliferative activity of HT-29 cells which survived the exposure to 5-FU, but differently regulated gene expression of these cells during their renewal. 1,25D2 and analogues (PRI-1907 and PRI-1917), as well as 1,25D3 and analogue PRI-2191, decreased the relative expression level of several stemness-related genes, such as NANOG, OCT3/4, PROM1, SOX2, ALDHA1, CXCR4, in HT-29/5-FU cells during their renewal, in comparison to untreated HT-29/5-FU cells. The other 1,25D2 analogues (PRI-1906 and PRI-1916) were not capable of downregulating the expression of these stemness-related genes as the analogues PRI-1907 and PRI-1917 did. All of the tested vitamin D analogues upregulated CDH1, the gene encoding E-cadherin associated with epithelial phenotype. Out of the series of analogues studied, side-chain branched analogues of 1,25D2 (PRI-1907, PRI-1917) and the analogue of 1,25D3 (PRI-2191) might be used to target cancer cells with stem-like phenotypes that survive conventional chemotherapy.

This study aimed to evaluate the capacity of hypocalcemic analogues of 1α,25-dihydroxyvitamin D2 (1,25D2) and 1α,25-dihydroxyvitamin D3 (1,25D3) to inhibit regrowth and regulate the stemness-related gene expression in colon cancer cells undergoing renewal after exposure to 5-fluorouracil (5-FU). All of the tested analogues of 1,25D2 equally potently decreased the clonogenicity and the proliferative activity of HT-29 cells which survived the exposure to 5-FU, but differently regulated gene expression of these cells during their renewal. 1,25D2 and analogues (PRI-1907 and PRI-1917), as well as 1,25D3 and analogue PRI-2191, decreased the relative expression level of several stemness-related genes, such as NANOG, OCT3/4, PROM1, SOX2, ALDHA1, CXCR4, in HT-29/5-FU cells during their renewal, in comparison to untreated HT-29/5-FU cells. The other 1,25D2 analogues (PRI-1906 and PRI-1916) were not capable of downregulating the expression of these stemness-related genes as the analogues PRI-1907 and PRI-1917 did. All of the tested vitamin D analogues upregulated CDH1, the gene encoding E-cadherin associated with epithelial phenotype. Out of the series of analogues studied, side-chain branched analogues of 1,25D2 (PRI-1907, PRI-1917) and the analogue of 1,25D3 (PRI-2191) might be used to target cancer cells with stem-like phenotypes that survive conventional chemotherapy.

Dendritic cells may be successfully used to induce in vivo-specific anti-tumor responses when combined with the appropriate antigen in the appropriate context. The purpose of this study was to evaluate efficacy of peptide-loaded DC vaccine in high-risk stage III melanoma patients after lymph node dissection (LND). HLA-A2+, -A1+, or -A3+ melanoma patients (N=22), stage III, N1b-N3, received 5–16 (median: 11) DC vaccines loaded with MHC class-I-restricted melanoma peptides respective to the patient’s haplotype, and with autologous tumor lysate, if available. Vaccinated patients were matched to unvaccinated stage III controls (22 of 869) by sex, number of metastatic lymph nodes, extracapsular involvement, LND type, Breslow stage, and ulceration. Vaccination elicited cutaneous delayed-type hypersensitivity (DTH) or/and IFN-γ-producing CD8+ cell response to melanoma peptides in 15 of 22 patients. Three-year overall survival (OS) rate was 68.2% in the vaccinated group versus 25.7% in the control group, P value accounting for matching: 0.0290. In a Cox regression model, hazard ratio (HR) for death of vaccinated patients was 0.31 [95% confidence interval (CI): 0.10–0.94]. The corresponding values for 3-year disease-free survival rate were 40.9 versus 14.5%, P=0.1083; HR of recurrence for vaccinated, 0.46 (95% CI: 0.18–1.22). There was no grade>1 toxicity. The DC/peptide vaccine was well tolerated and elicited immune responses to melanoma antigens. Vaccinated patients had significantly longer OS after LND than the matched controls, but a significant improvement in the primary endpoint DFS was not achieved.

The success of genome projects has provided us with a vast amount of information on genes of many pathogenic species and has raised hopes for rapid progress in combating infectious diseases, both by construction of new effective vaccines and by creating a new generation of therapeutic drugs. Proteomics, a strategy complementary to the genomic-based approach, when combined with immunomics (looking for immunogenic proteins) and vaccinomics (characterization of host response to immunization), delivers valuable information on pathogen-host cell interaction. It also speeds the identification and detailed characterization of new antigens, which are potential candidates for vaccine development. This review begins with an overview of the global status of vaccinology based on WHO data. The main part of this review describes the impact of proteomic strategies on advancements in constructing effective antibacterial, antiviral and anticancer vaccines. Diverse aspects of disease mechanisms and disease preventions have been investigated by proteomics.

We have previously found that ex vivo expanded human CD4

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1-TXN-p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.

Conventional anti-cancer drugs preferentially eliminate differentiated cancer cells but those cells that are spared (i.e. cancer stem cells: CSC), initiate recurrence. We tested whether drugs that target receptor tyrosine kinases (RTKs) involved in developmental signaling cascades and activated in CSC, could be used to silence and/or to eliminate colorectal cancer cells refractory to conventional treatment with cytoreductive drugs. A sequential treatment model was thereby developed with doxorubicin (DOX) and imatinib. CT-26 mouse colon carcinoma cells were pre-treated with DOX to select DOX-refractory cells with CSC properties, which were then subsequently treated with RTK inhibitor imatinib, where their regrowth was found to be inhibited. Under both normoxic and hypoxic conditions, imatinib potently inhibited clonogenicity of DOX-refractory CT-26 cells. Treatment with DOX did not eliminate tumorigenic CT-26 cells, since CT-26 cells pre-exposed to DOX in vitro, when inoculated subcutaneously, induced tumors in 90 % of mice, as opposed to a 100 % rate in the case of chemonaive CT-26 cells. In mice inoculated with chemonaive CT-26 cells, tumor formation was not prevented by imatinib. However, imatinib prevented tumor formation in 50 % of mice inoculated with CT-26 cells pre-exposed to DOX in vitro, with the remaining 50 % mice showing delayed tumor formation. These results suggest that the sequential use of the drug imatinib, as a drug targeting cancer cells expressing stem cell features after conventional cytoreductive treatment, is a promising future strategy for preventing tumor recurrence.

Marker genes, commonly used to detect circulating tumour cells in RT-PCR-based tests: squamous-cell carcinoma antigen, epidermal growth factor receptor, mammaglobin, small breast epithelial mucin, but not carbonic anhydrase 9, were shown to be expressed in normal, mitogen-stimulated peripheral blood mononuclear cells (PBMNC). Thus, considering the inflammatory reactions often accompanying cancer development, to reduce false-positive results of the metastatic tumour cell tests, molecular markers should be validated not against normal peripheral blood, but against activated lymphoid cells, such as in vitro mitogen-stimulated PBMNC.

Dendritic cell (DC) counts and function were assayed in peripheral blood of lymphoma and solid tumor patients before and after chemotherapy. The DC counts declined significantly within the first week from the start of chemotherapy, recovered in the second week, and exceeded the baseline values in the third week. DC recovery was usually similar after the first and after the last cycle of chemotherapy. DC1 and DC2 subsets followed the pattern of reconstitution found for the DC population as a whole. Monocytes and granulocytes recovered 1-2 weeks later than DC. The primary proliferative response to keyhole lympet hemocyanin (KLH), totally DC-dependent, declined within the first week from the start of chemotherapy, and in the majority of patients (including those initially unresponsive) recovered along with DC counts. The recovered responsiveness to KLH, but not to anti-CD3 antibody, disappeared at the end of chemotherapy in lymphoma and some solid tumor patients. Prolonged depletion of CD4+ T cells could contribute to the loss of responsiveness in lymphoma patients receiving multiple cycles of chemotherapy. However, in some solid tumor patients, the reactivity to KLH was absent, despite the reconstitution of both DC and CD4+ T-cell counts. Our data show that numerical reconstitution of DC is not necessarily accompanied by functional recovery. The early recovery of DC should be considered while designing protocols for DC collection for immunotherapy.

Opiate analgesics like morphine or fentanyl are the most widely used medicines for relieving severe acute or chronic pain, including cancer pain. Unfortunately, chronic pain treatment is associated with fast development of tolerance that creates the need to escalate the treatment doses. In addition, opiates may stimulate progression of cancer. Therefore, a new type of effective analgesic especially designed for chronic cancer pain treatment is needed. In this paper, a new opioid peptide analogue has been described as a new analgesic. The compound is characterized by very high agonist affinities to MOR and also high, but ten times lower affinity to DOR. Affinity to hNK1 as an antagonist is on the level of C-terminal hexapeptide fragment analogue of Substance P. The compound expressed reasonable antiproliferative properties toward various cancer cells. Interestingly, the peptide did not interfere with the proliferation of fibro-blasts. Therefore, the compound should be considered as a new analgesic for treatment of cancer-related pains with adjuvant anticancer properties which may support cancer treatments.

Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased in vitro dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.

This review presents the latest achievements in basic studies on stem-cell biology and on approaches aimed to design therapies based on stem cells and dendritic cells. Studies on stem-cell homeostasis are aimed to delineate the accessibility of these cells for therapeutic purposes. Hematopoietic stem cell transplantation has become a routine application of stem cells in the treatment of patients with cancer and with hematologic disorders. Perspectives of application of the mesenchymal stem cells for regenerative medicine and for tolerance induction following allogeneic transplantation are being extensively explored. Reprogramming of adult somatic cells to an undifferentiated pluripotent state in vitro by a transduction with just four genes encoding transcription factors opened the way for the generation of patient-specific pluripotent stem cells. Such induced pluripotent stem cells hold a great promise for replacement therapies of various so far incurable disorders. However, because of the tumorigenic potential of the retroviral vector transduction-induced pluripotent stem cells, before these cells become useful for clinical application, appropriate, safe methods of stem cell generation, selection, proliferation and differentiation need to be elaborated. There is a great potential for further developments of cancer immunotherapies and for controlling of post-transplantational reactions, if current basic studies on stem cells and on immunostimulatory and tolerogenic dendritic cells would be successfully translated to the clinic.

Glucocorticoids (GC) have been used for decades in the treatment of B-cell acute lymphoblastic leukemia (B-ALL) in children and adults. Induction of apoptosis is thought to be the principal effector mechanism of GC's action, but recent studies highlight the role of autophagy upstream of apoptotic cell death (Laane et al 2009). Resistance to GCs is a major adverse prognostic factor, however the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular mechanisms driving GC-resistance in precursor B-cell acute lymphoblastic leukemia cells and in vitro characterize the therapeutic potential of targeted intervention in these mechanisms. To identify molecular mechanisms involved in GC resistance, we performed gene set enrichment analysis of gene expression profiles GC-sensitive and -resistant B-ALL blasts using publicly available datasets and GenePattern program. Resistant cells exhibited significantly higher expression of MAPK/ERK pathway components (p Disclosures No relevant conflicts of interest to declare.

The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen-specific CD4+ T cells from previously unprimed individuals. Freshly isolated CD4+ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (M phi). In short-term (< 8 days) cultures, CD4+ T cells or their CD4+, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4+, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or M phi. KLH- and SWM-specific CD4+ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and M phi in medium supplemented with interleukin-2 and interleukin-4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen-specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4+ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long-term lines that retain their antigen specificity.

It has been previously shown that GM-CSF promotes differentiation and survival of human peripheral blood dendritic cells (DC) in vitro1. Human bone marrow progenitors of dendritic cells were identified in semi-solid cultures supplemented with conditioned media from PHA-stimulated leukocytes2. GM-CSF and TNF-α have been shown to cooperate in the regulation of DC growth from CD34+ progenitors in vitro3,4,5.

A feasibility of hybrid cell vaccination performed in remission following a standard chemotherapy (CHT) is currently being studied in patients with indolent B cell lymphomas at our institution under approval of the Ethics Committee. Adequate samples of fresh tumor tissue were collected from 16 patients. Seven patients were vaccinated with irradiated lymphoma/dendritic cell (DC) hybrids, and whenever the amount of collected viable lymphoma cells was not sufficient to complete vaccination with the use of cell hybrids, booster injections were alternatively performed with the use autologous DCs pulsed with tumor lysate. Autologous lymphoma cells were electrofused with autologous and/or allogeneic DCs, depending on accessibility of autologous DCs. Monocyte-derived DCs were basically generated from the peripheral blood adherent cells stimulated with GM-CSF and IL-4, and if available, from the bone marrow (BM) adherent cells. If only possible, autologous DCs were also generated from the BM non-adherent cells or from a sample of leukapheresis product collected for transplantation cultured with GM-CSF, TNF-α, SCF and FLT3-L to induce differentiation of DC progenitors. DC hybrids and/or DCs preincubated with tumor lysate were administered to uninvolved lymph nodes under USG guidance. Four patients failed to achieve a complete remission after initial therapy and the vaccination was discontinued due to disease progression (after 2, 2, 6, and 5 cycles). Three patients were vaccinated during remission following CHT. At present - 16, 18, and 10 months after starting the immunization, and after 8, 9, and after 7 cycles of vaccination, one patient with mantle cell lymphoma (MCL) is symptomless with persistent bone marrow (BM) involvement, one patient with MCL presenting with IgM paraproteinemia is disease-free with residual BM involvement (Fig 1), and one patient with follicular lymphoma is disease-free. Cutaneous delayed type hypersensitivity (DTH) response to DC/lymphoma hybrids exceeded the response to DCs alone in 3 of 7 vaccinated patients, and interferon-γ producing CD8+ cells specific to autologous lymphoma cells were demonstrated in one patient. In another patient, cutaneous DTH reaction was accompanied by transient pruritus and swelling of the injected lymph node two days after the sixth booster injection. No other adverse reactions to vaccinations were observed. Vaccination using the DC/lymphoma hybrids appears feasible, safe, and capable of inducing lymphoma cell specific immune responses. Further evaluation of efficacy in terms of disease control is mandatory. Fig 1. Disease and treatment course of a representative case Fig 1. Disease and treatment course of a representative case

Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease. The comparison of DLBCLs transcriptional profiles using multiple clustering algorithms led to the identification of distinct DLBCL subtypes reflecting tumor-intrinsic features. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation, a major source of potentially toxic reactive oxygen species (ROS). Therefore, we investigated the role of potential mechanisms attenuating ROS toxicity in this DLBCL subtype. We found significantly increased thioredoxin (TXN) mRNA abundance in DLBCLs classified as OxPhos subtype compared to other subtypes. The overall survival (OS) of patients with high TXN mRNA expression was significantly shorter than of those with low TXN mRNA expression, regardless of treatment regimen (R-CHOP and CHOP). TXN overexpression in OxPhos-DLBCLs was also confirmed in a cell line panel at protein level using immunoblotting. To explain transcriptional mechanisms responsible for differential TXN expression in different DLBCLs, we analyzed the TXN promoter and identified two BCL6 binding sites within 1kb upstream of TXN transcription start site. Unlike in other molecular subtypes, BCL6 does not exhibit a repressor activity in OxPhos-DLBCLs (PNAS, 2007; 104: 3207-12). Using luciferase reporter assays and shRNA-mediated gene expression knock-down, we demonstrated that relative differences in TXN abundance between DLBCL subtypes are at least in part caused by the lack of BCL6 transcription repressor activity. We next tested the consequences of TXN depletion in DLBCLs. We found that OxPhos cells with silenced TXN expression were uniformly more sensitive to apoptosis induced by ROS production than control cells. TXN inhibition sensitized all tested cell lines to doxorubicin, the fundamental drug used in DLBCL chemotherapy acting in part as a ROS inducer. In addition to its role in maintaining redox homeostasis, TXN also regulates transcriptional responses to ROS. For example, TXN reduces disulfide bonds between FOXO4 and acetyltransferase p300, which results in reduced FOXO4 acetylation and impaired proapoptotic signaling. For this reason, we assessed whether p300 and TXN are involved in acetylation of FOXO1, a major FOXO member expressed in DLBCLs. In cells with blocked TXN activity, FOXO1 acetylation was significantly higher compared to cells overexpressing wild-type TXN. TXN decreased p300-mediated FOXO1 acetylation, reduced its proapoptotic activity and expression of FOXO1-dependent genes (TRAIL, p27, BIM). We found that FOXO1 and p300 interact in redox and TXN-dependent manner and identified a conserved FOXO1's Cys612 to be responsible for FOXO1-p300 binding. We mutated FOXO1 C612 to Ala and found that when cotransfected with p300, C612A FOXO1 exhibited dramatically suppressed ROS-induced acetylation. Blockade of FOXO1 acetylation resulted in markedly lower expression of FOXO1 target genes, higher cellular proliferation and lower apoptosis. Furthermore, TXN inhibited FOXO1 nuclear translocation in response to oxidative stress in OxPhos-DLBCLs. Finally, silencing FOXO1 in OxPhos cells with knocked-down TXN expression markedly inhibited DLBCL cell line apoptosis in response to oxidative stress, suggesting that FOXO1 is an essential TXN-regulated sensor and effector of ROS toxicity in OxPhos-DLBCL cells. Taken together, these results demonstrate that TXN is overexpressed in a subset of DLBCLs, and high TXN mRNA abundance is related to shorter OS of DLBCL patients. TXN knock-down enhances oxidative stress toxicity in OxPhos cell lines at least in part by facilitating FOXO1 nuclear retention and increasing acetylation of this transcription factor, thus augmenting its proapoptotic activity. Disclosures No relevant conflicts of interest to declare.

Class I MHC-restricted CTLs are an important component of the host immune response against viral infections, and CTL effectors can often be isolated from infected individuals. However, the mechanism responsible for the induction of CTLs is incompletely understood because, in part, of the difficulty in generating such cells in vitro from naive precursors. In the present study we have used human peripheral blood dendritic cells (DCs), devoid of CD4+ T cells, to sensitize naive CD8+ T cells to exogenous Ags, resulting in the generation of Ag specific CTL effectors. With this system, Ag-specific CTL lines were generated to a complex glycoprotein, keyhole limpet hemocyanin, and to multiple small (9-15 amino acids) synthetic peptides derived from conserved regions of the HIV-1 gag and envelope proteins. The HIV-1-specific CTLs demonstrated potent HLA class I restricted killing of both Ag pulsed and virally infected target cells. In contrast to Ag-pulsed DCs, Ag-pulsed monocytes failed to sensitize CTL precursors although they could be used as feeders for purposes of CTL expansion and as target cells in cytolytic assays. With the use of the system described herein, a detailed analysis of the primary human T cell response to foreign Ags is now feasible, and CTL of desired specificity can be generated for potential clinical use in adoptive immunotherapy protocols.

Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR(+) cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34(+) cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor alpha (TNF-alpha). Transfection of CD34(+) cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.