Asc-Seurat: analytical single-cell Seurat-based web application.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of transcriptomes, arising as a powerful tool for discovering and characterizing cell types and their developmental trajectories. However, scRNA-seq analysis is complex, requiring a continuous, iterative process to refine the data and uncover relevant biological information. A diversity of tools has been developed to address the multiple aspects of scRNA-seq data analysis. However, an easy-to-use web application capable of conducting all critical steps of scRNA-seq data analysis is still lacking. We present Asc-Seurat, a feature-rich workbench, providing an user-friendly and easy-to-install web application encapsulating tools for an all-encompassing and fluid scRNA-seq data analysis. Asc-Seurat implements functions from the Seurat package for quality control, clustering, and genes differential expression. In addition, Asc-Seurat provides a pseudotime module containing dozens of models for the trajectory inference and a functional annotation module that allows recovering gene annotation and detecting gene ontology enriched terms. We showcase Asc-Seurat's capabilities by analyzing a peripheral blood mononuclear cell dataset. CONCLUSIONS: Asc-Seurat is a comprehensive workbench providing an accessible graphical interface for scRNA-seq analysis by biologists. Asc-Seurat significantly reduces the time and effort required to analyze and interpret the information in scRNA-seq datasets.

Inhibition and promotion of tumor growth with adeno-associated virus carcinoembryonic antigen vaccine and Toll-like receptor agonists.

Carcinoembryonic antigen (CEA) is a cancer vaccines' target. Several features of recombinant adeno-associated virus (rAAV) are attractive for vaccine applications. Combining other viral vector vaccines with Toll-like receptor (TLR) agonists enhances antitumor immunity. Wild-type and CEA transgenic (Tg) mice were immunized with rAAV-expressing CEA, the TLR9 agonist, oligodinucleotide (ODN)1826 and the TLR7 agonist, imiquimod. Mice were challenged with MC38 colon tumor cells and MC38 cells expressing CEA. rAAV-CEA immunization combined with ODN1826 or imiquimod enhanced CEA-specific T-helper 1 immunity and protected against tumor challenge in wild-type but not in CEA-Tg mice. In contrast, immunization with rAAV-CEA in CEA-Tg mice could abrogate the antitumor effects of ODN1826 and promote tumor growth. Compared to wild-type, CEA-Tg mice were characterized by a greater myeloid suppressor cell and T-helper 2 response to TLR agonists and to syngeneic tumors. Depleting PDCA1(+) plasmacytoid dendritic cells and Gr1(+) myeloid cells increased anti-CEA immune responses in CEA-Tg mice to rAAV-CEA-ODN1826 immunization, whereas depleting CD25(+) T cells did not. There are differences in the response of wild-type and CEA-Tg mice to rAAV-CEA, TLR agonists and syngeneic tumor. In CEA-Tg mice, tumor growth can be promoted with rAAV-CEA and TLR agonists. Dendritic and myeloid cells play a regulatory role.

Enhanced transduction of mouse bone marrow-derived dendritic cells by repetitive infection with self-complementary adeno-associated virus 6 combined with immunostimulatory ligands.

The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application of rAAV vectors encapsidated in serotype 2 capsid is hampered by poor transduction efficiency in many target tissues. These include ex vivo-generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for 7 days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to nontransduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and downregulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo-transduced and activated DC resulted in the development of CEA-specific antibody and T-helper 1-associated immune responses. Immunized mice also developed antibody to AAV6 capsid protein, which did not crossreact with AAV2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.

Coxsackievirus-adenovirus receptor genetically fused to anti-human CD40 scFv enhances adenoviral transduction of dendritic cells.

A promising approach to immunotherapy involves the loading of dendritic cells (DCs) with genetic material to facilitate sustained expression of a relevant antigen in this population of potent antigen presenting cells (APC). Viral vectors such as adenovirus (Ad) have been used for this purpose. Existing methods for DC infection are limited by lack of specificity and a requirement for DC exposure to high viral doses. Targeting of Ad to DCs with bispecific antibodies has significantly augmented levels of transgene expression. Genetic fusion of the extracellular portion of coxsackievirus-adenovirus receptor (CAR) to cell-specific ligands has also proved successful in targeting Ad to cells of interest. We report here the production and primary characterization of a new fusion protein comprising the ecto-domain of CAR connected to a single chain antibody (scFv) G28-5 against human CD40 present on the surface of DCs. We demonstrate that the fusion protein (CAR/G28) specifically interacts with both recombinant Ad fiber knob and the ecto-domain of human CD40 in a binding assay (ELISA). Finally, we show that the CAR/G28 fusion protein promotes highly efficient transduction of DCs of both rhesus monkey and human origin.

Infusional interleukin-2 and 5-fluorouracil with subcutaneous interferon-alpha for the treatment of patients with advanced renal cell carcinoma: a southwest oncology group Phase II study.

BACKGROUND: A Phase II trial was conducted to determine the response rate of patients with advanced renal cell carcinoma to a three-drug combination of 5-fluorouracil (5-FU), interleukin-2 (IL-2), and interferon-alpha-2b (IFN-alpha). METHODS: A 2-stage accrual plan was used that was designed to determine whether response to this regimen was consistent with a true response rate of >/= 30%. The regimen was comprised of 5 treatment days weekly for 4 weeks every 6 weeks. Each weekly treatment was comprised of 5-FU, 1750 mg/m(2), continuous intravenous (i.v.) infusion over 24 hours followed by IL-2, 6 MIU/m(2)/day, continuous i.v. infusion for 4 days. IFN-alpha, 6 MU/m(2), was given subcutaneously on Days 1, 2, and 5. RESULTS: Thirty-eight patients were entered on study, 3 of whom were ineligible. Among the 35 eligible patients there were 3 confirmed partial responses (PR) and 1 complete response (CR), for an overall response rate of 11% (95% confidence interval, 3-27%). One patient considered as having a PR had minimal evidence of residual disease and was free from disease progression at > 2.5 years of follow-up, as was the patient with CR. Three additional patients not qualified as having a PR were showing signs of response at the time they were removed from protocol, and another patient who was removed from protocol early for management of an infection subsequently responded to the same regimen off protocol. Thirteen patients were considered nonassessable (NASS) for response, many of whom had multiple poor risk features and were unable to complete 1 cycle of treatment. CONCLUSIONS: This multicenter study failed to confirm an advantageous overall response rate for this three-drug regimen. However, there were two durable responses and indications of responsiveness not scored as PRs among patients with more favorable risk factor patterns, and many poor risk NASS patients. For these reasons, the response rate reported in the current study may be a conservative reflection of the effectiveness of this regimen.

Prevention of mammary tumors with a chimeric HER-2 B-cell epitope peptide vaccine.

Synthetic peptide vaccines targeting B-cell epitopes of the extracellular domain of the HER-2 oncoprotein were evaluated for their capacity to elicit HER-2-specific antibodies with antiproliferative activity. Several HER-2 B-cell epitopes were identified by computer-aided analysis of protein antigenicity, and selected B-cell epitopes were synthesized colinearly with a promiscuous T-helper epitope (208-302) derived from the measles virus fusion protein at either the NH2 or COOH terminus linked via a four-residue turn sequence (GPSL). In addition, one epitope sequence, 628-647, was mutated to optimize disulfide pairing to mimic the native HER-2 receptor. All of the four selected epitopes elicited high-titered antibodies in outbred rabbits with exceptionally high titers for MVF-HER-2(628-647). These antibodies were cross-reactive with the native HER-2 receptor. Antibodies elicited by MVF HER-2(628-647) inhibited proliferation of human HER-2-overexpressing breast cancer cells in vitro and caused their antibody-dependent cell-mediated cytotoxicity. Furthermore, immunization with MVF-HER-2(628-647) prevented the spontaneous development of HER-2/neu-overexpressing mammary tumors in 83% of transgenic mice. The engineered, chimeric peptide B-cell immunogen MVF-HER-2(628-647) may have applications in the prevention of HER-2-overexpressing cancers.

B7.1 expression by the weakly immunogenic F98 rat glioma does not enhance immunogenicity.

Enhanced immunogenicity has been reported following transfection of a variety of immunogenic tumors with the B7.1 co-stimulatory molecule. The purpose of the present study was to determine if transfection of a weakly immunogenic rat brain tumor, the F98 glioma, with the gene encoding B7.1 could enhance its immunogenicity. F98 cells were transfected with a plasmid containing the B7.1 gene, and stable transfectants (F98/B7.1) were obtained. Flow cytometric analysis confirmed the expression of B7.1 and MHC class I antigens on the cell surface. To investigate the effects of B7.1 expression on the tumorigenicity of the F98 glioma, Fischer rats were implanted intracerebrally with either F98 (wild-type) or F98/B7.1 transfected cells. No significant differences in survival times were noted. Mean survival times of 21.8 and 24.0 days were observed for the respective groups at a challenge dose of 103 cells. These differences in survival time were not significant. To determine if expression of B7.1 enhanced the immunogenicity of the F98 glioma, rats were vaccinated weekly for 3 weeks with 107 mitomycin C-treated F98 or F98/B7.1 cells injected subcutaneously and then challenged intracerebrally with F98 cells 1 week later. Unvaccinated animals or those that received wild-type F98 cells as a vaccine had a survival time (mean +/- s.d.) of 22.3 +/- 1.5 days following tumor challenge versus 20.0 +/- 1.7 days for rats that had been vaccinated with F98/B7.1. Although we recognize that it might be possible to design more effective vaccination regimes, nevertheless, our data indicate that transfection of the B7.1 gene into the F98 rat glioma did not enhance its immunogenicity, and that other approaches will be required.

Intratumoral injection of dendritic cells derived in vitro in patients with metastatic cancer.

BACKGROUND: Dendritic cells (DCs) are potent initiators of immune responses, and the infiltration of DCs into tumors may confer an improved prognosis. Whether the injection of DCs directly into tumors can mediate biologic activity was examined. METHODS: Patients with metastatic dermal or subcutaneous tumors received granulocyte-macrophage-colony stimulating factor to increase the numbers of peripheral blood monocyte precursors. DCs were then generated from monocytes obtained by phlebotomy with granulocyte-macrophage-colony stimulating factor and interleukin-4 in autologous plasma. Tumors were injected at multiple sites with 30 million autologous DCs per tumor. RESULTS: Seven patients with melanoma and three patients with breast carcinoma were treated. Injections were well tolerated. Regression of the injected tumors, beginning as early as 4 days after injection, was observed in four patients with melanoma and in two patients with breast carcinoma. Biopsies of regressing lesions showed lymphocyte infiltration associated with DCs and necrosis. Neutrophils and macrophages were not evident. Lymphocytes expanded from the regressing tumors proliferated in response to heat shock proteins, HSP70 and gp96, derived from autologous tumor. The DCs injected produced interferon-alpha and expressed Fas ligand mRNA but did not exhibit cytolytic activity in vitro. Expression of the costimulatory molecule, B7-2 (CD86), decreased on DCs after intratumoral injection. CONCLUSIONS: This pilot study demonstrates that DCs derived in vitro can exist viably after intratumoral injection and can mediate biologic activity in situ. Tumor-derived heat shock proteins may be involved in the antitumor activity observed.

Modulation of CD4 cell cytokine production by colon cancer-associated mucin.

PURPOSE: Mucins have been implicated in tumor-associated immunosuppression. The possibility that colon cancer mucin (CCM) may modulate T-helper 1 (TH1) activity was evaluated by investigating its effect on the production of interleukin-2 (IL-2) by CD4+ cells, a process that requires antigen-specific and costimulatory signals. METHODS: CCM was purified from human colorectal cancer cells by gel-exclusion fast-pressure liquid chromatography. Cytokine production of purified CD4+ cells was evaluated at the protein and gene level in the presence of a phorbol ester or an anti-CD3 monoclonal antibody (mAb) plus mAb against the CD28 costimulatory receptor to mimic two-signal activation. RESULTS: Soluble CCM, which contains mucins MUC2 as well as MUC1, inhibited IL-2 mRNA expression and secretion of CD4+ stimulated with a phorbol ester or an anti-CD3 mAb plus anti-CD28 mAb. Pretreatment of CD4+ cells with anti-CD28 mAb abrogated the suppressive effects of CCM on IL-2 production, and flow cytometry showed decreased binding of anti-CD28 mAb to its receptor in the presence of mucin. In addition, Ca2+ mobilization after T cell receptor cross-linking with anti-CD3 mAb was maintained in the presence of CCM. Although interferon gamma production was also diminished, CCM did not induce a general inhibition of cytokine production, nor did it decrease cell viability. Macrophage inflammatory protein 1alpha production was up-regulated; the production of IL-10 and transforming growth factor beta was unchanged. CONCLUSIONS: The results indicate that CCM can alter TH1 activity and suggest that the modulation of costimulatory interactions is involved. They provide another mechanism of immunosuppression mediated by these highly expressed tumor products.

Cellular immunotherapy for patients with metastatic colorectal carcinoma using lymph node lymphocytes localized in vivo by radiolabeled monoclonal antibody.

BACKGROUND: The authors showed previously that radiolabeled monoclonal antibody (MoAb) and a hand-held, gamma-detecting probe can be used to localize tumor-reactive lymph nodes in vivo. The authors examined the feasibility, safety, and biologic effects of cellular immunotherapy using autologous cells expanded from these lymph nodes in patients with metastatic colorectal carcinoma. METHODS: Tumor-reactive lymph nodes containing radiolabeled MoAb were localized and excised from 32 patients with metastatic, unresectable colorectal carcinoma at laparotomy. Lymph nodes were dissociated, and cells were cultured ex vivo for 10-14 days. Patients received a single infusion of autologous, expanded cells with no systemic interleukin (IL)-2. RESULTS: A mean of 1.6 x 10(10) expanded autologous lymph node cells were infused with toxicity limited to occasional fevers or chills. The cells infused predominately were activated CD3+ T-cells that expressed genes for IL-4, IL-5, interferon-gamma, and granulocyte-macrophage colony stimulating factor (GM-CSF) by using reverse transcriptase-polymerase chain reaction. Indium-111 labeled cells were observed to traffic initially to the lungs, bone marrow, liver, and spleen. One patient on study achieved a partial response (>80% reduction), and mixed or minor responses were noted in 4 other patients. The responding patient's cell characteristics were notable for high levels of GM-CSF and IL-4 secretion on restimulation with immobilized anti-CD3 in vitro, and biopsies of the tumor were characterized by macrophage infiltration. The median survival of the cell-treated group compared favorably with a similar group of patients who underwent radioimmunoguided surgery without cell treatment (12.5 months vs. 5.8 months) CONCLUSIONS: The infusion of cells expanded from tumor-reactive lymph nodes localized with radiolabeled MoAb in vivo is reproducible and safe and has biologic activity, even in the absence of systemic IL-2 infusion. This approach represents a novel application of MoAb technology, in that MoAbs are used not to diagnose or treat disease directly but rather to identify lymph node cells with therapeutic potential.

Low-dose monoclonal antibody CC49 administered sequentially with granulocyte-macrophage colony-stimulating factor in patients with metastatic colorectal cancer.

The clinical and immunologic effects of murine monoclonal antibody (mAb) CC49 administered at a low dose sequentially with granulocyte-macrophage colony-stimulating factor (GM-CSF) were examined. Fourteen patients with metastatic colorectal cancer received 1 mg of unconjugated CC49 on day 1; on day 15 they began 125 micrograms/m2 GM-CSF by subcutaneous injection daily for 14 days, followed by 7 days of rest. Another 14 days of GM-CSF were then administered, followed by 7 days of rest. This 56-day cycle was repeated in patients whose cancer did not progress. Therapy was well tolerated; adverse allergic reactions were not observed. Objective tumor responses were not observed. Increases in antiidiotypic (T2) and anti-antiidiotypic (T3) cellular responses were observed, as were increases in human antimouse antibody levels. In contrast, the expression of Fc receptors on CD14+ peripheral blood monocytes decreased. This pilot study demonstrates idiotypic cellular immunologic effects of antitumor murine mAb, even at the doses used for imaging, and supports the sequential administration of GM-CSF as an adjuvant to mAb-based immunogens.

Human chorionic gonadotropin as a target for cancer vaccines.

Human chorionic gonadotropin (hCG) is expressed by common cancers and may play a role in cell transformation as well as angiogenic, metastatic, and immune escape phenomena that are central to cancer progression. Clinical trials with a vaccine targeting the carboxy-terminal peptide of -hCG have indicated that tolerance to this oncofetal antigen can be broken. Humoral responses that may modulate the biologic activity of tumor-associated hCG as well as cellular responses to hCG have been generated. Studies are in progress to further define the biologic significance of hCG in cancer and to develop a vaccine approach that will best target this expression.

Cellular immunotherapy of advanced human immunodeficiency virus type 1 infection using autologous lymph node lymphocytes: effects on chemokine production.

A pilot study was undertaken in patients with human immunodeficiency virus type 1 (HIV-1) infection to examine the effects of infusing autologous lymph node lymphocytes that had been cultured ex vivo in conditions designed to maximize the specific secretion of HIV-1-suppressive factors, including beta chemokines. Ten patients with CD4 cell counts between 119 and 436/microliter on antiretroviral drugs received a single infusion of CD4 and CD8 lymph node lymphocytes. There were no serious acute or chronic adverse clinical effects. Increases in serum levels of macrophage inflammatory protein 1beta (MIP-1beta) and increases in the production of MIP-1beta by peripheral blood lymphocytes in response to HIV-1 env were observed. Increases in CD4 and CD8 cell counts and skin test reactivity to recall antigens and decreases in HIV-1 virus load were also observed. This cellular immunotherapy can modulate beta chemokine production in patients with advanced HIV-1 infection and may contribute immunorestorative and antiviral activities.

HIV type 1-reactive chemokine-producing CD8+ and CD4+ cells expanded from infected lymph nodes.

The chemokines RANTES, MIP-1alpha, and MIP-1beta have been identified as HIV-1-suppressive factors produced by CD8+ T cells. We examined the possibility that HIV-1-specific, chemokine-releasing T cells could be expanded from the lymph nodes of patients with advanced infection. Lymphocytes, separated from lymph nodes of patients with peripheral blood CD4 counts less than 500/microl obtained at diagnostic biopsies, were activated with anti-CD3 monoclonal antibody, and cultured in vitro for up to 12 days with IL-2. The phenotype, proliferative response, chemokine production, and anti-HIV-1 activity of the expanded cells was examined. Cells expanded 2.4- to 49-fold from patients with as few as 15 CD4+ cells/microl in their peripheral blood. Expanded cells were a mixture of CD8+CD45RO+ and CD4+CD45RO+ T cells. The CD8+ cells were also CD30+CDw60+CD11b-. When challenged with autologous B cell targets expressing HIV-1 Env protein, unseparated expanded cells, and purified CD8+ and CD4+ T cell subsets, proliferated and secreted MIP-1alpha and RANTES. Expanded cells were negative for HIV-1 by PCR and by culture. Culture supernatants inhibited the replication of HIV-1 in CD4+ cells in vitro. These studies indicate that HIV-1 can stimulate chemokine release by CD8+ and CD4+ cells expanded from infected lymph nodes, even from individuals with advanced infection. The numbers of chemokine-releasing T cells produced in these short-term cultures may be sufficient to be applied therapeutically as an autologous cellular therapy for HIV-1.

Adoptive immunotherapy of feline leukemia virus infection using autologous lymph node lymphocytes.

Adoptive immunotherapy using autologous cells expanded ex vivo from lymph nodes was examined in cats infected with the retrovirus feline leukemia virus (FeLV). Cells were obtained from popliteal lymph nodes from 18 FeLV-antigen-positive cats without complications; a mean of 6.2 x 10(7) cells were obtained. Lymph node cells were cultured with 600 IU/ml interleukin-2 (IL-2) for 7 days. Cells expanded 0.8- to 11-fold (mean, 2.7; median, 2.4); were 80% +/- 8.0% CD3+, 29% +/- 8.1% CD4+, and 41% +/- 7.0% CD8+, and exhibited cytolytic activity against FeLV-transformed FL74 cells. Sixteen cats received a single intravenous infusion of 0.13 to 3.9 x 10(8) cells. Cell infusion was well tolerated; fever developed approximately 1 hour postinfusion. Clinical activity, antiviral activity, or both was observed in 10 cats. Nine cats had clinical responses with improvement in weight, activity, appearance, or a combination of these that began 2 to 4 weeks after cell infusion and that lasted for up to 13 or more months. FeLV antigen became undetectable in 4 cats. These results indicate that adoptive immunotherapy using autologous lymph node cells, activated and expanded ex vivo in short-term cultures with low concentrations of IL-2, can modulate the course of a retroviral infection.

Oncogene and cytokine expression of human colorectal tumors responding to immunotherapy.

Tumor samples from five patients with metastatic colorectal cancer who demonstrated tumor regressions in clinical trials of interleukin (IL)-1 beta, IL-2, and adoptive cellular therapy were analyzed for oncogene and cytokine mRNA expression. Tumors from eight nonresponding patients were also studied. Mutations of the ras protooncogene and overexpression of c-myc protooncogene were observed in both responding and nonresponding tumors. In contrast, none of the responding tumors expressed transforming growth factor (TGF)-beta 1 mRNA, whereas nonresponding tumors did. The expression of IL-1, IL-6, IL-8, IL-10, tumor necrosis factor-alpha, granulocyte macrophage-colony-stimulating factor, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, macrophage chemotactic protein, and RANTES was variable between responding and nonresponding patients. Although we cannot conclude that a pattern of oncogene and/or cytokine mRNA expression specifically characterizes sensitive colorectal cancers, these analyses-the assessment of TGF-beta 1 mRNA in particular-merit further evaluation as biomarkers prognostic of immunotherapy response.

Phenotypic and functional differences between human dendritic cells derived in vitro from hematopoietic progenitors and from monocytes/macrophages.

We compared dendritic cells (DC) derived from CD34+ hematopoietic progenitor cells with tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) to DC derived from monocytes/macrophages with interleukin-4 (IL-4) and GM-CSF. Monocyte/macrophage-derived DC demonstrated higher levels of CD1a, lower levels of CD14, greater stimulatory activity in mixed lymphocyte reactions, and greater capacity to present soluble protein antigen than CD34+ cell-derived DC. Lymphocytes stimulated with antigen-pulsed, monocyte/macrophage-derived DC produced more IL-10 than those stimulated with antigen-pulsed, CD34+-derived DC. Whereas CD1a+ DC could be derived from CD34+ cells in serum-free- and human-sera-containing cultures, the derivation of CD1a+ DC from monocytes/macrophages required the presence of fetal calf serum. The spectrum of cytokine mRNA expression, the presentation of peptide antigen, and the sensitivity to human immunodeficiency virus-1 infection of CD34(+)- and monocyte/macrophage-derived DC were comparable. Although cells derived by both methods are potent antigen-presenting cells, there are differences between DC derived in vitro from hematopoietic progenitors and from monocytes/macrophages that may influence their in vivo activity.