J-LEAPS vaccines initiate murine Th1 responses by activating dendritic cells.

The Ligand Epitope Antigen Presentation System (LEAPS) converts a peptide containing a T cell epitope as small as 8 amino acids into an immunogen and directs the nature of the subsequent response. Tandem synthesis of the J peptide (a peptide from the beta-2-microglobulin) with peptides of 15 or 30 amino acids from HSV-1 or HIV made them immunogenic and promoted Th1 immune responses. Immunization of A/J or C57BL/6 mice with J-LEAPS heteroconjugates containing an epitope from the HSV-1 glycoprotein D (JgD) or an epitope from the HIV gag protein (JH) emulsified with Seppic ISA51 induced increased levels of IL-12p70 by day 3 and increased levels of interferon gamma (IFN-gamma) on days 10 and 24. Interestingly, levels of IL-10, TNF-alpha, and IL-6 did not change. Neither the H nor the gD peptides alone elicited responses and only weak responses followed immunization with the J peptide. Bone marrow (BM) cells became CD86 and CD11c positive within 48 h of treatment with JgD or JH. JH or JgD treatment promoted IL-12p70 production and expression of CD8 denoting the maturation and activation of a subclass of myeloid DCs. Pure cultures of immature myeloid DCs also responded to JgD treatment, forming clusters, developing dendrites, and producing IL-12p70 within 24 h. The JH or JgD treated bone marrow cells (JgD-DC) were necessary and sufficient to activate splenic T cells to produce IFN-gamma and the JgD-DC provided an antigen specific booster response to T cells from JgD immunized mice. Adoptive transfer of JgD-DC was also sufficient to initiate protective antigen specific immunity from lethal challenge with HSV-1. The J-LEAPS vaccines appear to act as an adjuvant and immunogen on DC precursors in a unique manner to promote activation and maturation into IL-12p70 producing DCs which then can initiate sufficient Th1 immune responses to elicit protection without production of acute phase cytokines.

Radiation therapy and Toll-like receptor signaling: implications for the treatment of cancer.

The identification of pathogen-associated molecular patterns, conserved microbial structures that act on Toll-like receptors, has led to a novel avenue of investigation aimed at developing a new generation of cancer immunotherapies. Ligation of Toll-like receptors results in the induction of robust immune responses that may be directed against tumor-associated antigens. Recent data suggest that such strategies may result in enhanced antitumor immunity. Nonetheless, as clinically effective immunotherapy for cancer remains a somewhat distant goal, attention has shifted toward multimodality approaches to cancer therapy, sometimes combining novel immune interventions and conventional treatments. The traditional view of radiation therapy as immunosuppressive has now been challenged, prompting a re-evaluation of its potential as an adjunct to immunotherapy. Radiation therapy can enhance the expression of tumor-associated antigens, induce immune-mediated targeting of tumor stroma, and diminish regulatory T cell activity. Recent evidence suggests that radiation therapy may also activate effectors of innate immunity through TLR-dependent mechanisms, thereby augmenting the adaptive immune response to cancer. In this paper, we will review evidence for enhanced tumor-directed immunity resulting from radiation exposure and early promising data suggesting synergistic effects of radiation and TLR-targeted immunotherapies.

Multiple mechanisms for HSV-1 induction of interferon alpha production by peripheral blood mononuclear cells.

UV-inactivated, infectious, and other forms of herpes simplex virus 1 (HSV-1) induced interferon (IFN) production by different routes in myeloid origin mononuclear cells (MOMC) (consisting predominantly of monocytes). GM-CSF activated the MOMC (G-MOMC) to produce greater amounts of interferon while differentiation to DC, by the addition of granulocyte macrophage colony stimulating factor (GM-CSF) and calcium ionophore (GA-MOMC), reduced the levels of interferon production upon challenge with some HSV strains. UV-inactivated virus induced more interferon than infectious virus. L-fucose, an antagonist of the mannose receptor, inhibited the induction of IFN-alpha by UV-inactivated virus and gB(-) virus (defective in penetration) in MOMC and GA-MOMC but not G-MOMC. L-fucose had little effect on interferon induction by infectious HSV-1. The insensitivity of the G-MOMC to fucose inhibition distinguishes these interferon producing cells from the pDC2 cells previously described as natural interferon producing cells. The mannose receptor appears to be involved in the response to non-infectious forms of HSV but infectious virus appears to use a different pathway. These studies suggest that non-infectious virions and HSV infected cell debris effectively stimulate monocytes and pre-dendritic cells to produce IFN-alpha to initiate host protection against HSV infection.

Rapid lipopolysaccharide-induced differentiation of CD14(+) monocytes into CD83(+) dendritic cells is modulated under serum-free conditions by exogenously added IFN-gamma and endogenously produced IL-10.

We showed previously that about half of purified CD14(+) peripheral blood monocytes cultured under serum-free conditions and treated with GM-CSF and bacterial LPS rapidly (2 - 4 day) differentiate into CD83(+) dendritic cells (DC). The remaining cells retain the CD14(+)/CD83(-) monocyte/macrophage phenotype. In order to identify factors that influence whether monocytes differentiate into DC or remain on the monocyte/macrophage developmental pathway, we evaluated the effects of exogenously added IFN-gamma and endogenously produced IL-10 on the proportion and function of CD14(+) monocytes that adopt DC characteristics in response to LPS. IFN-gamma priming dramatically increased the proportion of monocytes that adopted stable DC characteristics in response to LPS, improved their T cell allosensitizing capacity, and enhanced levels of secreted IL-12 heterodimer. IFN-gamma priming also suppressed the production of IL-10, a cytokine known to have inhibitory effects on DC differentiation. When monocytes were treated with LPS plus IL-10-neutralizing antibodies, dramatically enhanced DC differentiation, IL-12 secretion, and T cell allosensitizing capacity were observed, mimicking in many respects the effects of IFN-gamma priming. IFN-gamma primed cells still displayed appreciable sensitivity to exogenously added IL-10, suggesting that attenuated IL-10 secretion is partially responsible for the enhancing effects of IFN-gamma. These studies therefore identify IFN-gamma as a DC differentiation co-factor for CD14(+) monocytes, and IL-10 as an autocrine/paracrine inhibitor of DC differentiation, linking these agents for the first time as mutually opposed regulators that govern whether CD14(+) cells differentiate into DC upon contact with LPS or remain on the monocyte/macrophage developmental pathway.

CD14+ monocytes as dendritic cell precursors: diverse maturation-inducing pathways lead to common activation of NF-kappab/RelB.

Dendritic cells are extremely potent antigen-presenting cells that are primarily responsible for the sensitization of naïve T cells to protein antigen in vivo. For this reason, dendritic cells are the focus of intense study. Despite this interest, relatively little information is available on the signal transduction pathways that regulate the development and activity of these cells. The last several years, however, have seen a steady accumulation of data regarding methods to cultivate large numbers of DC, the characterization of attendant signals that drive DC development from various precursor cells, and the induction of nuclear transcription factors that presumably direct alterations in gene expression that regulate aspects of DC development. In this review, we briefly summarize some of these findings, with emphasis on monocyte-derived dendritic cells and a discussion of two distinct types of signaling pathways that appear to regulate the final maturation of DC: one pathway calcium-dependent and cyclosporine A-sensitive, the other pathway CsA-insensitive. Although evidence suggests these signaling pathways are quite divergent in their upstream components, they both appear to activate NF-kappaB nuclear factors, particularly RelB.

T-cell adoptive therapy of tumors: mechanisms of improved therapeutic performance.

The T cells of many cancer patients are naturally sensitized to tumor-associated antigens (Ag), or they can readily be sensitized with vaccine maneuvers. In melanoma patients, the adoptive transfer of such T cells can often be causally linked to the objective regression of established tumors. So far, few patients have shown sustained clinical benefit from such therapy, but preclinical mouse studies have now clearly delineated the hurdles that must be overcome to render T-cell-based antitumor therapy effective. Contrary to earlier expectations, it is now established that remarkably potent CD4+ and CD8+ pre-effector T cells are naturally sensitized even in mice bearing progressive, weakly immunogenic tumors. However, such T cells often display signal transduction impairments as a consequence of the tumor environment, which limit their acquisition of optimal effector function. Extracorporealization and culture of these tumor-sensitized T cells with appropriate activation stimuli not only restores normal signal transduction, but also confers resolute effector activity that can often sustain tumor rejection upon reinfusion. In mouse studies, the L-selectin(low) fraction of T cells in tumor-draining lymph nodes (TDLN) constitutes the potent pre-effector population and comprises both CD4+ and helper-independent CD8+ T cells. Appropriate in vitro activation confers an apparently unrestricted trafficking capacity to this fraction, and even the ability to proliferate within the tumor bed, leading to unprecedented tumor rejection at anatomic sites (e.g., subcutaneous and intracranial) that were historically refractory to such treatment. Such results underscore the surprising capacity of appropriately activated effector T cells to withstand the immunosuppressive, tolerogenic, and apoptotic influences of the typical tumor environment. Given the increasingly appreciated and critical communications between T cells and host Ag-presenting cells (APC), which cross-present tumor Ag, it is likely that dendritic cell-based vaccine maneuvers that promote sensitization of T1-committed L-selectin(low) antitumor T cells will play an increasingly important role in adoptive therapy strategies.

Bacterial lipopolysaccharide, TNF-alpha, and calcium ionophore under serum-free conditions promote rapid dendritic cell-like differentiation in CD14+ monocytes through distinct pathways that activate NK-kappa B.

To facilitate the study of signaling pathways involved in myeloid dendritic cell (DC) differentiation, we have developed a serum-free culture system in which human CD14+ peripheral blood monocytes differentiate rapidly in response to bacterial LPS, TNF-alpha, or calcium ionophore (CI). Within 48-96 h, depending on the inducing agent, the cells acquire many immunophenotypical, morphological, functional, and molecular properties of DC. However, there are significant differences in the signaling pathways used by these agents, because 1) LPS-induced, but not CI-induced, DC differentiation required TNF-alpha production; and 2) cyclosporin A inhibited differentiation induced by CI, but not that induced by LPS. Nevertheless, all three inducing agents activated members of the NF-kappaB family of transcription factors, including RelB, suggesting that despite differences in upstream elements, the signaling pathways all involve NF-kappaB. In this report we also demonstrate and offer an explanation for two observed forms of the RelB protein and show that RelB can be induced in myeloid cells, either directly or indirectly, through a calcium-dependent and cyclosporin A-sensitive pathway.

Calcium ionophore activation of chronic myelogenous leukemia progenitor cells into dendritic cells is mediated by calcineurin phosphatase.

We have previously demonstrated that Ph+ myeloid progenitor cells of patients with chronic myeloid leukemia (CML) can acquire characteristics of mature dendritic cells (DC) following calcium mobilization with calcium ionophore (A23187, CI). In this study we characterize the intracellular signaling pathway by which CI induces the acquisition of DC features in these leukemic cells. CI-induced activation of CML cells is attenuated by the calcineurin phosphatase inhibitor cyclosporin A (CsA) as well as the calmodulin (CaM) antagonist W-7. These cause ablation of both the CI-induced immunophenotypic expression of DC markers and immunostimulatory properties in mixed leukocyte responses (MLR). Minimal blocking effect was observed when Ca(2+)/CaM kinase II (281-301) inhibitor was added to the cultures. These findings suggest a Ca(2+)-dependent mechanism for the CI-induced activation of CML cells into antigen-presenting cells (APC), which is primarily mediated through the CaM/calcineurin pathway.

Granulocyte-macrophage colony-stimulating factor, interleukin-2, and interleukin-12 synergize with calcium ionophore to enhance dendritic cell function.

The authors previously showed that monocytes treated with calcium ionophore (CI) acquire characteristics of mature dendritic cells (DC) in part through a calcineurin-dependent pathway. In this study, the authors evaluated the ability of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), and interleukin-12 (IL-12) alone or in combination with CI to induce DC characteristics in peripheral blood monocytes. Monocytes obtained by leukapheresis and countercurrent centrifugal elutriation were cultured with calcium, cytokines, or both, profiled by flow cytometry, and assessed for antigen uptake and sensitization of autologous CD8+ T cells to antigen. Monocytes treated with the combination of GM-CSF, IL-2, and IL-12 resulted in immunophenotypic and antigen uptake profiles typical of immature DC, including loss of surface CD14 expression, de novo low-level expression of B7.1, negligible CD83 expression, marked enhancement of CD40 and ICAM-1, and high major histocompatibility complex class I and II levels. A high level of antigen uptake by macro-pinocytosis was observed. In contrast, CI treatment significantly up-regulates B7.1, B7.2, CD40, CD54, and CD83 and substantially down-regulates CD14 and macro-pinocytosis, a profile consistent with mature DC. Many CI-induced modulations, but none resulting from cytokine treatment alone, were inhibited by the calcineurin phosphatase inhibitor cyclosporin A. Compared with monocytes treated with CI alone, combined treatment of monocytes with GM-CSF, IL-2, IL-12, and CI augmented B7.1 and CD83 expression and enhanced sensitization of autologous CD8+ T cells to melanoma-antigen-derived peptides. These results suggest that several independent pathways of DC activation can cooperatively enhance the function of monocyte-derived DC.

Calcium signaling induces acquisition of dendritic cell characteristics in chronic myelogenous leukemia myeloid progenitor cells.

Effective host T lymphocyte sensitization to malignant cells depends on successful antigen presentation. In this study, we examined the capacity of malignant myeloid progenitor cells of patients in the chronic phase of chronic myelogenous leukemia (CML) to acquire characteristics of activated dendritic cells (DCs) after intracellular calcium mobilization, thereby bypassing a need for third-party antigen-presenting cells. Treatment of purified CD33(+) CML cells from 15 patients with calcium ionophore (CI) consistently resulted in de novo expression of the costimulatory molecules CD80 (B7.1) and CD86 (B7.2), CD40 and the DC-specific activation marker CD83, as well as marked up-regulation of MHC class I and II molecules and the adhesion molecule CD54. Most of these changes occurred within 24 hr of treatment. Morphologically, CI-treated CML cells developed long dendritic projections similar to those seen in mature DCs. Functionally, CI-treated CML cells provided stimulation of allogeneic T lymphocytes 10- to 20-fold that of untreated CML cells or untreated monocytes. Fluorescent in situ hybridization of CI-activated CML cells confirmed their leukemic origin by displaying the typical bcr/abl fusion signal. No difference in bcr/abl translocation percentages between untreated and CI-treated CML nuclei was observed. These observations indicate that calcium mobilization may constitute a valuable approach for rapidly and reliably generating CML-derived DCs for immunotherapy of CML.

Calcium ionophore-treated myeloid cells acquire many dendritic cell characteristics independent of prior differentiation state, transformation status, or sensitivity to biologic agents.

We previously reported that treatment of human peripheral blood monocytes or dendritic cells (DC) with calcium ionophore (CI) led to the rapid (18 hour) acquisition of many characteristics of mature DC, including CD83 expression. We therefore investigated whether less-mature myeloid cells were similarly susceptible to rapid CI activation. Although the promyelocytic leukemia line HL-60 was refractory to cytokine differentiation, CI treatment induced near-uniform overnight expression of CD83, CD80 (B7.1), and CD86 (B7. 2), as well as additional characteristics of mature DC. Several cytokines that alone had restricted impact on HL-60 could enhance CI-induced differentiation and resultant T-cell sensitizing capacity. In parallel studies, CD34(pos) cells cultured from normal donor bone marrow developed marked DC-like morphology after overnight treatment with either rhCD40L or CI, but only CI simultaneously induced upregulation of CD83, CD80, and CD86. This contrasted to peripheral blood monocytes, in which such upregulation could be induced with either CI or rhCD40L treatment. We conclude that normal and transformed myeloid cells at many stages of ontogeny possess the capacity to rapidly acquire many properties of mature DC in response to CI treatment. This apparent ability to respond to calcium mobilization, even when putative signal-transducing agents are inoperative, suggests strategies for implementing host antileukemic immune responses.

Calcium mobilization in human myeloid cells results in acquisition of individual dendritic cell-like characteristics through discrete signaling pathways.

We have shown previously that calcium ionophore (CI) treatment of various myeloid origin cells results in rapid acquisition of properties associated with mature, activated dendritic cells. These properties include increased CD83 and costimulatory molecule expression, tendencies to form dendritic processes, loss of CD14 expression by monocytes, and typically an enhanced capacity to sensitize T lymphocytes to Ag. We here analyze the intracellular signaling pathways by which CI induces acquisition of such properties. Thapsigargin, which raises intracellular Ca2+ levels by antagonizing its sequestration, induced immunophenotypic and morphologic changes that paralleled CI treatment. CI-induced activation was broadly attenuated by the Ca2+ chelating compound EGTA and by calmodulin antagonists trifluoperazine dimaleate and W-7. However, antagonists of signaling pathways downstream to calmodulin displayed more selective inhibitory effects. Calcineurin antagonists cyclosporin A and the FK-506 analogue, ascomycin, diminished costimulatory molecule and CD83 expression, as well as formation of dendritic processes in CI-treated myeloid cells, and strongly attenuated the T cell allosensitizing capacity of CI-treated HL-60 cells. These calcineurin antagonists displayed minimal effect on CI-induced CD14 down-regulation in monocytes. In contrast, the calmodulin-dependent protein kinase antagonists, K252a and KT5926, while displaying only modest effects on CI-induced costimulatory molecule and CD83 expression, strongly blocked CD14 down-regulation. These results are consistent with a Ca2+-dependent mechanism for CI-induced differentiation of myeloid cells, and indicate that multiple discrete signaling pathways downstream to calcium mobilization and calmodulin activation may be essential in regulating this process.

Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells.

Human peripheral blood contains a small subpopulation of immature dendritic cells (iDC) distinguished from circulating monocytes by their low expression of CD14. We utilized leukapheresis and countercurrent centrifugal elutriation to obtain myeloid origin mononuclear cell (MOMC) fractions of monocytes and iDC for study. These subpopulations were ultrastructurally and immunophenotypically similar before culture. After a 20- to 96-h culture either alone, with recombinant human granulocyte-monocyte CSF, or with endotoxin, greater up-regulation of costimulatory molecule expression was observed among iDC than among monocytes, and only iDC expressed the activation molecule CD83. Treatment with rhIL-4 caused many MOMC to develop morphologic properties of dendritic cells within 96 h, but costimulatory molecule up-regulation and CD14 down-regulation were heterogeneous, and CD83 expression was infrequent. In contrast, calcium ionophore (CI) treatment induced rapid and consistent effects in MOMC from both healthy volunteers and cancer patients, including down-regulated CD14 expression, acquisition of dendritic cell morphologic properties, up-regulated MHC and costimulatory molecule expression, and de novo CD83 expression. Many such effects occurred within 20 h of treatment. CI treatment activated purified CD14+ monocytes and also enhanced the spontaneous activation of purified CD14-/dim iDC in culture. Unfractionated MOMC, purified monocytes, and purified iDC displayed equivalently enhanced T cell-sensitizing efficiency following CI treatment. CD4+ T cell sensitization to keyhole limpet hemocyanin and CD8+ T cell sensitization to MART-1 melanoma-associated peptide were achieved in a single culture stimulation. Therefore, circulating monocytes and iDC can be induced by CI to manifest properties of activated DC, providing large numbers of efficient, nontransformed autologous APC for T cell sensitization strategies.

Tandem use of PCR and synthetic peptides to map helper T-cell epitopes on 27-kDa sexual stage antigen of Plasmodium falciparum.

Monoclonal antibodies recognizing two overlapping linear epitopes (amino acid residues 10 to 25) on the 27-kDa sexual stage antigen of Plasmodium falciparum (Pfg 27) effectively reduce the infectivity of the parasites to mosquitoes. Although malaria transmission-blocking immunity is largely antibody-mediated, T cells play critical roles in the regulation of antibody-secreting B cells. In order to facilitate the development of a malaria transmission-blocking subunit vaccine, studies were undertaken to map epitopes on Pfg 27 recognized by T-helper lymphocytes. Pfg27-specific T-cell hybridoma clones were produced from rPfg27-immunized BALB/c (H-2d) and C57BL/6 (H-2b) mice, and used in studies to map antigenic determinants using PCR-generated Pfg27 gene fragments expressed in E. coli and synthetic peptides based on the Pfg27 sequence. We identified and mapped five distinct T-cell epitopes that are recognized by major histocompatibility complex (MHC) class II-restricted T-cell hybridoma clones. A single peptide (21 residues) was shown to contain two tandem or partially overlapping epitopes recognized by T-cell hybridomas in the context of I-Ad and I-Ab, respectively. Synthetic peptides representing epitopes recognized by T-cell hybridoma clones elicited strong IgG responses in immunized mice, suggesting that T-cells of the helper phenotype were stimulated in vivo by these peptides. These studies represent the first detailed T-cell epitope analysis of a malaria sexual-stage antigen.