NK cell activation by dendritic cell vaccine: a mechanism of action for clinical activity

Recent reports revealed that dendritic cell (DC)–natural killer (NK) cell interaction plays an important role in tumor immunity, but few DC vaccine studies have attempted to evaluate the non-specific, yet potentially clinically relevant, NK response to immunization. In this study, we first analyzed in vitro activation of NK cells by DCs similar to those used in clinical trials. Subsequently, NK cell responses were analyzed in a phase I clinical trial of a vaccine consisting of autologous DCs loaded with a fowlpox vector encoding CEA. The data were compared with the clinical outcome of the patients. DC enhances NK activity in vitro, partly by sustaining NK cell survival and by enhancing the expression of NK-activating receptors, including NKp46 and NKG2D. Among nine patients in our clinical trial, NK cytolytic activity increased in four (range 2.5–5 times greater lytic activity) including three who had increased NK cell frequency, was stable in two and decreased in three. NKp46 and NKG2D expression showed a good correlation with the patients’ NK activity. When patients were grouped by clinical activity (stable disease/no evidence of disease (stable/NE, n=5) vs progressive disease (N=4) at 3 months), the majority in the stable/NE group had increases in NK activity (P=0.016). Anti-CEA T cell response was enhanced in all the nine patients analyzed, but was not significantly different between the two groups (P=0.14). Thus, NK responses following DC vaccination may correlate more closely with clinical outcome than do T cell responses. Monitoring of NK response during vaccine studies should be routinely performed.     

Therapeutic vaccination against metastatic renal cell carcinoma by autologous dendritic cells: preclinical results and outcome of a first clinical phase I/II trial

In this study we have presented in vitro data and results of a preliminary clinical trial using dendritic cells (DC) in patients with progressive metastatic renal cell carcinoma. DC precursor cells were obtained from peripheral blood mononuclear cells (PBMC). DC were pulsed with autologous tumor cell lysate if available. In total, 15 patients were treated with a median of 3.95 x 10(6) DC administered and ultrasound-guided into a lymph node or into adjacent tissue. Seven patients remained with progressive disease (PD), 7 patients showed stable disease (SD), and one patient displayed a partial response (PR). Most interestingly, the patient who was treated with the highest number of DC (14.4 x 10(6) DC/vaccine) displayed a PR. Delayed-type hypersensitivity (DTH) reaction using autologous tumor lysate was positive in 3 out of 13 patients, including the patient with PR. Two out of 3 patients receiving additional treatment with keyhole limpet hemocyanin (KLH) showed reactivity to KLH after vaccination. CD3+CD4+ and CD3+CD28+ cells as well as the proliferation rate of peripheral blood lymphocytes (PBL) increased significantly in the blood of patients during therapy. In conclusion, our observations confirm the capability of tumor-lysate pulsed autologous DC vaccines to stimulate an immune response in patients with metastatic renal cell carcinoma even in the presence of a large tumor burden. The lack of adverse effects together with immunologic effects support further investigation of this novel therapeutic approach. Further studies are necessary to demonstrate clinical effectiveness in cancer patients, in particular in patients with less advanced disease.     

Induction of tumor-specific T-cell responses by vaccination with tumor lysate-loaded dendritic cells in colorectal cancer patients with carcinoembryonic-antigen positive tumors

Background Dendritic cells (DCs) are the most effective antigen-presenting cells. In the last decade, the use of DCs for immunotherapy of cancer patients has been vastly increased. High endocytic capacity together with a unique capability of initiating primary T-cell responses have made DCs the most potent candidates for this purpose. Although DC vaccination occasionally leads to tumor regression, clinical efficacy, and immunogenicity of DCs in clinical trials has not been yet clarified. The present study evaluated the safety and effectiveness of tumor-lysate loaded DC vaccines in advanced colorectal cancer (CRC) patients with carcinoembryonic antigen (CEA) positive tumors. Results Six patients HLA-A*0201-positive were vaccinated with autologous DCs loaded with tumor lysates (TL) together with tetanus toxoid antigen, hepatitis B, and influenza matrix peptides. Two additional patients were injected with DCs that were generated from their sibling or parent with one haplotype mismatch. All patients received the vaccines every 2 weeks, with a total of three intra-nodal injections per patient. The results indicated that DC vaccination was safe and well tolerated by the patients. Specific immune responses were detected and in some patients, transient stabilization or even reduction of CEA levels were observed. The injection of haplotype mismatched HLA-A*0201-positive DCs resulted in some enhancement of the anti-tumor response in vitro and led to stabilization/reduction of CEA levels in the serum, compared to the use of autologous DCs. Conclusion Altogether, these results suggest that TL-pulsed DCs may be an effective vaccine method in CRC patients. Elimination of regulatory mechanisms as well as adjustment of the vaccination protocol may improve the efficacy of DC vaccination. An erratum to this article can be found at     

Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial

Background aimsDendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2⁺ patients) or with autologous/allogeneic tumor lysate (HLA-A2^? patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-α2b.ResultsOf 46 patients who initiated treatment, 10 stopped treatment within 1–4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2⁺ patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25^high CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD.ConclusionsVaccination was feasible and safe. Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells.     

Dendritic cell vaccination in human melanoma: relationships between clinical effects and vaccine parameters

Eleven years have passed since the start of the first trial of dendritic cell (DC) vaccination for melanoma. A review of 54 trials was performed to evaluate the relationship between clinical effects and vaccine parameters. Significant differences were found between use of immature and mature DCs with regard to progressive disease (PD), between stage III and IV for clinical response, between use and non-use of adjuvants with regard to stable disease (SD) in treatment with tumor/tumor lysate-pulsed DCs, between positive and negative delayed-type hypersensitivity (DTH) for PD, and between increased and unchanged interferon (IFN)-γ-secreting T cells for clinical response. These results are consistent with the partial efficacy of vaccination with mature DCs in early stage melanoma and the partial correlation of efficacy with positive DTH and increased IFN-γ-secreting T cells. DC vaccination alone had a limited clinical effect and a modified regimen is needed to enhance antigen-specific cytotoxic T cells and decrease immunosuppression.     

Vaccination with p53 peptide-pulsed dendritic cells is associated with disease stabilization in patients with p53 expressing advanced breast cancer; monitoring of serum YKL-40 and IL-6 as response biomarkers

p53 Mutations are found in up to 30% of breast cancers and peptides derived from over-expressed p53 protein are presented by class I HLA molecules and may act as tumor-associated epitopes in cancer vaccines. A dendritic cell (DC) based p53 targeting vaccine was analyzed in HLA-A2+ patients with progressive advanced breast cancer. DCs were loaded with 3 wild-type and 3 P2 anchor modified HLA-A2 binding p53 peptides. Patients received up to 10 sc vaccinations with 5 x 10(6) p53-peptide loaded DC with 1-2 weeks interval. Concomitantly, 6 MIU/m(2) interleukine-2 was administered sc. Results from a phase II trial including 26 patients with verified progressive breast cancer are presented. Seven patients discontinued treatment after only 2-3 vaccination weeks due to rapid disease progression or death. Nineteen patients were available for first evaluation after 6 vaccinations; 8/19 evaluable patients attained stable disease (SD) or minor regression while 11/19 patients had progressive disease (PD), indicating an effect of p53-specific immune therapy. This was supported by: (1) a positive correlation between p53 expression of tumor and observed SD, (2) therapy induced p53 specific T cells in 4/7 patients with SD but only in 2/9 patients with PD, and (3) significant response associated changes in serum YKL-40 and IL-6 levels identifying these biomarkers as possible candidates for monitoring of response in connection with DC based cancer immunotherapy. In conclusion, a significant fraction of breast cancer patients obtained SD during p53-targeting DC therapy. Data encourage initiation of a randomized trial in p53 positive patients evaluating the impact on progression free survival.     

Patient-derived renal cell carcinoma cells fused with allogeneic dendritic cells elicit anti-tumor activity: in vitro results and clinical responses

Renal cell carcinoma (RCC) has been shown to be susceptible to immunotherapeutic treatment strategies. In the present study, patient-derived tumor cells were fused with allogeneic dendritic cells (DC) to elicit anti-tumor activity against RCC. DC from HLA-A2+ healthy donors were fused with primary RCC cells from ten patients. Phenotype of fusion cells were characterized by flow cytometer and confocal microscopy. In vitro, T cell proliferation, IFN-γ secretion and cytotocic T lymphocytes (CTL) activity elicited by allogeneic DC/RCC fusion cells were assessed. Clinically, ten patients were vaccinated with allogeneic DC/RCC fusion vaccine. The adverse effects and toxicity were observed. The clinical response was evaluated by CT scans. After fusion, the created hybrids expressed both tumor associated antigen and DC-derived molecules and could stimulate the proliferation and IFN-γ secretion of T cells as well as elicit strong CTL activity against RCC cells in vitro. In vivo, no serious adverse effects, toxicity, or signs of autoimmune disease were observed after vaccination therapy. Percentage of T lymphocyte subsets in peripheral blood of patients was increased significantly. One of ten patients exhibited a partial response with regression of lung metastases. Six patients showed stable disease with stabilization of previously progressive disease (follow up 1.5 years). The PR and SD responses, exhibited by 7/10 patients who received the allogeneic DC/RCC fusion vaccine treatment, suggest that this approach is safe and can elicit immunological responses in a significant portion of patients with RCC. J. Zhou and D. Weng contributed equally.     

Metastatic melanoma patients treated with dendritic cell vaccination, Interleukin-2 and metronomic cyclophosphamide: results from a phase II trial

Dendritic cells (DC) are the most potent antigen presenting cells and have proven effective in stimulation of specific immune responses in vivo. Competing immune inhibition could limit the clinical efficacy of DC vaccination. In this phase II trial, metronomic Cyclophosphamide and a Cox-2 inhibitor have been added to a DC vaccine with the intend to dampen immunosuppressive mechanisms. Twenty-eight patients with progressive metastatic melanoma were treated with autologous DCs pulsed with survivin, hTERT, and p53-derived peptides (HLA-A2(+)) or tumor lysate (HLA-A2(-)). Concomitantly the patients were treated with IL-2, Cyclophosphamide, and Celecoxib. The treatment was safe and tolerable. Sixteen patients (57?%) achieved stable disease (SD) at 1st evaluation and 8 patients had prolonged SD (7-13.7?months). The median OS was 9.4?months. Patients with SD had an OS of 10.5?months while patients with progressive disease (PD) had an OS of 6.0?months (p?=?0.048) even though there were no differences in prognostic factors between the two groups. Despite the use of metronomic Cyclophosphamide, regulatory T cells did not decrease during treatment. Indirect IFN-γ ELISPOT assays showed a general increase in immune responses from baseline to the time of 4th vaccination. Induction of antigen-specific immune responses was seen in 9 out of 15 screened HLA-A2(+) patients. In conclusion, the number of patients obtaining SD more than doubled and 6-month survival significantly increased compared to a previous trial without Cyclophosphamide and Celecoxib. A general increase in immune responses against the tested peptides was observed.     

Superior anti-tumor protection and therapeutic efficacy of vaccination with allogeneic and semiallogeneic dendritic cell/tumor cell fusion hybrids for murine colon adenocarcinoma

Cancer immunotherapy by dendritic cell (DC)/tumor cell fusion hybrids (DC/TC hybrids) has been shown to elicit potent anti-tumor effects via the induction of immune responses against multiple tumor-associated antigens. In the present study, we compared the anti-tumor effects of vaccinating Balb/c mice (H-2^d) with CT26CL25 colon carcinoma cells that had been fused with either syngeneic DCs from Balb/c mice, allogeneic DCs from C57BL/6 mice (H-2^b) or semiallogeneic DCs from B6D2F1 mice (H-2^b/d). Preimmunization with either semiallogeneic or allogeneic DC/TC hybrids induced complete protection from tumor challenge, whereas mice preimmunized with syngeneic DC/TC hybrids were only partially protected (75% tumor rejection). The average number of pulmonary metastases after intravenous tumor injection decreased significantly following immunization with semiallogeneic or allogeneic DC/TC hybrids (8.3 ± 7.9 or 16.3 ± 3.5, mean ± SD) relative to syngeneic DC/TC hybrids (67.8 ± 6.3). These data demonstrate that vaccination with semiallogeneic DC/TC hybrids resulted in the greatest anti-tumor efficacy. Anti-tumor effects showed by in vivo studies were virtually accomplished by the frequency of induced CTLs specific to both gp70 and β-galactosidase assessed by using pentameric assay. Among the fusion vaccines tested, semiallogeneic DC/TC hybrids induced the highest ratio of Th1 cytokine IFN-γ to Th2 cytokine IL-10. In addition, allogeneic or semiallogeneic DC/TC hybrids elicited a significantly stronger NK activity than syngeneic DC/TC hybrids. These findings suggest that in clinical settings, DCs derived from a healthy donor (which are generally characterized as more semiallogeneic than allogeneic) may be more capable than autologous DCs of inducing promising anti-tumor effects in vaccinations with DC/TC hybrids.     

Blood Dendritic Cell Frequency Declines in Idiopathic Parkinson’s Disease and Is Associated with Motor Symptom Severity

The role of inflammation in Parkinson’s Disease (PD) is well appreciated, but its underlying mechanisms are still unclear. Our objective was to determine whether dendritic cells (DC), a unique type of migratory immune cells that regulate immunological response and inflammation have an impact on PD. In a case-control study including 80 PD patients and 80 age- and gender-matched healthy control subjects, the two main blood subsets of plasmacytoid and myeloid DC were defined by flow cytometry analysis. Clinical evaluation of subjects consisting of cognition and depression assessment was performed using the Mini Mental State Examination and the Beck Depression Inventory. The severity of motor symptoms was measured using the Unified Parkinson’s Disease Rating Scale-Part III. Comparison between patient and control DC measures and their relationships with clinical assessments were evaluated.The following main results were obtained: 1) the level of circulating DC (mainly the myeloid subset) was significantly reduced in PD patients in comparison with healthy controls; 2) after controlling for depressive and cognitive characteristics, the frequency of myeloid DC was confirmed as one of the independent determinants of PD; 3) the number of both myeloid and plasmacytoid DC was negatively associated with motor symptom severity. Overall, the decline of blood DC, perhaps due to the recruitment of immune cells to the site of disease-specific lesions, can be considered a clue of the immune alteration that characterizes PD, suggesting innovative exploitations of DC monitoring as a clinically significant tool for PD treatment. Indeed, this study suggests that reduced peripheral blood DC are a pathologically-relevant factor of PD and also displays the urgency to better understand DC role in PD for unraveling the immune system contribution to disease progression and thus favoring the development of innovative therapies ideally based on immunomodulation.     

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.     

Changes in circulating dendritic cells and IL-12 in relation to the angiogenic factor VEGF during IL-2 immunotherapy of metastatic renal cell cancer

Angiogenesis and immunosuppression are the main biological mechanisms responsible for cancer progression. Moreover, recent observations suggesting a negative influence of angiogenesis on anticancer immunity have shown that some angiogenic factors, such as VEGF, may induce immunosuppression. In addition, the evidence of abnormally high blood levels of VEGF has been proven to be associated with resistance to IL-2 immunotherapy. The present study was performed to establish a possible relation ship between the efficacy of IL-2 cancer immunotherapy and changes in circulating levels of VEGF, IL-12, mature and immature dendritic cells (DC). The study included 25 metastatic renal cell cancer patients who underwent subcutaneous low-dose IL-2 immunotherapy (6 MIU/day for 6 days/week for 4 weeks). Immature and mature DCs were identified as CD123+ and CD11c+ cells, respectively. The clinical response consisted of partial response (PR) in five, stable disease (SD) in 11 and progressive disease (PD) in the remaining nine patients. The mean IL-12 levels observed during IL-2 immunotherapy were significantly higher in patients with PR or SD than in those with PD, whereas the mean VEGF concentrations were significantly higher in patients who had PD than in those with PR or SD. Finally, a significant increase in the mean number of circulating mature DCs occurred only in patients with PR or SD, whereas no significant change was seen in patients with PD. By contrast, no significant change was observed in the mean number of immature DCs. This study shows that the efficacy of IL-2 immunotherapy is associated with a significant increase in circulating mature DCs and IL-12, without any concomitant increase in VEGF concentrations. Further studies will be required to better define the relationship between activation of anticancer immunity and control of angiogenesis-related mechanisms.     

Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate

The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20 months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10 months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation. Margarita Salcedo and Nadège Bercovici both contributed equally to this work     

HSV-2 Specifically Down Regulates HLA-C Expression to Render HSV-2-Infected DCs Susceptible to NK Cell Killing

Both NK cells and CTLs kill virus-infected and tumor cells. However, the ways by which these killer cells recognize the infected or the tumorigenic cells are different, in fact almost opposite. CTLs are activated through the interaction of the TCR with MHC class I proteins. In contrast, NK cells are inhibited by MHC class I molecules. The inhibitory NK receptors recognize mainly MHC class I proteins and in this regard practically all of the HLA-C proteins are recognized by inhibitory NK cell receptors, while only certain HLA-A and HLA-B proteins interact with these receptors. Sophisticated viruses developed mechanisms to avoid the attack of both NK cells and CTLs through, for example, down regulation of HLA-A and HLA-B molecules to avoid CTL recognition, leaving HLA-C proteins on the cell surface to inhibit NK cell response. Here we provide the first example of a virus that through specific down regulation of HLA-C, harness the NK cells for its own benefit. We initially demonstrated that none of the tested HSV-2 derived microRNAs affect NK cell activity. Then we show that surprisingly upon HSV-2 infection, HLA-C proteins are specifically down regulated, rendering the infected cells susceptible to NK cell attack. We identified a motif in the tail of HLA-C that is responsible for the HSV-2-meduiated HLA-C down regulation and we show that the HLA-C down regulation is mediated by the viral protein ICP47. Finally we show that HLA-C proteins are down regulated from the surface of HSV-2 infected dendritic cells (DCs) and that this leads to the killing of DC by NK cells. Thus, we propose that HSV-2 had developed this unique and surprising NK cell-mediated killing strategy of infected DC to prevent the activation of the adaptive immunity.     

Dynamic monitoring the TCR CDR3 spectratypes in patients with metastatic CRC treated with a combination of bevacizumab, irinotecan, fluorouracil, and leucovorin

In the present study, either modified IFL regimen (modified irinotecan, fluorouracil and leucovorin, mIFL) alone or in combination with bevacizumab was used to treat patients with metastatic colorectal cancer (CRC). Treatment efficacy was assessed using coupled tomography imaging diagnosis. The toxicity accompany with treatment was evaluated, as well as T cell receptor (TCR) repertoire before and several cycles after therapy was dynamically monitored by analyzing the complementarity-determining region 3 (CDR3) length distribution within CD4⁺ and CD8⁺ T cell subsets. The degrees of normalization of the T cell repertoire in CRC patients treated with the two methods were compared. The results showed that mIFL combined with bevacizumab was more effective in treating patients with metastatic CRC, and was accompanied by an increase in side effects such as proteinuria and hematuria. An even more restricted CDR3 profile in patients with metastatic CRC compared with healthy control has been detected. A prominent usage of TCR β chain variable (BV) gene BV12 and BV16 families within the CD4⁺ T cell subset and BV19 and BV21 families within the CD8⁺ T cell subset have been found before treatment. Moreover, CD8⁺ T cells showed more restricted patterns than CD4⁺ T cells, especially in patients before treatment. For patients with stable disease (SD) or partial remission (PR) after treatment, a less restricted CDR3 profile in post-treatment compared with pre-treatment has been found, but the opposite result was observed for patients with progressive disease (PD). The less restricted CDR3 pattern suggested a trend toward normalization of the TCR repertoire. The normalization of TCR repertoire significantly increased in patients treated with mIFL in combination with bevacizumab, but slightly in patients treated with mIFL alone. The results demonstrate a positive correlation between post-therapy TCR repertoire normalization and remission of metastatic CRC.     

Relevance of C1 and C2 epitopes for hemopoietic stem cell transplantation: role for sequential acquisition of HLA-C-specific inhibitory killer Ig-like receptor

Killer Ig-like receptors (KIR) and HLA class I ligands were studied in unrelated hemopoietic stem cell transplantation for chronic myeloid leukemia (n = 108). Significantly improved overall survival was observed in patients, which were homozygous for HLA-C-encoded group 1 (C1) ligands compared with those with group 2 (C2) ligands. Favorable outcome in the former patient group was an early effect that was highly significant in patients transplanted with G-CSF-mobilized peripheral blood and patients with advanced disease stages. In contrast, presence of C1 ligands in the donor was associated with significantly reduced patient survival. The differential roles of the two HLA-C ligands are explained in the context of a biased NK cell reconstitution, which is generally dominated by the presence of C1- but absence of C2-specific NK cells. The clinical observations are corroborated by in vitro experiments showing that NK cells derived from hemopoietic progenitor cells generally acquire the C1-specific inhibitory KIR2DL2/3 at earlier time points and with higher frequency than the C2-specific KIR2DL1. These findings define a novel determinant for understanding the role of NK cells in clinical hemopoietic stem cell transplantation.     

Testing the safety of clinical-grade mature autologous myeloid DC in a phase I clinical immunotherapy trial of CML

BACKGROUND: We conducted a phase I clinical immunotherapy trial of CML to evaluate the safety of a clinical-grade leukemic DC product standardized for purity and mature phenotype. METHODS: We injected autologous DC into patients in late chronic or accelerated phases of CML. The patients received mature CD83+ and bcr-abl+ DC prepared from CD14+ cells. Two cohorts of three patients received four injections each of 3 x 10(6) DC and 15 x 10(6) DC/injection, respectively. The first patient was studied before imatinib mesylate (IM) was available, four patients were treated concurrently with IM therapy and one did not tolerate the IM and was off the drug at the time of DC therapy. IM effects on WBC counts precluded DC preparation in numbers sufficient for further dose escalation. The first patient received DC s.c. and all subsequent patients received DC into a cervical lymph node under ultrasound guidance. RESULTS: DC injections were well tolerated. We observed no clinical responses. T cells drawn later in the course of therapy were more sensitive to stimulation by CML DC in vitro. DISCUSSION: The increase in T-cell sensitivity to CML-specific stimulation that accompanied active immunization by CML DC justifies further clinical studies, possibly with modifications such as an increased frequency and number of DC injections.     

Autologous dendritic cell vaccine for estrogen receptor (ER)/progestin receptor (PR) double-negative breast cancer

Purpose

A wealth of preclinical information, as well as a modest amount of clinical information, indicates that dendritic cell vaccines have therapeutic potential. The aim of this work was to assess the immune response, disease progression, and post-treatment survival of ER/PR double-negative stage II/IIIA breast cancer patients vaccinated with autologous dendritic cells pulsed with autologous tumor lysates.

Methods

Dendritic cell (DC) vaccines were generated from CD14+ precursors pulsed with autologous tumor lysates. DCs were matured with defined factors that induced surface marker and cytokine production. Individuals were immunized intradermally four times. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and lymphocyte subsets were determined for the evaluation of the therapeutic efficiency. Overall survival and disease progression rates were analyzed using Kaplan–Meier curves and compared with those of contemporaneous patients who were not administered DC vaccines.

Results

There were no unanticipated or serious adverse effects. DC vaccines elicited Th1 cytokine secretion and increased NK cells, CD8+ IFN-γ+ cells but decreased the percentage of CD3+ T cells and CD3+ HLA-DR+ T cells in the peripheral blood. Approximately 58% (18/31) of patients had a DTH-positive reaction. There was no difference in overall survival between the patients with and without DC vaccine. The 3-year progression-free survival was significantly prolonged: 76.9% versus 31.0% (with vs. without DC vaccine, p?Conclusion Our findings strongly suggest that tumor lysate-pulsed DCs provide a standardized and widely applicable source of breast cancer antigens that are very effective in evoking anti-breast cancer immune responses.     

Perioperative IFN-alpha to avoid surgically induced immune suppression in colorectal cancer patients

Surgical treatment of colorectal cancer is associated with postoperative immunosuppression, which might facilitate dissemination of tumor cells and outgrowth of minimal residual disease/(micro) metastases. Minimal residual disease has been shown to be of prognostic relevance in colorectal cancer. Therefore, stimulation of (anti-tumor) immune responses may be beneficial in the prevention of metastases formation. Important anti-tumor effector cells, which serve this function, are natural killer (NK) cells, CD8+ lymphocytes (CTL), dendritic cells (DC) and macrophages. In this review the immunomodulating properties of IFN-alpha are discussed, with a particular focus on perioperative stimulation of immune function in cancer patients. IFN-alpha is known to enhance innate immune functions such as stimulation of NK cells, transition from innate to adaptive responses (activation of DC) and regulating of CD8+ CTL activity and memory. Moreover, it exerts direct antitumor effects by regulating apoptosis and cell cycle. In several clinical trials, perioperative administration of IFN-alpha has indeed been shown to improve T cell responsiveness, prevent impairment of NK cell cytotoxicity and increase expression of activation markers on NK, T and NKT cells. In a clinical pilot study we showed in colorectal cancer patients that received perioperative IFN-alpha enhanced activation markers on T cells and NK cells, combined with better-preserved T cell function as indicated by phytohemaggluttinin skin tests. In the liver of these patients significantly more CD8+ T cells were found. In conclusion, IFN-alpha provides an effective adjuvant in several forms of cancer and improves several postoperative immune functions in perioperative administration. However, larger clinical trials are necessary to investigate effects on disease-free and overall survival.     

Epitope-dependent avidity thresholds for cytotoxic T-lymphocyte clearance of virus-infected cells

Cytotoxic T lymphocytes (CTLs) are crucial for immune control of viral infections. "Functional avidity," defined by the sensitizing dose of exogenously added epitope yielding half-maximal CTL triggering against uninfected target cells (SD(50)), has been utilized extensively as a measure of antiviral efficiency. However, CTLs recognize infected cells via endogenously produced epitopes, and the relationship of SD(50) to antiviral activity has never been directly revealed. We elucidate this relationship by comparing CTL killing of cells infected with panels of epitope-variant viruses to the corresponding SD(50) for the variant epitopes. This reveals a steeply sigmoid relationship between avidity and infected cell killing, with avidity thresholds (defined as the SD(50) required for CTL to achieve 50% efficiency of infected cell killing [KE(50)]), below which infected cell killing rapidly drops to none and above which killing efficiency rapidly plateaus. Three CTL clones recognizing the same viral epitope show the same KE(50) despite differential recognition of individual epitope variants, while CTLs recognizing another epitope show a 10-fold-higher KE(50), demonstrating epitope dependence of KE(50). Finally, the ability of CTLs to suppress viral replication depends on the same threshold KE(50). Thus, defining KE(50) values is required to interpret the significance of functional avidity measurements and predict CTL efficacy against virus-infected cells in pathogenesis and vaccine studies.