Autologous Immature Dendritic Cells Research Articles

A phase I prospective, non-randomized trial of autologous dendritic cell-based cryoimmunotherapy in patients with metastatic castration-resistant prostate cancer

Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 108) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.

Combined immunotherapy and radiation induce cellular and humoral immune responses to tumor-associated antigens and prolong survival of patients with advanced cancers.

Immunotherapy is effective in treating patients with a variety of cancers We developed and employed an immunotherapeutic protocol that combined intra-tumor injection of autologous immature dendritic cells , with standard of care radiation to treat patients with a variety of different advanced solid tumors. Pre and post treatment peripheral blood mononuclear cells ( PBMC) were assayed for their capacity to kill autologous tumor cell lines that had been established from patients with different advanced solid tumors. PBMC obtained post treatment were cytotoxic to autologous but not allogeneic tumor cell lines. Cytotoxic activity increased as relative numbers of CD8+-CD56+ dual positive lymphoid cells increased in PBMC obtained post treatment. The autologous tumor cell line directed cytotoxicity was shown to be mediated by both innate and adaptive immune mechanisms. Cytotoxicity of PBMC obtained post treatment was enhanced after co-culture with autologous and not allogeneic tumor cell line lysates. Antibody activity to mesothelin, a tumor associated antigen expressed in a variety of solid tumors, increased in post treatment serum samples from lung cancer patients. These sera mediated antibody directed cellular cytotoxicity ( ADCC) to mesothelin expressing tumor cell lines. SSurvival of 96 patients with a variety advanced cancers patients treated on this protocol over a 3 year period and followed for 1000+ days was significantly increased in patients with low tumor burden ( lesion at <5 sites with diameters < 3cm) (Km= <0.0001) regardless of malignancy. These findings support application of this combination of immunotherapy and radiation in the treatment of cancer patients with advanced disease

Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells.

Introduction: Adaptive regulatory T cells (Tr1) are induced in the periphery by environmental stimuli. CD73 expression and adenosine (ADO) production by tumor cells may influence Tr1 generation and their immunosuppressive activity. Material and Methods: Tr1 were generated in co-cultures of CD4+CD25neg T cells, autologous immature dendritic cells (iDC), and irradiated ADO-producing CD73+ or non-producing CD73neg breast cancer (BrCa) cell lines (TU). The expression of ectonucleotidases and other surface markers on Tr1 was determined by flow cytometry. Tr1-mediated suppression of proliferation was evaluated in CFSE-based assays. Luciferase-based ATP detection assays and mass spectrometry were used to measure ATP hydrolysis and ADO levels. Cytokine levels were measured by ELISA or Luminex. CD73 expression on tumor cells or T cells in TU tissues was assessed by immunofluorescence. Results: CD73+ TU induced higher numbers of Tr1 cells (p < 0.01) than CD73neg TU. Tr1TU73+ hydrolyzed more exogenous ATP, produced more ADO, and mediated higher suppression than Tr1TU73neg (p < 0.05 for all). ARL67156, an ectonucleotidase inhibitor, and ZM241385, A2A receptor antagonist, reduced suppression of proliferation mediated by Tr1TU73+ cells (p < 0.01). Basal-like primary BrCa cells expressed higher levels of ectonucleotidases and induced more Tr1 than less aggressive primary luminal-like BrCa. Conclusion: BrCa producing ADO (CD73+ TU) favor the induction of Tr1, which expresses CD39 and CD73, hydrolyzes ATP to ADO, and effectively suppresses anti-tumor immunity.

Abstract CT066: Dendritic cell based cryoimmunotherapy associates with clinical variables and changes in T-cell receptor expression in a prospective phase I trial of metastatic castration resistant prostate cancer

Abstract Interim analysis data are presented for 13 patients with metastatic castration resistant prostate cancer treated with dendritic cell based cryoimmunotherapy (CryoIT) and at least three months follow-up (FU). In CryoIT autologous immature dendritic cells (iDCs) mature in cryoablated tumor tissue and engulf tumor associated antigens and thereafter migrate to lymph nodes to instruct systemic immune attacks on heterogenous cancer cells. Primary endpoint of the trial is to investigate safety of CryoIT while secondary aims include clinical, radiological and immunological responses to treatment. Adverse events (AEs) and blood analyses were registered at all visits and imaging performed before and three months post-treatment. Norwegian regulatory authorities have approved the study. CryoIT of the prostate was performed as a freeze-thaw process under anesthesia and autologous iDCs injected after last thawing cycle. Radiologic responses according to RECISTv1.1 could categorize participants into either Group 1; stable disease (n=5) or Group 2; progressive cancer (n=4). Three patients showed mixed response and one had no follow-up imaging. Hematologic and immunologic values were evaluated according to response group. To identify peripheral blood leukocyte subsets and circulating tumor cells (CTCs), ultradeep T-cell receptor (TCR) β-chain sequences of complementarity-determining region 3, we utilized flow / mass cytometry, Cellsearch System, and TCRSafe Technology, respectively. At inclusion median age was 69 (62-73) years, BMI 25.9 (25-35), ECOG score 0 (0-1) and median PSA 8 (5-11). Group 2 demonstrated higher PSA (p=0.03), median 3.9 (IQR 2.7-6.8) and 74.6 (IQR 47.4-99.0) for Group 1 and Group 2, respectively. In total, 13 possible iDC-related AEs were reported by 10 individuals, whereof one severe and 12 mild-to-moderate, with urinary retention in seven subjects. All but one resolved by use of indwelling urinary catheter for median 8 (1-40) days. Participants with high CTC numbers (&amp;gt;5) before therapy had higher rates of progression, moderate pre-treatment CTC counts (3-5) decreased and no patient acquired CTCs after therapy. Ultradeep TCR-sequencing showed more prevalent and higher expression (&amp;gt;5-fold) of new TCR clonotypes 2-6 weeks after treatment in Group 1. The treatment seems safe and well tolerated. Higher PSA indicates more advanced disease at baseline for Group 2. The data indicate immune responses to treatment with higher increases in TCR clonotypes in radiologic stable disease. Clinical trial ID: NCT02423928. Citation Format: Liv Cecilie V. Thomsen, Alfred Honorè, Bjarte Almås, Lars A. Reisæter, Jannicke Frugård, Einar K. Kristoffersen, Guro Melve, Torjan Haslerud, Jarle Rørvik, Martin Biermann, Svein Inge Helle, Gunnar Kvalheim, Waqas Azeem, Jan Roger Olsen, Ole Johan Halvorsen, Lars Akslen, Duke K. Bahn, Klaus Pantel, Sabine Riethdorf, Haakon Ragde, Bjørn Tore Gjertsen, Anne M. Øyan, Karl-Henning Kalland, Christian Beisland. Dendritic cell based cryoimmunotherapy associates with clinical variables and changes in T-cell receptor expression in a prospective phase I trial of metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT066.

PERSPECTIVES IN ACTIVE SPECIFIC IMMUNOTHERAPY WITH AUTOLOGOUS IMMATURE DENDRITIC CELLS COMBINED WITH PHOTODYNAMIC THERAPY AND CYCLOPHOSPHAMIDE IN PATIENTS WITH DISSEMINATED MELANOMA RESISTANT TO STANDARD THERAPY

There are described the results of clinical and immunological efficacy assessment of active specific immunotherapy with autologous immature dendritic cells (DC) combined with photodynamic therapy (PDT) and cyclophosphamide (C) in disseminated melanoma patients, resistant to standard therapy. 27 patients treated in the N.N. Petrov Research Institute of Oncology were included in the study from 2007 till 2016. Immunotherapy was conducted in a 21-day cycles. Therapy included following steps: 1) preparation of individual vaccine preparation from bone-marrow derived DC with immune phenotype CD34-/CD14VCD1a+/CD83VCD80-/+/CD86-/+/HLA-DR+; 2) Intramuscular 300 mg C injection in day 1 of treatment cycle for elimination T-lymphocytes with immunosuppressing activity; 3) PDT with chlorin salts at day 4 six hours before start of vaccinotherapy; 4) Daily intralesional injections of DC vatccine in irradiated lesions in the dose 1х106 DC cells/ kg. Clinical and immunological efficacy was assessed in 27 patients. Fourteen (52%) patients received 1-2 cycles of therapy, 13(48%) received 3 or more cycles. No complete response was seen. Partial response (RECIST 1.1) was found in 2 (7,4%) patients, stable disease in 8 (29,6%) patients. Seventeen (63%) patients progressed. Median time to progression (TTP) was 2.5 month, median overall survival (OS) 8.4 month. One-year survival was 5% and 37% for TTP and OS, respectively. No adverse events (AE) of grade 4-5 (CTC AE v4) were seen. Grade 3 fever was registered in 4% of patients. Grade 1-2 AE were found in 54% of patients. Immunologic assessment revealed significant decline of immunoregulatory index (CD/ CD8) caused by prevalence of cytotoxic T-lymphocytes in peripheral blood of responding patients (patients with clinical benefit). Tendency for elevation of absolute number of activates T-helpers and cytotoxic T-lymphocytes together with low T-regulatory cells concentration was also found. Combination therapy using immunomodulatory effects of C, PDT and DC vaccine in 21-day treatment cycles produce promising activity and favorable toxicity profile in heavily pretreated disseminated melanoma patients.

Clinical response of advanced cancer patients to cellular immunotherapy and intensity-modulated radiation therapy

Patients afflicted with advanced cancers were treated with the intratumoral injection of autologous immature dendritic cells (iDCs) followed by activated T-cell infusion and intensity-modulated radiation therapy (IMRT). A second round of iDCs and activated T cells was then administered to patients after the last radiation cycle. This complete regimen was repeated for new and recurring lesions after 6 weeks of follow-up. One year post therapy, outcome analyses were performed to evaluate treatment efficacy. Patients were grouped according to both the number and size of tumors and clinical parameters at treatment initiation, including recurrent disease after standard cancer therapy, Stage IV disease, and no prior therapy. Irrespective of prior treatment status, 23/37 patients with ≤ 5 neoplastic lesions that were ≤ 3 cm in diameter achieved complete responses (CRs), and 5/37 exhibited partial responses (PRs). Among 130 individuals harboring larger and more numerous lesions, CRs were observed in 7/74 patients that had received prior SCT and in 2/56 previously untreated patients. Some patients manifested immune responses including an increase in CD8+CD56+ lymphocytes among circulating mononuclear cells in the course of treatment. To prospectively explore the therapeutic use of these cells, CD8+ cells were isolated from patients that had been treated with cellular immunotherapy and IMRT, expanded in vitro, and injected into recurrent metastatic sites in 13 individuals who underwent the same immunoradiotherapeutic regimens but failed to respond. CRs were achieved in 34 of 58 of such recurrent lesions while PRs in 17 of 58. These data support the expanded use of immunoradiotherapy in advanced cancer patients exhibiting progressive disease.

Generation and immunosuppressive functions of p53-induced human adaptive regulatory T cells

Inducible regulatory T cells (iTregs, also called Tr1 cells) are generated in the periphery (circulation or tissue) of cancer patients upon the encounter of naïve CD4+ T cells with tumor-associated antigens. As p53 is often inactivated by genetic or epigenetic events during oncogenesis, p53-induced Tr1 cells might play a key role in establishing immunosuppressive networks in cancer patients. Tr1 cells were generated by co-culturing circulating CD4+CD25− T cells with autologous immature dendritic cells pulsed with a wild-type (WT) p53-derived peptide or an unrelated peptide derived from mucin 1 (MUC1). The Tr1 phenotype and the specificity for p53 of these cells were confirmed by multicolor flow cytometry. Moreover, the Tr1 cell-mediated suppression of T-cell proliferation was evaluated by CFSE-based flow cytometry, while their ability to alter the T-cell cytokine profile by ELISA and Luminex assays. The capacity of p53-induced Tr1 cells to suppress the generation and function of cytotoxic T lymphcoytes (CTLs) was assessed by flow cytometry and ELISPOT. Of note, low doses of the p53-derived peptide (p53low) induced greater numbers of Tr1 cells than the same peptide employed at high doses (p53high). Moreover, Tr1/p53low cells not secreted higher levels of interleukin-10 and transforming growth factor β1, but also mediated more robust suppressive effects on CTL proliferation than Tr1/p53high cells. Tr1/p53low cells, Tr1/p53high cells, as well as Tr1 cells generated with low doses of an unrelated MUC1-derived peptide were equally effective in suppressing the expansion and antitumor activity of p53-reactive CTLs. p53low induced the expansion of highly suppressive p53-reactive Tr1 cells. However, the capacity of these Tr1 cells to suppress the generation and function of p53-reactive CTLs was independent of their antigen-specificity.

The Signal Peptide of the Tumor-shared Antigen Midkine Hosts CD4+ T Cell Epitopes

The CD4 T cell response to the tumor antigen Midkine was unknown. Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.

Generation of Human iNKT Cell Lines

Natural killer T (NKT) cells comprise an important immunoregulatory T cell subset and express cell surface proteins characteristic of both natural killer cells and T cells. Invariant NKT (iNKT) cells are activated by lipid antigen presented in the context of CD1d molecules, in contrast to classic T cell subsets, which recognize peptide antigens presented by MHC molecules. Following activation, iNKT cells rapidly secrete large amounts of cytokines and can lyse tumor cells and virally infected cells; however, iNKT cells are reduced in patients with autoimmune disease and cancer. The potential to characterize and investigate the prospective use of iNKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human iNKT cells. In this protocol, we describe a method to generate and propagate primary human iNKT cells. Specifically, primary iNKT cells were isolated from human peripheral blood mononuclear cells (PBMC), and then expanded periodically with irradiated α-GalCer loaded autologous immature dendritic cells (DC) in the presence of human IL-2.

Dendritic Cell Editing by Activated Natural Killer Cells Results in a More Protective Cancer-Specific Immune Response

Over the last decade, several studies have extensively reported that activated natural killer (NK) cells can kill autologous immature dendritic cells (DCs) in vitro, whereas they spare fully activated DCs. This led to the proposal that activated NK cells might select a more immunogenic subset of DCs during a protective immune response. However, there is no demonstration that autologous DC killing by NK cells is an event occurring in vivo and, consequently, the functional relevance of this killing remains elusive. Here we report that a significant decrease of CD11c+ DCs was observed in draining lymph nodes of mice inoculated with MHC-devoid cells as NK cell targets able to induce NK cell activation. This in vivo DC editing by NK cells was perforin-dependent and it was functionally relevant, since residual lymph node DCs displayed an improved capability to induce T cell proliferation. In addition, in a model of anti-cancer vaccination, the administration of MHC-devoid cells together with tumor cells increased the number of tumor-specific CTLs and resulted in a significant increase in survival of mice upon challenge with a lethal dose of tumor cells. Depletion of NK cells or the use of perforin knockout mice strongly decreased the tumor-specific CTL expansion and its protective role against tumor cell challenge. As a whole, our data support the hypothesis that NK cell-mediated DC killing takes place in vivo and is able to promote expansion of cancer-specific CTLs. Our results also indicate that cancer vaccines could be improved by strategies aimed at activating NK cells.

Altered dendritic cell–natural killer interaction in Kenyan sex workers resistant to HIV-1 infection

Natural killer (NK) cells are members of the innate immune system that play an important role in the defense against viral infection. They are also involved in the regulation of adaptive immune responses through cytokine secretion and the interaction with antigen-presenting cells. However, their role in HIV infection is only partially understood. Here we studied the phenotype and function of NK cells of highly HIV-exposed but seronegative (HESN) uninfected commercial sex workers from Kenya who can be epidemiologically defined as relatively resistant to HIV infection. The purpose of this study was to gain insight into the role of NK cells in mediating resistance to HIV-1. This information can be used to better understand protection from infection which can be used for informing future design of effective prophylactics and therapeutics for HIV. Whole blood samples were collected from study participants and isolated NK cells and dendritic cells were used in assays for phenotyping and cell function. Activated NK cells from resistant women killed autologous immature dendritic cells more efficiently and also secreted more interferon (IFN)-γ than those of uninfected, susceptible women. Interestingly, NK cells from HIV-resistant women were significantly more effective in inducing secretion of IL-12 in immature dendritic cells. These data suggest that an altered NK cell-dendritic cell interaction plays an important role in the protection from infection with HIV-1.

IRX-2, a novel biologic, favors the expansion of T effector over T regulatory cells in a human tumor microenvironment model

IRX-2, a natural cytokine biological with multiple components, has been used in preclinical and clinical studies to promote antitumor activity of T lymphocytes. To define cellular mechanisms responsible for antitumor effects of IRX-2, its ability to induce effector T cells (Teff) was examined in a model simulating the tumor microenvironment. An in vitro model containing conventional CD4+CD25− cells co-cultured with autologous immature dendritic cells, irradiated tumor cells, and cytokines was used to study differentiation and expansion of regulatory T cells (Treg) and Teff in the presence and absence of IRX-2. Phenotype, suppressor function, signaling, and cytokine production were serially measured using flow cytometry, Western blots, CFSE-based suppressor assays, and Luminex-based analyses. The presence of IRX-2 in the co-cultures promoted the induction and expansion of IFN-γ+Tbet+ Teff and significantly (p < 0.01) decreased the induction of inducible IL-10+TGF-β+ Treg. The responsible mechanism involved IFN-γ-driven T cell polarization towards Teff and suppression of Treg differentiation. In an in vitro model simulating the human tumor microenvironment, IRX-2 promoted Teff expansion and antitumor activity without inducing Treg. Thus, IRX-2 could be considered as a promising component of future antitumor therapies.

Human Vδ1-T cells regulate immune responses by targeting autologous immature dendritic cells

Recent studies suggest that tissue resident Vδ1-T cells may downregulate immune responses in human beings. However, the function of peripheral blood Vδ1-T cells and their mechanisms of action remain largely unknown because of their limited numbers and the difficulties encountered in expanding these cells. In this study, we provide direct evidence demonstrating that peripheral human Vδ1-T cells can abrogate adaptive immune responses by direct killing of autologous dendritic cells through a perforin-mediated pathway. These findings advance our basic understanding of this unique T-cell subset.

Adenosine and Prostaglandin E2 Cooperate in the Suppression of Immune Responses Mediated by Adaptive Regulatory T Cells

Adaptive regulatory T cells (Tr1) are induced in the periphery upon encountering cognate antigens. In cancer, their frequency is increased; however, Tr1-mediated suppression mechanisms are not yet defined. Here, we evaluate the simultaneous involvement of ectonucleotidases (CD39/CD73) and cyclooxygenase 2 (COX-2) in Tr1-mediated suppression. Human Tr1 cells were generated from peripheral blood mononuclear cell-derived, sorted CD4(+)CD25(-) T cells and incubated with autologous immature dendritic cells, irradiated COX-2(+) or COX-2(-) tumor cells, and IL-2, IL-10, and IL-15 (each at 10-15 IU/ml) for 10 days as described (Bergmann, C., Strauss, L., Zeidler, R., Lang, S., and Whiteside, T. L. (2007) Cancer Immunol. Immunother. 56, 1429-1442). Tr1 were phenotyped by multicolor flow cytometry, and suppression of proliferating responder cells was assessed in carboxyfluorescein diacetate succinimidyl ester-based assays. ATP hydrolysis was measured using a luciferase detection assay, and levels of adenosine or prostaglandin E(2) (PGE(2)) in cell supernatants were analyzed by mass spectrometry or ELISA, respectively. Intracellular cAMP levels were measured by enzyme immunoassay. The COX-2(+) tumor induced a greater number of Tr1 than COX-2(-) tumor (p < 0.05). Tr1 induced by COX-2(+) tumor were more suppressive, hydrolyzed more exogenous ATP (p < 0.05), and produced higher levels of adenosine and PGE(2) (p < 0.05) than Tr1 induced by COX-2(-) tumor. Inhibitors of ectonucleotidase activity, A(2A) and EP(2) receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE(4) inhibitor, increased the intracellular cAMP level in responder cells and their susceptibility to Tr1-mediated suppression. Tr1 present in tumors or the peripheral blood of head and neck squamous cell carcinoma patients co-expressed COX-2, CD39, and CD73. A concomitant inhibition of PGE(2) and adenosine via the common intracellular cAMP pathway might be a novel approach for improving results of immune therapies for cancer.

Differential effects of ifosfamide on dendritic cell-mediated stimulation of T cell interleukin-2 production, natural killer cell cytotoxicity and interferon-γ production

Ifosfamide is a DNA-alkylating agent used frequently in chemotherapy of human malignancies. Ifosfamide and its major decomposition products deplete intracellular glutathione (GSH). Glutathione is the major intracellular thiol reductant that protects cells against oxidative injury. Ifosfamide depletion of intracellular GSH in human dendritic cells (DC), T cells and natural killer (NK) cells impairs their functional activity which can be restored by reconstituting GSH. Here we assessed the effect of ifosfamide on DC-mediated stimulation of NK cell proliferation via T cells and on direct DC stimulation of NK cell cytotoxicity and interferon (IFN)-gamma production. Indirect DC stimulation of NK cell proliferation via T cells and T cell-derived interleukin (IL)-2 were reduced by ifosfamide treatment of DC and reconstitution of GSH in DC restored both responses. When DC and NK cells were treated with ifosfamide, DC could overcome the negative effect of ifosfamide on NK cytotoxic function whereas NK cell IFN-gamma production was less efficiently restored. The ability of IL-2 activated NK cells to kill autologous immature DC or to induce DC maturation was reduced moderately by treatment of both cell types with ifosfamide. Overall, our results suggest that DC may stimulate anti-tumour effector cells in patients even if they had received treatment with chemotherapeutic agents such as ifosfamide.

Evaluation of T cell immune responses in multi-drug-resistant tuberculosis (MDR-TB) patients to Mycobacterium tuberculosis total lipid antigens.

Mycobacterium tuberculosis lipid antigens produce significant T cell responses in healthy tuberculin reactor [purified protein derivative (PPD-positive] individuals. In the present study, proliferation and interferon (IFN)-gamma/interleukin (IL)-4 responses were analysed to M. tuberculosis total lipid antigens in T lymphocytes from 25 patients with multi-drug-resistant tuberculosis (MDR-TB). The obtained results were compared with those of 30 asymptomatic healthy PPD-positive and 30 healthy tuberculin skin test negative (PPD-negative) subjects. Peripheral blood mononuclear cells (PBMCs) and T cells (CD4(+) and CD8(+)) were stimulated using autologous immature dendritic cells. Proliferation responses were assessed using 3-{4,5-dimethylthiazol-2-yl}-2,5 diphenyl tetrazolium bromide (MTT). IFN-gamma/IL-4 concentrations in the supernatant of the CD4(+) and CD8(+)T cells were measured by enzyme-linked immunosorbent assay. Proliferation assay showed that the peripheral blood mononuclear cells and CD4(+) T cells from the MDR-TB patients responded significantly less to the M. tuberculosis total lipid antigens than to the CD4(+) T cells in the PPD-positive subjects. Total lipid antigen-specific proliferative responses in the CD8(+) T cells from the MDR-TB patients were minimally detected and the responses were similar to those of the PPD-positive subjects. IFN-gamma production by the CD4(+) T cells stimulated by total lipid antigens from the MDR-TB patients was decreased significantly compared with the PPD-positive individuals, whereas IL-4 production in the patients was elevated. IFN-gamma and IL-4 production in the CD8(+) T cells of the MDR-TB patients was similar to those of the PPD-positive subjects. In conclusion, it is suggested that stimulated CD4(+) T cells by M. tuberculosis total lipid antigens may be shifted to T helper 2 responses in MDR-TB patients.

Intratumoral injection of dendritic cells in combination with local hyperthermia induces systemic antitumor effect in patients with advanced melanoma

Dendritic cells (DC) are potent antigen-presenting cells that can present tumor antigens chaperoned by heat shock proteins (HSPs), while local hyperthermia (LHT) can increase the expression of HSPs. In this study, we determine if intratumoral injection of immature DC after LHT (LHT+IT-DC) induces systemic antitumor immunity in patients with advanced melanoma, and investigate the potential immunological mechanisms involved in the treatments. Patients were randomly assigned to intratumoral administration of autologous immature DC triweekly, with (LHT+IT-DC, arm A, n = 9) or without (IT-DC, arm B, n = 9) LHT. Our results showed that there were no grade 3/4 toxicities. The time to progress (TTP) of arm A was 5 months, significantly longer than that in arm B (2 months, p < 0.05). However, the overall survival time had no statistical difference (13 months vs. 6 months, p > 0.05) between the 2 groups. Our ELISPOT assay showed a significantly increased melanoma-specific IFN-gamma production in arm A, suggesting that LHT+IT-DC was more effective in the induction of cytotoxic T lymphocytes (CTL) than IT-DC alone. Furthermore, we detected an increased HSPs expression 4 hr after the first LHT, an enhanced Th1/Th2 chemokines production 24 hr after the first LHT+IT-DC treatment, a promoted migration of DC to afferent lymph nodes, and a decreased infiltration of regulatory T cells (CD4(+)CD25(+)) and an increased infiltration of active CTL (CD8(+)CD28(+)) 48 hr after the third DC injection in arm A patients. Therefore, LHT+IT-DC can induce effective specific antitumor immunity and facilitate a Th1-polarized immune response in patients with advanced melanoma.

The maturation potential of NK cell clones toward autologous dendritic cells correlates with HMGB1 secretion

Interaction of NK cells with autologous immature dendritic cells (iDCs) results in reciprocal activation. We have previously reported that NK cells trigger iDC to polarize and secrete IL-18; in turn, DC-activated NK cells secrete the nuclear protein/proinflammatory cytokine high mobility group box protein 1 (HMGB1), which induces DC maturation and prevents DC from lysis. However, activated NK cells can also kill iDC. To investigate whether effector and maturative properties may coexist or segregate in different NK subsets, human NK cell clones were generated and analyzed for their effects on iDC. We found that the ability of different NK cell clones to induce iDC maturation is unlinked to their phenotypic and cytolytic features but correlates with the relocation of HMGB1 from nucleus to cytoplasm. "Maturative" NK cell clones secrete HMGB1 spontaneously. It is interesting that secretion is strongly enhanced by engagement of the surface molecule NKp30 but only slightly induced by triggering of the activating NK receptor CD16. However, culturing freshly isolated NK cells for 1 week with low doses of anti-CD16 triggers the relocation of HMGB1 from nucleus to cytoplasm and its spontaneous secretion, resulting in a stronger maturation potential of the NK cells. Together, our data indicate that NK cells comprise functionally different subsets, endowed with different capacities to secrete HMGB1 and to induce maturation of autologous iDC. Nonetheless, maturation properties can be modulated by different stimuli. This suggests that depending on the environmental stimuli, NK/iDC interaction can lead to different outcomes, thus influencing immune response.

NK/iDC interaction results in IL-18 secretion by DCs at the synaptic cleft followed by NK cell activation and release of the DC maturation factor HMGB1

AbstractInteraction of natural killer (NK) cells with autologous immature dendritic cells (DCs) results in reciprocal activation; however, the underlying mechanisms are so far elusive. We show here that NK cells trigger immature DCs to polarize and secrete interleukin 18 (IL-18), a cytokine lacking a secretory leader sequence. This occurs through a Ca2+-dependent and tubulin-mediated recruitment of IL-18-containing secretory lysosomes toward the adhering NK cell. Lysosome exocytosis and IL-18 secretion are restricted at the synaptic cleft, thus allowing activation of the interacting NK cells without spreading of the cytokine. In turn, DC-activated NK cells secrete the proinflammatory cytokine high mobility group B1 (HMGB1), which induces DC maturation and protects DCs from lysis. Also HMGB1 is a leaderless cytokine that undergoes regulated secretion. Differently from IL-18, soluble HMGB1 is consistently detected in NK/DC supernatants. These data point to secretion of leaderless cytokines as a key event for the reciprocal activation of NK cells and DCs. DCs initiate NK cell activation by targeted delivery of IL-18, thus instructing NK cells in the absence of adaptive-type cytokines; in turn, activated NK cells release HMGB1, which promotes inflammation and induces DC maturation, thus favoring the onset of the adaptive immune response. (Blood. 2005;106:609-616)

Combination of Conformal Radiotherapy and Intratumoral Injection of Adoptive Dendritic Cell Immunotherapy in Refractory Hepatoma

A phase 1 study was conducted to assess the safety and immunologic response induced by direct injection of autologous immature dendritic cells (DCs) into tumor under radiotherapy in advanced hepatoma patients. Patients with advanced/metastatic stage hepatoma not suitable for surgery or transarterial embolization were enrolled. Groups of patients received two vaccinations. Each vaccination consisted of intratumoral injections of autologous immature DCs in four dose cohorts of 5 x 10(6), 1.5 x 10(7), 3 x 10(7), and 5 x 10(7) cells 2 days after a single fraction of conformal radiotherapy of 8 Gy. The second vaccination was performed 3 weeks later. Of the 14 patients entered, 12 completed two cycles of vaccination. The treatment was well tolerated at any of the dose levels. Six patients had mild transient fever (grade 1-2) with chill reactions, three patients developed grade 1 fatigue, and one patient developed mild myalgia and arthralgia after DC injections. There was no evidence of clinically manifested autoimmune disease. There were two partial responses and four minor responses. A decrease in the alpha-fetoprotein (AFP) level of more than 50% was found in three patients. Ten patients had completed immunologic response evaluation 2 weeks after the second cycle of vaccination. The AFP-specific immune response was evident in eight patients examined by cytokine release assay and in seven patients by ELISPOT assay. Six patients showed an increased NK cell cytotoxic activity after vaccination. These data suggest that the combination of intratumoral injection of DCs and conformal radiotherapy is safe and can induce tumor-specific and innate immunity.