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The development of vaccines against infectious diseases is one of the greatest achievements of modern medicine. However, the results of clinical trials of vaccines against established malignancies have been disappointing, clearly demonstrating a compelling need to discover novel nontoxic vaccine adjuvants as well as to develop innovative vaccination protocols that combine other therapies to create synergistic effects. We have previously shown that a polyphenylpropenoid-polysaccharide complex (PPC) derived from the cones of Pinus sylvestris induces the differentiation and maturation of dendritic cells (DCs) in vitro and enhances DC vaccine-induced antigen-specific CD8+ T-cell responses by oral administration. In the present study, we explored the potential of a combination chemoimmunotherapy, including PPC, a DC-based vaccine and low-dose cyclophosphamide (CTX), to eradicate established large tumors in mice. TC-1 tumor cells were injected subcutaneously into C57BL/6 mice (five mice per group) on day 0. TC-1 is a mouse tumor cell line expressing the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV16). Bone marrow-derived DCs (BMDCs) were prepared by culturing murine BM cells in the presence of GM-CSF. These BMDCs were pulsed with the synthetic tumor antigen peptide HPV16 E743-77. Peptide-pulsed DCs were activated further ex vivo with PPC (PPC-DC(E7)) and then injected into tumor-bearing mice on days 13, 20 and 27. Vaccination with PPC-DC(E7) was followed by oral administration (via gavage) of PPC. Low-dose CTX was administered intraperitoneally 3 days prior to each vaccination with PPC-DC(E7). We observed that 1) all of the untreated control mice died by day 39 post TC-1 injection; 2) treatment with low-dose CTX alone significantly suppressed tumor growth compared to untreated mice (p = 0.0132, day 28) but did not eradicate large established tumors (these mice died by day 56); 3) therapy with low-dose CTX plus oral administration of PPC more strongly suppressed TC-1 tumor growth (p = 0.0008, day 28), and two out of five mice had complete tumor regression by day 42 (these mice were tumor-free on day 231); 4) five out of five mice treated with low-dose CTX and PPC-DC(E7) vaccine had complete tumor regression by day 39, but two of these mice developed tumors again on days 70 and 196 and died on days 98 and 222, respectively (the remaining three mice were tumor-free on day 231); and 5) five out of five mice treated with low-dose CTX and PPC-DC(E7) vaccine plus oral administration of PPC had complete tumor regression by day 39, and all of these mice remained tumor-free on day 231. These results show that therapy with PPC (a DC-activating vaccine adjuvant) and a DC-based vaccine, in combination with low-dose CTX, induced complete eradication of established large tumors in 100% of mice. This may comprise a promising strategy for the design of a safe and effective vaccination protocol to treat established malignancies. Citation Format: Soichi Haraguchi, William Guy Bradley, Akiko Tanaka. A combination chemoimmunotherapy with a dendritic cell-based vaccine activated by a polyphenylpropenoid-carbohydrate complex and low-dose cyclophosphamide completely eradicates established large tumors in mice [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B10.

Studies of the effects of retroviruses on the immune system, which date back through thirty years of investigations, are reviewed. In the earliest published studies in the 1960s, it was demonstrated that mice infected with oncogenic viruses were immunosuppressed. Since then, numerous articles have been published describing profound immunodeficiencies observed in vivo in humans infected with human immunodeficiency virus and in animals such as cats infected with the feline immunodeficiency virus. In vitro investigations have shown that inactivated retroviruses or transmembrane envelope protein pl5E as well as a synthetic 17-amino acid peptide (CKS-17) impressively conserved within the transmembrane envelope protein of several animal or human retroviruses are highly immunosuppressive. More recently, dysfunction of cytokines produced by CKS-17 at both a cellular and molecular level have been found to mimic influences observed in vivo in patients infected with the human immunodeficiency virus. CKS-17 has also been shown to induce cAMP in vitro. The significance of these observations to understanding the immunological disturbances observed in malignancy, cytokine biosynthesis, and modulations of immune functions through cAMP is discussed.

The mechanism(s) by which retroviral proteins exert immunosuppressive influences has remained enigmatic. Here, Soichi Haraguchi, Robert Good and Noorbibi Day propose that induction of intracellular cAMP by a synthetic, immunosuppressive, retroviral envelope peptide causes a shift in the cytokine balance, leading to suppression of cell-mediated immunity by upregulation of interleukin 10 (IL-10) and downregulation of IL-2, IL-12 and tumor necrosis factor α production. This may be a crucial step towards generation of immune dysfunction.

A synthetic heptadecapeptide, CKS-17, represents the highly conserved amino acid sequences occurring within the transmembrane envelope protein of many animal and human retroviruses. CKS-17 has been demonstrated to exhibit suppressive properties for numerous immune functions. We have recently shown that CKS-17 acts as an immunomodulatory epitope causing an imbalance of human type 1 and type 2 cytokine production and suppression of cell-mediated immunities. cAMP, an intracellular second messenger, plays an important role in regulation of cytokine biosynthesis--i.e., elevation of intracellular cAMP levels selectively inhibits type 1 cytokine production but has no effect or enhances type 2 cytokine production. Here, we demonstrate that CKS-17 induces dramatic rises in the intracellular cAMP levels of a human monocyte cell line and of human peripheral blood mononuclear cells in a time- and dose-dependent manner. A peptide corresponding to the reverse sequence of CKS-17, used as control, has no effect on intracellular cAMP levels. The cAMP-inducing ability of CKS-17 is significantly blocked by SQ-22536, an inhibitor of adenylate cyclase. These results indicate that CKS-17, a highly conserved component of the transmembrane proteins of immunosuppressive retroviruses, induces increased intracellular levels of cAMP via activation of adenylate cyclase and suggest that this retroviral envelope peptide may differentially modulate type 1 and type 2 cytokine production through elevation of intracellular cAMP levels.

Prolactin plays a key role in the regulation and growth of mammary cells, and influences tumor promotion. We have shown that chronic energy restriction intake depresses prolactin levels, inhibits production of MMTV proviral DNA and proto-oncogene expression in mammary glands and prevents development of mammary tumors. Since the expression and proto-oncogene activation of MMTV are regulated by promoter/enhancer elements within its long terminal repeat (LTR), in the present study we used a chloramphenicol acetyl transferase (CAT) reporter gene system and gene transfection methods to study the effect of prolactin on MMTV LTR using a human ductal carcinoma cell line T47D stably or transiently transfected with a plasmid consisting of the LTR upstream of CAT gene. Human prolactin or dexamethasone induced, respectively, a 2-fold or 6-fold increase in CAT activity compared with background CAT activity in the absence of hormones. However, the combination of human prolactin and dexamethasone strongly enhanced (20-fold) induction of the LTR compared with the control. Human prolactin also showed a synergistic effect with progesterone on LTR induction. Both LTR and CAT genes needed to be linked for induction of CAT activity by prolactin and dexamethasone. Our results indicate that human prolactin can act synergistically with steroid hormones to regulate MMTV LTR-directed gene expression in transfected T47D cells. © 1992 Wiley-Liss, Inc.

We investigated the influence of CKS-17, a synthetic heptadecapeptide that corresponds to a highly conserved domain of the immunosuppressive retroviral envelope protein p15E, on staphylococcal enterotoxin B (SEB)-induced TNF-alpha gene expression in human peripheral blood mononuclear cells and highly purified human monocyte preparations, as well as the production of TNF-alpha protein, using human peripheral blood mononuclear cells. RNA hybridization studies show that CKS-17 inhibits SEB-induced TNF-alpha mRNA accumulation in human peripheral blood mononuclear cells and human monocytes. CKS-17 is also shown to be highly suppressive for SEB-induced production of TNF-alpha proteins. Similarly, CKS-17 inhibits expression of SEB-induced IFN-gamma mRNA in human peripheral blood mononuclear cells. These results suggest that CKS-17 down-regulates both TNF-alpha and IFN-gamma production at mRNA level.

CKS-17, a synthetic amino acid peptide homologous to a highly conserved region of retroviral transmembrane protein exerts a suppressive action on staphylococcal enterotoxin A (SEA)-induced the production of IFN-γ by human peripheral blood mononuclear cells (PBMC) (Ogasawara et al., J. Immunol. 141 , 615, 1988). This action has been shown in the present study to be preceded by dramatic clustering of PBMC. Clusters appear within 3 hr of exposure of PBMC to CKS-17; they are dose dependent, inhibited by cycloheximide, and require a temperature of 37 °C. The cells in the clusters are predominantly monocytes. Although it has been previously shown that CKS-17 inhibits monocyte-mediated killing by inactivating IL-1 (Kleinerman et al., J. Immunol. 139 , 2329, 1987) and production of IL-2 by murine thymoma cells treated with IL-1 (Gottlieb et al., J. Immunol. 142 , 4321, 1989), in the present study we show that IL-1 does not prevent clustering of PBMC by CKS-17. Using CKS-17 and highly purified monocytes or lymphocytes, profound alterations occur only with monocytes, as revealed by light or electron microscopy. SEA- or staphylococcal enterotoxin B-induced production of IFN-γ is inhibited when highly purified monocytes pretreated with CKS-17 are cocultured with highly purified T lymphocytes. Thus, CKS-17 induces dramatic clustering of cells apparently by inducing alterations of monocytes but not lymphocytes, suggesting that CKS-17 may interfere with the capacity of monocytes to facilitate production of IFN-γ by T lymphocytes.

Immunization with dendritic cell (DC)-based vaccines is a promising vaccination strategy to provide an effective personalized immunotherapy against cancer and other diseases. However, the results of clinical cancer immunotherapy trials carried out using DC-based vaccines to date have shown only modest clinical outcomes. We have previously demonstrated that a polyphenylpropenoid-polysaccharide complex (PPC), a lignin-carbohydrate complex derived from the cones of Pinus sylvestris, induces in vitro the differentiation and maturation of human mononuclear cell-derived DCs (Bradley et al., Int Immunopharmacol 2003;3) and murine bone marrow-derived DCs (BMDCs) (An et al., Anticancer Res 2010;30). Furthermore, oral administration of PPC enhanced a DC vaccine-induced melanoma antigen-specific CD8+ T-cell response (Burrows et al., BMC Complement Altern Med 2009;9) and the proliferative phase of a primary T cell response (Bradley et al., BMC Complement Altern Med 2014;14). In the present study, using a human papillomavirus (HPV)-induced mouse tumor model, we investigated whether ex vivo treatment of BMDCs with the synthetic lignin-like polyphenol polymers poly-caffeic acid (pCA) and poly-coumaric acid (pCoA), as well as PPC, improves the efficacy of DC-based vaccines. Polymerization of the phenylpropenoic acids caffeic acid (CA) and poly-coumaric acid (CoA) was performed by horseradish peroxidase/hydrogen peroxide-mediated enzymatic reactions. BMDCs were prepared by culturing murine bone marrow cells in the presence of GM-CSF for 8-10 days. These BMDCs were pulsed with synthetic tumor antigen peptides HPV16 E743-62 or HPV16 E743-77 that contain both a cytotoxic T lymphocyte (CTL) and a helper T cell epitope. Peptide-pulsed DCs were treated further ex vivo with synthetic polyphenol polymers or PPC and then injected twice into C57BL/6 mice. In addition, splenocytes obtained from immunized mice were restimulated with HPV16 E749-57 peptide (E7 CTL epitope) for 5 days. Tumor antigen-specific CD8+ T cell responses were assessed by HPV16 E749-57/H-2Db pentamer staining. We observed that immunization with PPC-treated HPV16 E743-77 peptide-loaded BMDCs significantly enhanced the induction of pentamer-positive CD8 positive cells in splenocytes (2- to 3-fold increase). Moreover, ex vivo restimulation with E7 CTL epitope peptide dramatically increased the number of pentamer-positive CD8 positive cells in splenocytes obtained from mice immunized with PPC-treated, peptide-loaded DCs (6- to 7-fold increase) when compared to splenocytes from mice immunized with PPC-nontreated, peptide-loaded DCs. Experiments using pCA and pCoA are in progress. We conclude that 1) ex vivo treatment with the natural PPC complex significantly potentiates the in vivo efficacy of DC-based HPV vaccines and 2) tumor antigen-specific CD8+ T cells are significantly expanded ex vivo by restimulating splenocytes from mice primed in vivo with PPC-treated, peptide-loaded BMDCs. In addition, our preliminary studies show that PPC, pCA and pCoA significantly increase the expression levels of CD8α, XCR1 and/or CLEC9A on murine BMDCs that have been demonstrated to be essential for the induction of CTL responses. Our approaches will provide innovative strategies for the design of more effective DC-based vaccines and adaptive T-cell therapies for the treatment of patients with cancer and other immune disorders. Citation Format: Soichi Haraguchi, Tina Pastoor, William Guy Bradley, Akiko Tanaka. Improving the efficacy of dendritic cell-based human papillomavirus vaccines in mice using a natural lignin-carbohydrate complex and/or synthetic lignin-like polyphenol polymers. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B29.

Interleukin-12 (IL-12), an important cytokine in host defense against microbial pathogens, regulates natural killer and T-cell function(s) including the induction of γ-interferon production. The major cellular sources of IL-12 are monocytes/macrophages. Bacteria, bacterial products, and intracellular parasites are the most efficient inducers of IL-12 production. In the present study we show that a signal transduction pathway sensitive to rapamycin may have an important role in the regulation/suppression of Staphylococcus aureus–induced IL-12 production in vitro. Human peripheral blood mononuclear cells, monocytes, or a human monocytic cell line THP-1 were stimulated with S. aureus Cowan strain 1 (SAC) in the presence or absence of rapamycin and investigated for production of IL-12 protein by enzyme-linked immunosorbent assay and IL-12 p40 mRNA accumulation by RNase protection assay or real-time quantitative polymerase chain reaction. The results show that rapamycin significantly enhances SAC-induced IL-12 p70 protein production and IL-12 p40 mRNA accumulation. Further the results demonstrate that wortmannin enhances SAC-induced IL-12 p40 mRNA accumulation, whereas Ly294002 does not. These data indicate that a rapamycin-sensitive signaling pathway may act as a negative feedback cascade in the regulatory mechanisms of IL-12 production.

Background/Aims: Proteinuria in idiopathic minimal lesion nephrotic syndrome (IMLNS) is presumed to be due to the effect of circulating factors on glomerular permeability to plasma proteins. This study examines the expression of messenger ribonucleic acid (mRNA) for cytokines thought to mediate glomerular inflammation during different stages of the nephrotic syndrome. Methods: Messenger RNA expression and stability from peripheral blood mononuclear cells of IMLNS patients in relapse and in remission, and age matched normal controls were measured using a ribonuclease protection assay (RPA). The spontaneous and Interleukin 2 (IL-2) stimulated mRNA expression were studied. Results: Spontaneous mRNA expression for Interleukin 8 (IL-8) from IMLNS patients in relapse was significantly increased when compared to IMLNS patients in remission and normal controls (p < 0.05). After 14 h of IL-2 stimulation, mRNA IL-8 levels expressed by IMLNS PBMC patients in remission were not different from those observed in normal controls. However, after 5 days of PBMC incubation, a significant increase in mRNA for IL-8 in IMLNS patients compared to controls was found (p < 0.01). Stability assay demonstrated that IL-8 mRNA transcript from the nephrotic patients remained higher than those from controls and showed a significantly prolonged life t1/2 (p = 0.02). Conclusions: IL-8 mRNA expression is increased in IMLNS patients in relapse. Moreover, stability studies show that IL-8 mRNA life t1/2 is prolonged due to altered post-transcriptional regulation. This finding may explain the elevated serum IL-8 levels observed in these patients during relapse and may have pathogenic significance since IL-8 has been shown to induce proteinuria in the experimental animal.

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.

A rat hepatoma cell line, FF101, established in serum-free, protein-free medium, synthesizes a growth factor (FF-GF). FF-GF was purified by gel filtration chromatography, cation-exchange chromatography and isoelectric focusing. Purified FF-GF was revealed as a single band on SDS-gel electrophoresis and its molecular weight was estimated to be 70 KDa. FF-GF stimulated DNA synthesis of various cells from different origins. The growth-promoting activities of FF-GF were abolished by treating with protease, dithiothreitol, acid and heating, wherease its activity was not inhibited by antibodies against acidic and basic fibroblast growth factors. These results indicate that FF-GF is a novel growth factor.

Early studies with the Gross passage A leukemia virus demonstrated that retroviral infection suppresses cellular and humoral immune responses. In extensive studies of the feline leukemia (FeLV) virus, which can induce profound immunodeficiency disease, are generative anemia and lymphoid, myeloid and erythroid neoplasia, the immunosuppressive effects of this retrovirus could be attributed to the actions of the retroviral envelope protein p15E. We found that a highly conserved, synthetic 17 amino acid peptide synthesized by Cianciolo and co-workers that is homologous to the hydrophilic portion of the otherwise hydrophobic transmembrane envelope protein can suppress polyclonal activation of B-cells, impair production of gamma- and alpha-interferon, inhibit production of interleukin-2, inhibit expression of IL-2 receptors, and suppress responses of cytotoxic lymphocytes. In analyses with inactivated preparations of the human immunodeficiency virus, with Pahwa et al. we demonstrated that purified non-infectious retrovirus and also retroviral proteins, in particular gp120, appeared to produce some of the immunosuppressive properties of HIV, particularly suppression of B-cell activation in response to known B-cell stimulants irrespective of T-cell influence, suppression of T-helper cell functions essential to B-lymphocyte responsiveness, and impaired function of immunoglobulin-secreting cells. Other investigators have also reported strong immunosuppressive or immunostimulatory influences for components of the HIV retrovirus and also gp120 through yet poorly elucidated but certainly complex actions on both T- and B-lymphocyte-mediated immune functions.