Whole blood transcriptomics reveals granulocyte colony-stimulating factor as a mediator of cardiopulmonary bypass-induced systemic inflammatory response syndrome.

Objectives: Systemic inflammatory response syndrome (SIRS) is a frequent complication of cardiopulmonary bypass (CPB). SIRS is associated with significant morbidity and mortality, but its pathogenesis remains incompletely understood, and as a result, biomarkers are lacking and treatment remains expectant and supportive. This study aimed to understand the pathophysiological mechanisms driving SIRS induced by CPB and identify novel therapeutic targets that might reduce systemic inflammation and improve patient outcomes. Methods: Twenty-one patients undergoing cardiac surgery and CPB were recruited, and blood was sampled before, during and after surgery. SIRS was defined using the American College of Chest Physicians/Society of Critical Care Medicine criteria. We performed immune cell profiling and whole blood transcriptomics and measured individual mediators in plasma/serum to characterise SIRS induced by CPB. Results: Nineteen patients fulfilled criteria for SIRS, with a mean duration of 2.7 days. Neutrophil numbers rose rapidly with CPB and remained elevated for at least 48 h afterwards. Transcriptional signatures associated with neutrophil activation and degranulation were enriched during CPB. We identified a network of cytokines governing these transcriptional changes, including granulocyte colony-stimulating factor (G-CSF), a regulator of neutrophil production and function. Conclusions: We identified neutrophils and G-CSF as major regulators of CPB-induced systemic inflammation. Short-term targeting of G-CSF could provide a novel therapeutic strategy to limit neutrophil-mediated inflammation and tissue damage in SIRS induced by CPB.

CSL362 potently and specifically depletes pDCs invitro and ablates SLE-immune complex-induced IFN responses.

Systemic lupus erythematosus (SLE) is an autoimmune disease with significant morbidity and mortality. Type I interferon (IFN) drives SLE pathology and plasmacytoid dendritic cells (pDCs) are potent producers of IFN; however, the specific effects of pDC depletion have not been demonstrated. We show CD123 was highly expressed on pDCs and the anti-CD123 antibody CSL362 potently depleted pDCs in vitro. CSL362 pre-treatment abrogated the induction of IFNα and IFN-induced gene transcription following stimulation with SLE patient-derived serum or immune complexes. RNA transcripts induced in pDCs by ex vivo stimulation with TLR ligands were reflected in gene expression profiles of SLE blood, and correlated with disease severity. TLR ligand-induced protein production by SLE patient peripheral mononuclear cells was abrogated by CSL362 pre-treatment including proteins over expressed in SLE patient serum. These findings implicate pDCs as key drivers in the cellular activation and production of soluble factors seen in SLE.

UpStreAm doxycycline in ST-eLeVation myocArdial infarction: targetinG infarct hEaling and ModulatIon (SALVAGE-MI trial).

AIMS: Experimental studies demonstrate protective effects of doxycycline on myocardial ischaemia-reperfusion injury. The trial investigated whether doxycycline administered prior to reperfusion in patients presenting with ST-elevation myocardial infarction (STEMI) reduces infarct size (IS) and ameliorates adverse left ventricular (LV) remodelling. METHODS AND RESULTS: In this randomized, double-blind, placebo-controlled trial, patients presenting with STEMI undergoing primary percutaneous coronary intervention (PPCI) were randomized to either intravenous doxycycline or placebo prior to reperfusion followed by 7 days of oral doxycycline or placebo. The primary outcome was final IS adjusted for area-at-risk (fIS/AAR) measured on two cardiac magnetic resonance scans ∼6 months apart. Of 103 participants, 50 were randomized to doxycycline and 53 to placebo and were matched for age (59 ± 12 vs. 60 ± 10 years), male sex (92% vs. 79%), diabetes mellitus (26% vs. 11%) and left anterior descending artery occlusion (50% vs. 49%), all P > 0.05. Patients treated with doxycycline had a trend for larger fIS/AAR [0.79 (0.5-0.9) vs. 0.61 (0.47-0.76), P = 0.06], larger fIS at 6 months [18.8% (12-26) vs. 13.6% (11-21), P = 0.08], but similar acute IS [21.7% (17-34) vs. 19.4% (14-27), P = 0.19] and AAR [26% (20-36) vs. 24.7% (16-31), P = 0.22] compared with placebo. Doxycycline did not ameliorate adverse LV remodelling [%Δend-diastolic volume index, 1.1% (-3.8-8.4) vs. -1.34% (-6.1-5.8), P = 0.42] and was independently associated with larger fIS (regression coefficient = 0.175, P = 0.03). CONCLUSION: Doxycycline prior to PPCI neither reduced IS acutely or at six months nor attenuated adverse LV remodelling. These data raise safety concerns regarding doxycycline use in STEMI for infarct modulation and healing.

A randomised preclinical trial of adrenaline use during cardiac arrest in mice.

Background: Adrenaline is routinely administered during cardiac arrest resuscitation. Using a novel murine model of cardiac arrest, this study evaluates the effects of adrenaline use on survival and end-organ injury. Methods: A total of 58 mice, including cardiac arrest (CA) and sham (SHAM) groups received intravenous potassium chloride either as a bolus (CA) or slow infusion (SHAM), inducing ECG-confirmed asystole (in CA only) for 4-minutes prior to intravenous adrenaline (+ADR;250 ul,32 ug/ml) or saline (-ADR;250 ul) and manual chest compressions (300 BPM) for 4-minutes. Mice with return of spontaneous circulation (ROSC) were assessed at 24- or 72-h timepoints. Results: Among animals that underwent CA, rates of ROSC (n = 21 (95 %) vs n = 14 (82 %), P = 0.18) and survival to the planned endpoint (n = 11 (50 %) vs n = 12 (71 %), P = 0.19) were similar when comparing those treated with (CA+ADR) and without (CA-ADR) adrenaline. However, in CA animals that initially achieved ROSC, subsequent mortality was approximately 3-fold greater with adrenaline treatment (48 % vs 14 %, P = 0.042). Among SHAM animals, adrenaline use had no impact on survival rates or other endpoints. Greater myocardial injury occurred in CA+ADR vs CA-ADR, with increased Hs-Troponin levels measured at 24- (26.0 ± 0.9 vs 9.4 ± 5.3 ng/mL, P = 0.015) and 72-h (20.9 ± 8.3 vs 5.0 ± 2.4 ng/mL, P = 0.012), associated with increased expression of pro-inflammatory and fibrotic genes within cardiac and renal tissue. Conclusion: Adrenaline did not improve ROSC or overall survival but following successful ROSC, its use resulted in 3-fold greater mortality rates. Adrenaline was also associated with increased myocardial injury, end-organ inflammation, and fibrosis. These findings underscore the need for further preclinical evaluation of alternate pharmacologic adjuncts for cardiopulmonary resuscitation that improve survival and limit end-organ injury.

A pilot study of peripheral blood BDCA-1 (CD1c) positive dendritic cells pulsed with NY-ESO-1 ISCOMATRIX™ adjuvant.

AIM: Pilot clinical trial of NY-ESO-1 (ESO) protein in ISCOMATRIX™ adjuvant pulsed onto peripheral blood dendritic cells (PBDC), to ascertain feasibility, evaluate toxicity and assess induction of ESO-specific immune responses. PATIENTS & METHODS: Eligible participants had resected cancers expressing ESO or LAGE-1 and were at high risk of relapse. PBDC were produced using CliniMACS®plus, with initial depletion of CD1c+ B cells followed by positive selection of CD1c+ PBDC. Patients received three intradermal vaccinations of ESO/IMX-pulsed PBDC at 4-week intervals. RESULTS: The process was feasible and safe. No vaccine-induced immune responses were detected. Assays of immunomodulatory cells did not correlate with outcomes. One patient had a long lasting complete remission. CONCLUSION: This method was feasible and safe but was minimally immunogenic.

A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus.

To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE.

Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib.

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8+ T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.

Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia.

In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14-38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T-cell therapy in high-risk AML, and demonstrates durable in vivo persistence.

The immunostimulatory effect of lenalidomide on NK-cell function is profoundly inhibited by concurrent dexamethasone therapy.

Lenalidomide combined with dexamethasone is an effective treatment for refractory/relapsed multiple myeloma (MM). Lenalidomide stimulates natural killer (NK) cells and enhances antitumor responses. We assessed NK cell number and function in 25 patients with MM participating in a clinical trial of lenalidomide and dexamethasone. NK cell numbers increased from a mean of 2.20 ± 0.05 × 10(5)/mL (baseline) to a mean of 3.90 ± 0.03 × 10(5)/mL (cycle 6; P = .05); however, in vitro NK-cell-mediated cytotoxicity decreased from 48.9% ± 6.8% to 27.6% ± 5.1% (P = .0028) and could not be rescued by lenalidomide retreatment. Lenalidomide increased normal donor NK-cell cytotoxicity in vitro from 38.5% to 53.3%, but this was completely abrogated by dexamethasone. Dexamethasone suppression of NK cell-mediated cytotoxicity was partially reversed by a 3-day washout, but these cells remained refractory to lenalidomide-induced enhanced function. Lymphocyte subset depletion experiments revealed that lenalidomide's enhancement of NK cell-mediated cytotoxicity was mediated by CD4(+) T-cell production of interleukin 2 and that dexamethasone acted by suppressing interleukin-2 production. Similarly, the reduced ability of NK cells from patients with MM to respond to lenalidomide was also due to impaired CD4 T-cell function. Our findings indicate that lenalidomide immunostimulatory effects on patient NK cells are severely blunted by concurrent dexamethasone administration.

Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients.

Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10-specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.

Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1.

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

Characterization of antigen-specific CD8+ T lymphocyte responses in skin and peripheral blood following intradermal peptide vaccination.

Immune responses to cancer vaccines are commonly tested by measuring cutaneous reactions to intradermal (i.d.) antigen. When well-characterized peptide epitopes are injected i.d., infiltrates of CD4+ and CD8+ T lymphocytes are frequently seen. In this study, we have further characterized T cells derived from vaccine-infiltrating lymphocyte (VIL) responses. We found that the infiltrates capable of producing IFN-gamma and cytolytic activity could recognize vaccine peptide, as well as antigen-positive melanoma cells. We studied antigen-specific T cell responses from VILs and peripheral blood in 10 patients who participated in a clinical trial. All patients received systemic Flt3 ligand (20 microg/kg/d) and i.d. peptides: Three NY-ESO-1 peptides, SLLMWITQCFL (157-167), SLLMWITQC (157-165), QLSLLMWIT (155-163); tyrosinase internal peptide YMDGTMSQV (368-376); Melan-A/MART-1 analogue peptide ELAGIGILTV (26-35, E27L substitution); and influenza matrix peptide GILGFVFTL (58-66). In 54 paired VIL and peripheral blood analyses, a good correlation was found between responses in skin and in blood. These cells could be rapidly expanded in a short-term assay and thus appear to be memory T cells. The demonstrated presence of antigen-specific T cells at vaccination sites validates this method of assessing the immune response to i.d. vaccines.

Dissecting cytotoxic T cell responses towards the NY-ESO-1 protein by peptide/MHC-specific antibody fragments.

NY-ESO-1 is a germ cell antigen aberrantly expressed by different tumor types that elicits strong humoral and cellular immune responses, representing one of the most promising candidates for vaccination of cancer patients. A detailed analysis of CD8(+) T cells generated in vaccine trials using NY-ESO-1-derived peptides (157-165 and 157-167) revealed that the dominant immune response was directed against a cryptic epitope (159-167) diverting the immune response from tumor recognition. Only CTL reactivity to the NY-ESO-1(157-165) peptide appeared to be capable of lysing NY-ESO-1/HLA-A0201-expressing tumor cells. To study the process of NY-ESO-1 peptide presentation by tumor cells in more detail we generated a high-affinity (K(D)=60 nM) antibody fragment that specifically recognizes the NY-ESO-1(157-165) peptide/HLA-A0201 complex. Peptide variants such as the NY-ESO-1(157-167) peptide or the cryptic NY-ESO-1(159-167) peptide were not recognized. The antibody fragment blocked in a dose-dependent fashion the recognition of NY-ESO-1/HLA-A2-positive tumor cells by NY-ESO-1(157-165) peptide-specific CD8(+) T cells. This antibody fragment is a novel reagent that binds with TCR-like specificity to the NY-ESO-1(157-165)/HLA-A2 complex thus distinguishing between CTL responses against immunological meaningful or cryptic NY-ESO-1-derived peptides. It may therefore become a useful monitoring tool for the development of NY-ESO-1-based cancer vaccines.

The impact of imiquimod, a Toll-like receptor-7 ligand (TLR7L), on the immunogenicity of melanoma peptide vaccination with adjuvant Flt3 ligand.

Dendritic cells (DCs) show promise as adjuvants in anticancer immunotherapeutic strategies. Flt3 ligand (FL) is a hematopoietic growth factor that increases the number of immature DCs in the blood and other tissues. We treated 27 patients with metastatic or high-risk resected melanoma with s.c. FL daily for 14 d in three 28 d cycles. Eighteen of these patients also received vaccination with influenza (Flu), Melan-A (Mel), tyrosinase (Tyr), and NY-ESO-1 peptides. To induce local DC maturation, 8 of the vaccinated patients had imiquimod, a Toll-like receptor-7 ligand (TLR7L), applied topically to their vaccine sites. Patients were monitored for clinical and hematological effects. Immune responses were assessed by cutaneous reactivity to vaccination and by the induction of peptide-specific CD8+ T-cells. Eight patients did not complete the protocol due to adverse events related to their cancer. The treatment was generally safe and well tolerated, although some patients developed clinically significant toxicities related to FL. FL induced increases in immature CD11c+ and CD123+ peripheral blood (PB) DCs. Other hematological effects included monocytosis, granulocytosis, and thrombocytosis, which were marked in some patients. Cutaneous reactions to peptide vaccination and circulating peptide-specific CD8+ T-cells were more frequent in imiquimod-treated patients. FL treatment of melanoma patients has pleiotropic clinical and hematological effects. In vivo maturation of FL-generated DCs using imiquimod may increase immune responses to tumor antigens.

A robust human T-cell culture method suitable for monitoring CD8+ and CD4+ T-cell responses from cancer clinical trial samples.

Many tumor antigenic determinants have been identified and included in cancer clinical trials. Due to low T-cell frequencies even after vaccination, few T-cell responses can be revealed ex vivo without in vitro stimulation. Various expansion protocols have been employed for this purpose and the outcomes tend to be quite variable, partly due to the high complexity involved in the protocols. Here we systematically studied various common culture conditions including sera, cytokines and feeders and describe a reliable "bulk" culture method that is robust, simpler and more economical. We demonstrated that fetal calf serum (FCS) supported T-cell proliferation better than multiple commercially available pooled human AB sera. IL-2 is critical in our cultures, but IL-7, IL-15 and anti-CTLA-4 in combination with IL-2 did not further enhance T-cell expansion. We typically achieve more than a 40-fold expansion within a 10-day culture period for antigen-specific T cells measured by HLA-peptide tetramer before and after culture. This method was not only validated by multiple operators as a standard operating procedure for monitoring T-cell responses but was also successfully used for discovering novel CD8+ and CD4+ T cells specific to previously unknown epitopes from the NY-ESO-1 tumor antigen.

Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4(+) and CD8(+) T cell responses in humans.

NY-ESO-1 is a "cancer-testis" antigen expressed in many cancers. ISCOMATRIX is a saponin-based adjuvant that induces antibody and T cell responses. We performed a placebo-controlled clinical trial evaluating the safety and immunogenicity of recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant. Forty-six evaluable patients with resected NY-ESO-1-positive tumors received three doses of vaccine intramuscularly at monthly intervals. The vaccine was well tolerated. We observed high-titer antibody responses, strong delayed-type hypersensitivity reactions, and circulating CD8(+) and CD4(+) T cells specific for a broad range of NY-ESO-1 epitopes, including known and previously unknown epitopes. In an unplanned analysis, vaccinated patients appeared to have superior clinical outcomes to those treated with placebo or protein alone. The vaccine is safe and highly potent immunologically.

Immunodominant CD4+ responses identified in a patient vaccinated with full-length NY-ESO-1 formulated with ISCOMATRIX adjuvant.

There is increasing evidence showing the involvement of CD4(+) T cells in initiating and maintaining antitumor immune responses. NY-ESO-1 is expressed by various tumors but not normal tissues except testis. We conducted a cancer clinical trial by using full-length NY-ESO-1 protein formulated with ISCOMATRIX adjuvant and injected into patients intramuscularly. Autologous dendritic cells pulsed with NY-ESO-1 ISCOMATRIX in combination with overlapping synthetic peptides were used to identify immunodominant T cells from a vaccinated patient. We show here the identification and characterization of two novel CD4(+) T cell epitopes. T cells specific to these epitopes not only recognized autologous dendritic cells loaded with NY-ESO-1 but also NY-ESO-1-expressing tumor cell lines treated with IFN-gamma. One of the two responses identified was greater than the previously identified immunodominant HLA-DP4-restricted response and correlated with NY-ESO-1-specific CD8(+) T cell induction after vaccination. This T cell response was vaccinated in most patients who expressed HLA-DR2. This study has systematically surveyed patients vaccinated with full-length tumor antigen for a vaccinated CD4 helper T cell response.

NY-ESO-1 protein formulated in ISCOMATRIX adjuvant is a potent anticancer vaccine inducing both humoral and CD8+ t-cell-mediated immunity and protection against NY-ESO-1+ tumors.

NY-ESO-1 is a 180 amino-acid human tumor antigen expressed by many different tumor types and belongs to the family of "cancer-testis" antigens. In humans, NY-ESO-1 is one of the most immunogenic tumor antigens and NY-ESO-1 peptides have been shown to induce NY-ESO-1-specific CD8(+) CTLs capable of altering the natural course of NY-ESO-1-expressing tumors in cancer patients. Here we describe the preclinical immunogenicity and efficacy of NY-ESO-1 protein formulated with the ISCOMATRIX adjuvant (NY-ESO-1 vaccine). In vitro, the NY-ESO-1 vaccine was readily taken up by human monocyte-derived dendritic cells, and on maturation, these human monocyte-derived dendritic cells efficiently cross-presented HLA-A2-restricted epitopes to NY-ESO-1-specific CD8(+) T cells. In addition, epitopes of NY-ESO-1 protein were also presented on MHC class II molecules to NY-ESO-1-specific CD4(+) T cells. The NY-ESO-1 vaccine induced strong NY-ESO-1-specific IFN-gamma and IgG2a responses in C57BL/6 mice. Furthermore, the NY-ESO-1 vaccine induced NY-ESO-1-specific CD8(+) CTLs in HLA-A2 transgenic mice that were capable of lysing human HLA-A2(+) NY-ESO-1(+) tumor cells. Finally, C57BL/6 mice, immunized with the NY-ESO-1 vaccine, were protected against challenge with a B16 melanoma cell line expressing NY-ESO-1. These data illustrate that the NY-ESO-1 vaccine represents a potent therapeutic anticancer vaccine.

Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli.

Dendritic cells (DCs) are a family of leukocytes that initiate T- and B-cell immunity against pathogens. Migration of antigen-loaded DCs from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. In humans, the major peripheral blood DC (PBDC) types, CD1c+ DCs and interleukin 3 receptor-positive (IL-3R+) plasmacytoid DCs, are significantly expanded in vivo with the use of Flt3 ligand (FL). DC-like cells can also be generated from monocyte precursors (MoDCs). A detailed comparison of the functional potential of these types of DCs (in an autologous setting) has yet to be reported. Here, we compared the functional capacity of FL-expanded CD1c+ PBDCs with autologous MoDCs in response to 3 different classes of stimuli: (1) proinflammatory mediators, (2) soluble CD40 ligand trimer (CD40L), and (3) intact bacteria (Escherichia coli). Significant differences in functional capacities were found with respect to changes in phenotype, migratory capacity, cytokine secretion, and T-cell stimulation. MoDCs required specific stimuli for the expression of functions. They responded vigorously to CD40L or E coli, expressing cytokines known to regulate interferon-gamma (IFN-gamma) in T cells (IL-12p70, IL-18, and IL-23), but required prostaglandin E2 (PGE2) during stimulation to migrate to chemokines. In contrast, PBDCs matured in response to minimal stimulation, rapidly acquired migratory function in the absence of PGE2-containing stimuli, and were low cytokine producers. Interestingly, both types of DCs were equivalent with respect to stimulation of allogeneic T-cell proliferation and presentation of peptides to cytotoxic T lymphocyte (CTL) lines. These distinct differences are of particular importance when considering the choice of DC types for clinical applications.