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Prepublished online as a Blood First Edition Paper on January 30, 2003; DOI 10.1182/blood-2002-07-2274.
Blood, 1 July 2003, Vol. 102, No. 1, pp. 36-42 Intranodal injection of semimature monocyte-derived dendritic cells induces T helper type 1 responses to protein neoantigenFrom the Department of Dermatology, University of Zurich Medical School, Zurich, Switzerland; the Basel Institute for Immunology, Basel, Switzerland; and the Clinical Cooperation Unit for Dermatooncology (DKFZ) at the Klinikum Mannheim, Heidelberg, Germany.
Dendritic cells (DCs) represent the most potent antigen-presenting cells of the immune system capable of initiating primary immune responses to neoantigens. Here we characterize the primary CD4 T-cell immune response to protein keyhole limpet hemocyanin (KLH) in 5 metastatic melanoma patients undergoing a tumor peptide–based dendritic cell vaccination trial. Monocyte-derived dendritic cells displaying a semimature phenotype, as defined by surface markers, were loaded ex vivo with antigen and injected intranodally at weekly intervals for 4 weeks. All patients developed a strong and long-lasting delayed-type hypersensitivity reactivity to KLH, which correlated with the induction of KLH-dependent proliferation of CD4 T cells in vitro. Secondary in vitro stimulation with KLH showed significant increase in interferon-  and interleukin-2 (IL-2) but not IL-4, IL-5, nor IL-10 secretion by bulk T cells. On the single-cell level, most TH1 cells among in vitro–generated KLH-specific T-cell lines confirmed the preferential induction of a KLH-specific type 1 T helper immune response. Furthermore, the induction of KLH-specific antibodies of the IgG2 subtype may reflect the induction of a type 1 cytokine profile in vivo after vaccination. Our results indicate that intranodal vaccination with semimature DCs can prime strong, long-lasting CD4 T-cell responses with a TH1-type cytokine profile in cancer patients. (Blood. 2003;102:36-42)
Dendritic cells (DCs) play a pivotal role in the initiation of T-cell–mediated immune responses, making them an attractive cellular adjuvant for use in cancer vaccines.1 DCs have been shown to promote protective and therapeutic antitumor immunity in several animal models.2-4 These studies have formed the basis for the clinical use of DCs in vaccination strategies against human tumor Ags. Current pilot DC vaccination studies have indeed been able to elicit specific antitumoral immune responses and some clinical responses. Nevertheless, differences in the study design, including the type of DC preparation such as DC origin, their maturation stage, the nature and duration of activation, remain a matter of debate.5 Human myeloid DCs derived from monocytes,6-8 CD34+ stem cells,9 and CD11c+ cells10 all have been able to induce some degree of immune response in vivo. Antigen-specific immunity also has been induced using DCs at different stages of their differentiation/maturation such as mature DCs,7,8 semimature DCs,6,9 and immature DCs.11,12 However, a recent study showed inhibition of effector T-cell function using immature DCs in 2 volunteers.13 In addition, the strength of the Ag-specific immune response induced also may depend on the route of vaccine administration and the vaccination schedule.14 It is therefore of critical importance to test the immunogenicity of DC preparation in the context of the study protocol. To this aim we added protein keyhole limpet hemocyanin (KLH) as helper epitope to our DC preparation by the exogenous pathway of Ag presentation. KLH is a snail-derived neoantigen and a large globular protein that consists of a wide array of immunogenic CD4 T-cell determinants and can therefore be used as a tracer molecule to easily and reproducibly monitor the induction of a primary immune response. Furthermore, as a helper protein in a peptide-based vaccination trial, KLH also may favor the induction of cytotoxic T lymphocyte (CTL) responses through the generation of bystander CD4 T-cell help.15-18 In the context of a peptide-based vaccination trial,6 semimature monocyte-derived DCs generated in fetal calf serum–supplemented medium and pulsed with KLH were injected intranodally at weekly intervals. Here we present a quantitative and qualitative analysis of the primary immune response to KLH.
Reagents The complete medium (CM) used throughout consisted of RPMI 1640 (Gibco, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS, Life Technologies, Eggenstein, Germany), 10 U/mL penicillin/streptomycin (Gibco), and 50 µg/mL L-glutamine (Seromed, Basel, Switzerland). Keyhole limpet hemocyanin (KLH) from megathura crenulata (Calbiochem, Bad Soden, Germany) was solubilized and passed 4 times through a purification column to remove endotoxin contamination (Detoxigel column, Pierce, Rockford, IL). After purification less than 1 pg/mL endotoxin was detected, as assessed by the limulus amebocyte lysate (LAL) test (sensitivity level of 1 pg/mL). The procedure was defined according to the limit requested by our study protocol. Patient selection All metastatic melanoma patients treated with DC vaccination at our institution according to our clinical protocol during a defined period of time (June 1996 to January 1997) were included in this study. The DC vaccination study was approved by the Zürich University Hospital institutional review board; informed consent was provided according to the Declaration of Helsinki. Inclusion criteria for the vaccination trial and patient's characteristics have been published previously.6 Corresponding patient numbers to the previous publication are given in parenthesis: 1 = (5), 2 = (16), 3 = (9), 4 = (14), and 5 = (8). Clinical responses included 1 complete response (patient 3), 1 partial remission (patient 5), 1 minor response (patient 2), and 2 progressive diseases (patients 1 and 4).6 Generation of DCs Peripheral blood mononuclear cells (PBMCs) were separated from fresh patient blood (100 mL) using Ficoll-Hypaque density centrifugation, resuspended in CM, and allowed to adhere to plastic dishes. After 2 hours at 37°C, the nonadherent cells were removed and the adherent cells were subsequently cultured in FCS containing CM supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) (800 U/mL, kindly provided by U. Haus, Sandoz, Nürnberg, Germany) and interleukin-4 (IL-4) (500 U/mL, PharMingen, Hamburg, Germany). After 7 days of culture nonadherent cells were collected. Production of DC vaccine DCs were pulsed for 2 hours at 37°C with 50 µg/mL KLH protein and either HLA-A2 binding melanoma-associated peptides for tyrosinase, Melan-A/melanoma antigen recognized by T cells (MART)–1, and gp100 (patients 1-4) or HLA-A1–binding peptides derived from melanoma antigen encoding gene (MAGE)–1 and MAGE-3 (patient 5), depending on the HLA type of the patient. Before injection 1 x 106 DCs were washed 3 times in sterile phosphate-buffered saline (PBS) and resuspended in a total volume of 0.5 mL PBS. Injection into an uninvolved inguinal lymph node was performed under ultrasound guidance as published.6 All injected DC preparations were negative for bacterial and fungal contamination. Immunization protocol Antigen-loaded DCs were repetitively administered into the same uninvolved inguinal lymph node according to a schedule of 4 vaccinations at weekly intervals. At weeks 6 and 10, 2 additional immunizations were given. Delayed-type hypersensitivity Delayed-type hypersensitivity (DTH) skin tests were performed before vaccination, at week 4 and week 10 of the vaccination protocol by intradermal injection of KLH (5 µg in 200 µL PBS) into the forearm. Negative control was PBS alone. A positive skin test was defined as erythema and induration of more than 5 mm at 48 hours after intradermal injection. KLH-dependent proliferation in vitro PBMCs from patients had been collected before vaccination, at week 4 and week 10 of the vaccination protocol and frozen. For the assays described in this paragraph, samples were thawed and assayed together. Recall proliferation was assessed by stimulating 1 x 105 peripheral blood leukocytes (PBLs) for 4 days with 20 µg/mL KLH. Phytohemagglutinin (PHA) stimulation (10 µg/mL, Gibco) was done in parallel to produce a positive control for cell viability and responsiveness. During the final 6 hours of the incubation period cells were pulsed with 1 µCi (0.037 MBq)/well of [3H]-thymidine (Amersham, Little Chalfont, United Kingdom), harvested onto glass microfiber filter strips using a cell harvester, and placed in a liquid scintillation counter. Values are expressed as mean counts per minute (cpm) ± standard deviation (SD) of triplicate wells. Blocking of major histocompatibility complex (MHC) class II–mediated T-cell proliferation was performed by addition of human leukocyte antigen (HLA)–DR antibody-containing supernatants derived from cultured L243-hybridoma (ATCC HB-55) cells or the corresponding isotype control antibody. Recall cytokine production
1 x 106 PBMCs were stimulated for 4 days with either KLH (20 µg/mL) or PHA (10 µg/mL, Gibco) in 1 mL CM at 37°C. After centrifugation, cell-free supernatants were collected and stored at -80°C until the interferon (IFN)- Generation of KLH-specific T-cell lines and intracellular cytokine staining
PBMCs were cultured in 96-well flat-bottom plates (Costar, Cambridge, MA) in RPMI 1640 supplemented with 5% human serum (HS; Swiss Red Cross Laboratory, Bern, Switzerland) and 2 mM L-glutamine, 1% nonessential amino acids, 1% pyruvate, 50 µg/mL kanamicin, and 5 x 10-5 M 2-ME (Gibco Laboratories, Grand Island, NY) in the presence of KLH (10 µg/mL). Recombinant IL-2 (20 U/mL, kindly provided by Dr A. Lanzavecchia, Basel Institute for Immunology, Switzerland) was added at day 6. T-cell lines were expanded in medium containing IL-2 and analyzed at day 20. For intracellular cytokine staining KLH-specific T cells (1 x 106) were stimulated with 10-7 M phorbol myristate acetate (PMA) and 1 µg/mL ionomycin (Sigma Chemicals, St Louis, MO) in 1 mL 10% FCS-RPMI in 24-well plates. After 2 hours of incubation 10 µg/mL brefeldin A (Sigma) was added to the culture for the last 2 hours. Cells were washed twice in PBS, fixed with PBS containing 2% paraformaldehyde (Merck, Darmstadt, Germany) for 15 minutes at 4°C, washed with PBS-1% FCS-0.5% saponin (Sigma), and incubated in the same buffer for 20 minutes at room temperature before adding fluorescein isothiocyanate–labeled anti–IFN- Detection of KLH-specific antibodies Maxisorp 96-wells (Nunc Brand Products, Wiesbaden, Germany) were coated with 50 µg/mL KLH in sodium carbonate buffer, pH 9.6, overnight at 4°C. After washing (PBS/0.05 Tween 20) and blocking with PBS/1% bovine serum albumin (BSA) for 2 hours at room temperature, serial dilutions of serum were added to the wells and incubated overnight at 4°C. Biotin-conjugated monoclonal antihuman IgG1, IgG2, IgG3, and IgG4 (Sigma) were incubated for 2 hours at room temperature, followed by the addition of peroxidase-conjugated avidin (DAKO Diagnostics, Zug, Switzerland) for 30 minutes at 37°C. For color development tetramethylbenzidine (TMB) one-step substrate system (DAKO) was used. Plates were measured by photometer, and results were indicated as absorption (A) at wavelength 620 nm.
Generation of CD1a+ semimature DCs DCs were differentiated from plastic-adherent peripheral blood monocytes in the presence of medium containing 10% FCS and supplemented with IL-4 and GM-CSF as described in "Patients, materials, and methods." After 7 days of culture, harvested nonadherent cells consisted routinely of more than 90% CD1a+ DCs expressing high levels of MHC class II products and intermediate levels of CD86 and CD80 (Figure 1A). Consistent with a phenotype of intermediate maturity, generated DCs expressed low levels of maturation marker CD83 (Figure 1A). No expression of activation marker CD25 was detected (Figure 1). Generated DCs had the ability to produce large amounts of IL-12 upon stimulation with CD40-ligand transfected L cells (> 1000 pg/mL, n = 2). No marked difference in the maturation stage of DCs derived from patients was detected, according to their surface expression of CD80 and CD83 (Table 1). Interestingly, an additional 4 hours' stimulation with soluble protein KLH (containing < 1 pg/mL LPS) but not with medium alone as control significantly enhanced the number of CD83+ DCs (Figure 1C), indicating a strong tendency of generated DCs to mature. Therefore, the vaccine preparation for intranodal injection consisted of DCs exhibiting a semimature phenotype.
Intranodal injection of KLH-pulsed semimature DCs induces strong DTH reactivity to KLH The standard in vivo assay for CD4 T-cell–mediated immunity is injection of Ag into the skin and assessment of the subsequent reaction. Ag-specific memory CD4 T cells will mediate an infiltration at site of challenge that is clinically detected within 24-72 hours as so-called delayed-type hypersensitivity (DTH) reaction. None of the patients showed DTH reactivity to KLH before vaccination (week 0). After 4 cycles of vaccination (week 4) intracutaneous challenge with soluble KLH in PBS but not PBS alone elicited strong DTH reactions, as shown by the erythematous induration more than 10 mm in diameter in all the patients (Figure 2). No significant changes in DTH diameter were observed at week 10 after 2 additional booster immunizations. Representative biopsies showing massive infiltration of CD45RO-positive memory T cells confirmed the presence of a DTH reaction. Comparable DTH reactions to KLH also were elicited in patients who were not challenged with soluble KLH at week 0 (n = 2, data not shown), confirming that the immune response to KLH was generated by the intranodal DC vaccination.
KLH-dependent T-cell proliferation in vitro is MHC class II restricted and correlates with the induction of DTH reactivity to KLH in vivo Secondary proliferative T-cell responses in vitro were obtained by culturing prevaccination and postvaccination PBMCs in the presence or absence of KLH for 4 days. Before vaccination, KLH-dependent T-cell proliferation of patients 1, 2, 3, and 5 was not significantly different from background proliferation of PBLs cultured in CM alone (< 2000 cpm for all patients) (Figure 3A-B). Only in patient 5 was a PBL proliferation of 7825 ± 78 cpm before vaccination observed. At week 4 of the vaccination protocol, all patients demonstrated a significant increase of the proliferative activity to KLH stimulation (Figure 3A), while background proliferation in CM alone remained less than 2000 cpm (Figure 3B). No significant changes were observed at week 10 after 2 additional booster immunizations (Figure 3A). To assess the involvement of an MHC class II–restricted antigen presentation, we added HLA-DR–specific blocking antibody to the proliferation assay. Figure 3B shows that the KLH-specific PBL proliferation was indeed inhibited by anti–HLA-DR mAb, indicating that the KLH-specific response was predominantly mediated by MHC class II–restricted CD4 T cells. A highly significant correlation between the KLH-dependent T-cell proliferation in vitro and the diameter of the KLH-specific DTH reaction in vivo confirmed the validity of the in vitro assay to assess the KLH-specific immune response (coefficient of regression 0.976, P < .01, Figure 3C).
Generated KLH-specific T-cell immunity is long-lived We assessed the duration of the KLH-specific immune response by testing DTH reactivity to KLH at various time points after the last DC vaccination. Patients 2, 3, and 5, who responded to therapy, were tested after 9, 4, and 5 months, respectively, before receiving additional booster immunizations. Progressing patient 4 was tested at 12 months, after 4 cycles of additional chemotherapy. Patient 1 died 6 months after vaccination and could not be tested. The DTH response persisted at all times tested, without evidence of a significant decline of the KLH-specific immunity (Figure 4). The in vivo data correlated with enhanced KLH-specific PBL proliferation in vitro (data not shown).
Preferential induction of a T helper 1–type cytokine profile
Next we analyzed the T-cell cytokine profile induced by vaccination with KLH-pulsed DCs. In vitro restimulation of total PBMCs with KLH demonstrated a preferential KLH-dependent TH1-type cytokine profile with enhanced secretion of IFN-
Production of KLH-specific IgG2 antibodies in vivo
Dendritic cells can retain native unprocessed antigen and transfer it to naive B lymphocytes to initiate class switching in a primary T-cell–dependent response.19 It has been suggested that a difference in the balance of Th1 and Th2 cytokines at the site of B-cell activation accounts for a differential production of IgG subclasses. In rodents this relation is well defined: a switch of an antigen-specific antibody subclass in response to immunization can be used as a reliable indicator of the cytokines produced in vivo. Vaccination of KLH-pulsed murine DCs induced anti-KLH mAb of the IgG2a subtype, characteristic of a TH1 response.20 In humans it has been reported that IgG2 production depends on the presence of IFN-
In this study we demonstrate that repetitive intranodal injection of semimature human monocyte–derived DCs pulsed with protein neoantigen KLH induces strong and long-lasting cellular immune responses to KLH, as demonstrated by DTH reactivity in vivo and KLH-dependent T-cell proliferation in vitro. Interestingly, maximal KLH-specific T-cell responses were induced after the initial 4 cycles of vaccination, could not be further enhanced by additional immunizations (week 6 and 10), and persisted for several months without the need for additional boosters. Long-lived immune responses to protein Ags usually require the use of granuloma-forming complete Freund adjuvant by favoring the in vivo persistence of Ag and therefore keeping CD4 T cells on a constant level of activation.26 Since in vivo persistence of KLH in our study is unlikely, the persistence of the response may reflect the magnitude of expanded KLH-specific T cells induced by DC vaccination.27
Several groups have reported that immunization with soluble KLH protein in adjuvant induces Th0/Th2-type immune response28-30 independently of the route and the schedule of immunization.31 To assess the role of DCs in the TH cell polarization in vivo, we performed a qualitative analysis of the KLH-specific CD4 T-cell immune response by a framework of assays, including the measurement of KLH-dependent cytokine production by bulk T cells in vitro, the cytokine profile of in vitro–generated KLH-specific T-cell lines, and the identification of KLH-specific anti-body subclass induced in vivo. Assessment of the type of CD4 T-cell immune response is of key importance, since dendritic cells, in addition to their unique role in the generation of protective immunity, also have been implicated in the induction of T-cell tolerance by promoting TH2 responses, T regulatory cells, and anergic T cells. This functional plasticity among monocyte-derived DCs depends on their stage of maturation, the type, and the duration of activation. Mature CD40 ligand–activated monocyte-derived DCs secrete large amounts of IL-12, induce T helper (TH)–1 cell differentiation, and have therefore been called DC1.32 DC1 also can be induced by activation signals such as LPS, proinflammatory cytokines IL-1, TNF-
It has been proposed that bystander CD4 T helper response is an essential requirement for the induction of CD8 T-cell responses to weak antigens such as tumor antigens (which are normally encountered outside an inflammatory context). T helper cells may promote the induction of primary CD8 T-cell response either directly through the secretion of TH1 cytokines (such as IFN- In conclusion, we show that FCS-derived DCs are semimature, and we demonstrated in 5 melanoma patients that intranodal injection of semimature DCs induces a strong and long-lasting CD4 T-cell response with a preferential TH1-type cytokine profile. Since subtle differences in the study design are critical for the induction of a therapeutic immune response, the use of a protein neoantigen as helper epitope allows one to reproducibly trace and characterize the immune response elicited during DC vaccination trials.
Submitted July 26, 2002; accepted January 6, 2003.
Prepublished online as Blood First Edition Paper, January 30, 2003; DOI 10.1182/blood-2002-07-2274.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Frank O. Nestle, Department of Dermatology, University of Zürich, Gloriastr 31, 8091 Zürich, Switzerland; e-mail: nestle{at}derm.unizh.ch.
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Abstract
Introduction
indicates patient 1; 
, patient 2; 
, patient 3; 
, patient 4; and 
, patient 5. (B) Immunhistochemical characterization of the lymphocytic infiltrate at the DTH site. Alkaline-phosphatase-antialkaline phosphatase (APAAP) staining with a CD45RO mAb was performed, demonstrating a strong perivascular and epidermal infiltration of CD45RO+ memory T cells. Original magnification, x 100.
indicates CM; 
indicates DC vaccination cycle; 
, time of death; CR, complete response, PR, partial response; MR, minor response; and PD, progressive disease.
, IgG3; and 
, and IL-6, CpG, poly I:C, and viruses.
(TGF-