Autologous Lysate-pulsed Dendritic Cell Research Articles

Abstract PR13: Adjuvant TLR-3 administration enhances proinflammatory immune responses and is associated with extended survival in glioblastoma patients treated with dendritic cell vaccination

Abstract Malignant glioma, associated with a poor prognosis, is the most common primary malignant brain tumor in adults. We and others have documented immune responses following dendritic cell (DC) vaccination as an active immunotherapeutic treatment for these patients. In this phase II clinical trial, we randomized malignant glioma patients to receive autologous tumor lysate pulsed DC vaccination with and without adjuvant Toll-like receptor (TLR) agonists. TLRs are present on dendritic cells and serve to modulate immune responses. Twenty-three patients with WHO grade III or IV glioma were treated with three intradermal injections of autologous tumor lysate-pulsed DC on days 0, 14, and 28 followed by an adjuvant placebo, TLR-7 agonist (resiquimod), or TLR-3 agonist (Poly ICLC). Mass cytometry (CyTOF) was used to analyze immune cell populations of patient peripheral blood mononuclear cells (PBMC) before and following treatment. DC-vaccinated patients who received adjuvant Poly ICLC treatment had a significantly improved median survival of 54 months over placebo (11 months) and adjuvant resiquimod (17 months) groups (P<0.01). CyTOF and single-cell RNA sequencing (scRNseq) gene expression analysis of systemic PBMCs from patients demonstrated increased immune cell activation and expression of proinflammatory genes. Notably, the expansion of a heterogenous myeloid cell population was noted to correlate with increased survival, corroborating preclinical evidence that these cells modulate adaptive immunity in the tumor microenvironment. Overall, these findings suggest that adjuvant Poly ICLC treatment improves outcomes with autologous lysate-pulsed DC vaccine treatment via modulation of proinflammatory pathways. This abstract is also being presented as Poster B27. Citation Format: Joseph P. Antonios, Richard G. Everson, Aaron Mochizuki, Sara Khattab, Horacio Soto, Prashant Romiyo, Matthew Z. Sun, Diana Moughon, Emma Billingslea-Yoon, Sylvia Odesa, Gang Li, Eric Kawaguchi, Alex Salazar, William Yong, Jason Schlossman, Benjamin Ellingson, Anthony C. Wang, Timothy Cloughesy, Robert M. Prins, Linda M. Liau. Adjuvant TLR-3 administration enhances proinflammatory immune responses and is associated with extended survival in glioblastoma patients treated with dendritic cell vaccination [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR13.

A phase II trial of autologous dendritic cell vaccination and radiochemotherapy following fluorescence-guided surgery in newly diagnosed glioblastoma patients

BackgroundPrognosis of patients with glioblastoma multiforme (GBM) remains dismal, with median overall survival (OS) of about 15 months. It is therefore crucial to search alternative strategies that improve these results obtained with conventional treatments. In this context, immunotherapy seems to be a promising therapeutic option. We hypothesized that the addition of tumor lysate-pulsed autologous dendritic cells (DCs) vaccination to maximal safe resection followed by radiotherapy and concomitant and adjuvant temozolomide could improve patients’ survival.MethodsWe conducted a phase-II clinical trial of autologous DCs vaccination in patients with newly diagnosed patients GBM who were candidates to complete or near complete resection. Candidates were finally included if residual tumor volume was lower than 1 cc on postoperative radiological examination. Autologous DCs were generated from peripheral blood monocytes and pulsed with autologous whole tumor lysate. The vaccination calendar started before radiotherapy and was continued during adjuvant chemotherapy. Progression free survival (PFS) and OS were analyzed with the Kaplan–Meier method. Immune response were assessed in blood samples obtained before each vaccines.ResultsThirty-two consecutive patients were screened, one of which was a screening failure due to insufficient resection. Median age was 61 years (range 42–70). Karnofsky performance score (KPS) was 90–100 in 29%, 80 in 35.5% and 60–70 in 35.5% of cases. MGMT (O6-methylguanine-DNA-methyltransferase) promoter was methylated in 45.2% of patients. No severe adverse effects related to immunotherapy were registered. Median PFS was 12.7 months (CI 95% 7–16) and median OS was 23.4 months (95% CI 16–33.1). Increase in post-vaccination tumor specific immune response after vaccines (proliferation or cytokine production) was detected in 11/27 evaluated patients. No correlation between immune response and survival was found.ConclusionsOur results suggest that the addition of tumor lysate-pulsed autologous DCs vaccination to tumor resection and combined radio-chemotherapy is feasible and safe. A multicenter randomized clinical trial is warranted to evaluate the potential survival benefit of this therapeutic approach.Trial registration This phase-II trial was registered as EudraCT: 2009-009879-35 and ClinicalTrials.gov Identifier: NCT01006044 retrospectively registered

Abstract 2491: Treatment with tumor lysate-pulsed autologous dendritic cells prolongs survival in patients with recurrent glioblastoma multiforme

Abstract Background The survival rate in recurrent Glioblastoma multiforme (rGBM) patients has not meaningfully changed in the past several decades. We have treated two cohorts of rGBM patients, one consisting of patients with early progression, and one of patients with progression following several cycles of adjuvant temozomolide chemotherapy, with autologous dendritic cells pulsed with autologous tumor cell lysate (DCVax®-L). Such treatment is intended to activate the immune system against the tumor cells, so that the ensuing immune attack may delay progression and time to death. Methods Disease progression in patients with GBM was determined through MRI. Progression needed to be determined by independent review on 2 consecutive scans for early progressor classification to avoid enrolling patients with pseudoprogression. Assessment of progression was done using modified RANO criteria. Results Nineteen (19) patients diagnosed with GBM were determined to have recurrence immediately following radiation therapy with concomitant temozolomide chemotherapy. Median overall survival in this cohort from initial GBM diagnosis is 15.1 months (95% CI: 10.5-17.2), and the range is 8.1->31 months. A literature search revealed 6 publications with comparable populations of patients. The table below demonstrates that these patients typically have a life expectancy of 8-10 months. Reference (n)PopulationMedian Survival (95% CI)DCVax-L (19)PD post RT+chemo, confirmed 2 months later15.1 months (10.5 - 17.2)Brandes et al. 2008 (18)PD at 4 weeks pos RT+chemo, confirmed after two more tmz cycles10.2 months (n.a.)Roldan et al. 2009 (10)PD at 4-6 weeks post RT+chemo, confirmed after ≥1 more tmz cycle(s)9.1 months (4.9 - 19.1)Kang et al. 2010 (10)PD at 2 consecutive scans post RT+chemo10.8 (n.a.)Sanghera et al. 2010 (29)PD at 2 consecutive scans within 8 weeks post RT+chemo8.3 months (n.a.)Gunjur et al. 2011 (27)PD at 2 consecutive scans within 3 months post RT+chemo, or clinical deterioration10.4 months (n.a.)Linhares et al. 2013 (13)PD at 2 consecutive scans within 3 months post RT+chemo9.0 months (3.7 - 14.3) A second cohort of patients with rGBM consisted of 8 patients with recurrence following several adjuvant temozolomide treatment cycles. Median OS for these patients is 14.7 months from the time of surgery for first recurrence. The comparator for this cohort is derived from Friedman et al. (2009), who reported mOS of 8.7-9.3 months from the time of first recurrence for rGBM patients treated with Bevacizumab with or without Irinotecan. Results and Conclusion These results suggest that treatment with DCVax-L can extend survival by 5 months or more in patients with rGBM. Citation Format: Marnix Bosch, Robert Prins, Linda Liau. Treatment with tumor lysate-pulsed autologous dendritic cells prolongs survival in patients with recurrent glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2491. doi:10.1158/1538-7445.AM2015-2491

ITOC2 – 017. Prolonged survival for patients with recurrent glioblastoma multiforme who are treated with tumour lysate-pulsed autologous dendritic cells

<h3>Background</h3> Recurrent glioblastoma multiforme (rGBM) is a life threatening condition, with a mortality rate of approximately 100%. Despite recent advances in therapy, the survival rate in rGBM patients has not meaningfully changed in the past several decades. We have treated two cohorts of rGBM patients, one consisting of patients with early progression, and one consisting of patients with progression following several cycles of adjuvant temozomolide chemotherapy, with autologous dendritic cells pulsed with autologous tumour cell lysate (DCVax®-L). Such treatment is intended to activate the immune system against the tumour cells, with the expectation that the ensuing immune attack may delay progression and time to death. <h3>Aim</h3> To determine survival characteristics of patients with rGBM who are treated with DCVax-L. <h3>Methods</h3> Disease progression in patients with GBM was determined through magnetic resonance imaging. Progression needed to be determined by independent review on two consecutive scans for early progressor classification to avoid enrolling patients with pseudoprogression. Assessment of progression was done using modified RANO criteria. <h3>Results</h3> Nineteen (19) patients diagnosed with GBM were determined to have rapid recurrence following radiation therapy with concomitant temozolomide chemotherapy. The life expectancy for such patients is typically poor, and they were provided the DCVax-L treatment as part of a compassionate use approach in parallel to ongoing late stage clinical trials. Median overall survival in this cohort from initial GBM diagnosis is 15.1months (95% confidence interval (CI): 10.5–17.2), and the range is 8.1–>31months. A literature search revealed six publications with comparable populations of patients (Brandes et al., 2008; Roldan et al., 2009; Sanghera et al., 2010; Kang et al., 2010; Gunjur et al., 2011; Linhares et al., 2013). The table below demonstrates that these patients typically have a life expectancy of 8–10months. Reference (<i>n</i>) Population Median Survival (95% CI) DCVax-L (19) post RT+chemo, confirmed 2months later 15.1months (10.5–17.2) Brandes et al. (2008) (18) PD at 4weeks pos RT+chemo, confirmed after two more tmz cycles 10.2 months (n.a.) Roldan et al. (2009) (10) PD at 4–6weeks post RT+chemo, confirmed after <mml:math><mml:mrow><mml:mo>⩾</mml:mo></mml:mrow></mml:math>1 more tmz cycle(s) 9.1months (4.9–19.1) Kang et al. (2010) (10) PD at two consecutive scans post RT+chemo 10.8 (n.a.) Sanghera et al. (2010) (29) PD at two consecutive scans within 8weeks post RT+chemo8.3months (n.a.) Gunjur et al. (2011 (27) PD at two consecutive scans within 3months post RT+chemo, or clinical deterioration 10.4months (n.a.) Linhares et al. (2013) (13) PD at two consecutive scans within 3months post RT+chemo 9.0 months (3.7–14.3) A second cohort of patients with rGBM consisted of eight patients with recurrence following several adjuvant temozolomide treatment cycles. Median overall survival (OS) for these patients is 14.7months from the time of surgery for first recurrence. The comparator for this cohort is derived from Friedman et al. (2009), who reported mOS of 8.7–9.3months from the time of first recurrence for rGBM patients treated with Bevacizumab with or without irinotecan. <h3>Results and conclusion</h3> The results obtained in two cohorts of patients with rGBM suggest that treatment with autologous DC pulsed with autologous tumour cell lysate can extend survival by 5months or more.

Abstract 1071: Adoptive transfer of T cells overcomes barriers of homing to the tumor microenvironment in ovarian cancer

Abstract Objectives: Chemokine-dependent molecular mechanisms could play a key role in controlling tumor-specific T cell trafficking and the clinical success of adoptive transfer of ex vivo expanded T cells. We thus characterized the chemokine microenvironment in advanced stage serous ovarian cancer and evaluated whether CD3/CD28 co-stimulation of lysate vaccine-primed autologous T cells prepared for adoptive transfer led to the expansion of tumor antigen-reactive T cells able to home to ovarian cancer microenvironment. Methods: The expression of all known human chemokines was analyzed on a gene expression microarray from 63 patients with primary ovarian cancer and was validated on publicly available gene expression dataset and on tissue microarray constructed from a matching cohort of patients. Five HLA-A2 patients with recurrent ovarian cancer, who had previously received vaccination with autologous lysate-pulsed dendritic cell vaccine, underwent apheresis to harvest peripheral blood T cells, which were then expanded using beads coated with anti-CD3/anti-CD28 antibodies and IL-2. T cells were analyzed for 10 chemokine receptors that matched the chemokines expressed in ovarian cancer. Tumor-specific T cells were identified by a HER2/neu pentamer staining. Chemotaxis assays were performed to assess the functionality of these receptors. Results: Chemokines in general were found to be expressed at low level in advanced stage ovarian cancer, with the 10 most highly expressed ones being CCL2, CCL4, CCL5, CCL28, CXCL5, CXCL6 CXCL10, CXCL12, CXCL16 and CX3CL1. The two most commonly expressed chemokine receptors on the expanded cells were CXCR3 and CXCR4 (receptors for CXCL10 and CXCL12). At the end of expansion, 70% of output CD4+ and CD8+ cells expressed CXCR3 and CXCR4, compared to 20% of input T cells. The higher expression of CXCR3 and CXCR4 in output T cells resulted in significantly increased chemotaxis toward CXCL10 and CXCL12, suggesting a better homing ability to the tumor microenvironment. HER2/neu-specific T cells were detected in the expanded T cell population and showed migration toward CXCL10 and CXCL12. Conclusions: Adoptive transfer of ex vivo expanded tumor specific T cells that are capable of tumor engraftment holds a highly promising treatment approach for recurrent ovarian cancer. Chemokines are key regulators of circulation, homing and retention of these cells. Our data confirm that T cells prepared for adoptive transfer through CD3/CD28 bead expansion comprise tumor-specific cells, which upregulate appropriate chemokine receptors and show enhanced migration towards chemokines highly expressed by ovarian cancer. Citation Format: Emese Zsiros, Priyanka Duttagupta, Thomas Garrabrant, Janos L. Tanyi, Lana E. Kandalaft, George Coukos. Adoptive transfer of T cells overcomes barriers of homing to the tumor microenvironment in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1071. doi:10.1158/1538-7445.AM2014-1071

Sequential administration of chemotherapy and tumor lysate pulsed autologous dendritic cells induce stronger anti-tumor activity in treatment of metastatic colorectal cancer: immunologic results and the underlying mechanisms from a phase II trial. (156.3)

Abstract We conducted a randomized phase II trial to determine whether sequential administration of chemoherapy plus DC vaccine is superior to chemotherapy alone. Our studies indicated that appropriate doses of oxaliplatin(OXA) and/or 5-Fu could enhance HLA-A2 and Fas expression, depressed FasL expression on SW480 cell surface and stimulate higher counts of IFN-γ secretion antigen-specific T cells, decrease the productions of immunosuppression cytokines such as IL-10, TGF-β, and VEGF in supernatants of treated SW480 cells, in peripheral blood of patients with metastatic colorectal cancer(MCRC) and CT26 liver metastasis mice models. The proportions of CD4+CD25+ Treg were observed to reduce in PBMCs from MCRC patients or in splenocytes from CT26 liver metastasis mice, as well as CD11b+Gr-1+ MSCs after the chemotherapy. Sequential antigen-pulsed DC administration could stabilize above-mentioned results afterwards. We also found that the production of Hsp70, a new Th1 polarization adjuvant which is a chaperone important to the DCs’ cross-presentation, significant increased in peripheral blood of MCRC patients after OXA and 5-Fu chemotherapy. The survival time of CT26 liver metastasis mice had been significant prolonged and specific anti-tumor immune response induced by receiving sequential immuno-chemotherapy. We hope that these results may provide new strategies and methods in clinical trials revolving immunotherapy.

Generation in vitro of B-cell chronic lymphocytic leukaemia-proliferative and specific HLA class-II-restricted cytotoxic T-cell responses using autologous dendritic cells pulsed with tumour cell lysate.

Immunotherapy using dendritic cells has shown encouraging results in both haematological and non-haematological malignancies. In this study, monocyte-derived dendritic cells from patients with B-CLL were cultured for 6 days in the presence of IL-4 and GM-CSF. Autologous B-CLL T-cells were cultured alone or with B-CLL lysate-pulsed and unpulsed autologous dendritic cells. IFN-gamma secretion was assessed using ELISA. Cytotoxicity was assessed, after 21 days in culture and re-stimulation, using flow cytometry with and without blockade by anti-HLA class I, anti-HLA class II, anti-CD4, anti-CD8 and anti-TCRalphabeta monoclonal antibodies. B-CLL T cells stimulated with B-CLL lysate-pulsed autologous dendritic cells showed a significant (P = 0.0004) increase in IFN-gamma secretion and a significant (P = 0.0008) increase in specific cytotoxicity to autologous B-cell targets, but none to autologous T cell or B cell targets from healthy individuals. B-CLL T cells cultured with (non-B-CLL) B-cell lysate-pulsed B-CLL dendritic cells showed no significant response. Pulsing dendritic cells from healthy volunteers with an autologous (non-B-CLL) B-cell lysate did not stimulate proliferation, cytokine production or cytotoxicity by autologous T cells. Pulsing B-CLL dendritic cells with allogeneic B-CLL lysates and culturing with autologous T-cells elicited cytotoxicity against autologous B-CLL targets in some cases, but not in others. Cytotoxicity was significantly reduced by blocking with anti-HLA class II (P = 0.001), anti-TCRalphabeta (P = 0.03) and anti-CD4 (P = 0.046) antibodies. Phenotyping of the responding T-cell population demonstrated the majority to be CD4 positive. Our data demonstrate that HLA class II-restricted proliferative and cytotoxic T-cell responses to B-CLL can be generated using autologous dendritic cells pulsed with tumour cell lysate.

Importance of [18F]fluorodeoxyglucose-positron emission tomography scanning for the monitoring of responses to immunotherapy in follicular lymphoma

We prospectively investigated the use of [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) to kinetically monitor cell activation in six follicular lymphoma patients vaccinated with tumor lysate-pulsed autologous dendritic cells. PET revealed additional initial nodes suggestive of lymphoma, that were of less than 10 mm of diameter on computed tomography, and documented disease progression with sensitivity, even within loci with no significant variations of CT findings. Although tracer fixation was not observed in the inguinal nodes draining the dendritic cell intradermal injection sites in the six patients, a transient marked increase in FDG uptake within malignant nodes was observed early after vaccine administration in a patient who achieved complete remission. In non-responding patients, we observed a continuous increase of FDG uptake associated with an increase in the size and number of pathological nodes/locus, or of involved loci. FDG-PET is a non-invasive imaging procedure that might be of crucial interest to detect cell responses induced by immunotherapies.

Interest of FDG-PET Scanning for the Monitoring of Immunotherapies for Follicular Lymphoma.

Except in the subset of patients with localized disease, follicular lymphoma (FL), the most common type of indolent non-Hodgkin's lymphoma, should still be regarded as an incurable malignancy with a relentless relapsing course. New therapeutic approaches have been developed including anti-tumor vaccination strategies with induced humoral and cellular immune responses revealed by sensitive biological analyses but no imaging of the lymphocyte activation developed so far. Because [18F]fluorodeoxyglucose (FDG) is sequestred in metabolically active cells, we prospectively investigated the use of FDG-PET (positron emission tomography) to kinetically monitor cell activation in 7 FL patients vaccinated by repeated intradermal (ID) and intravenous injections of tumor lysate-pulsed autologous dendritic cells (DC-lysate). Patients were administered 5 vaccines and all patients developed a positive delayed-type hypersensitivity reaction at the site of DC-lysate ID injections after several vaccines administration, testifying of the induction of a biological response. Among the 4 patients with measurable disease at time of vaccination, 2 complete remissions (CR) and 2 disease progressions were observed. Three patients with initial normal computed tomographies (CT) were still in CR 12 months after the last vaccine. PETs were performed before vaccination, 14 days and 1 month after the last vaccine. PET revealed initial malignant nodes infracentimetric on concomitant CT, and was very sensitive in documenting disease progression even in patients with no significant variations of CT findings. A transient marked increase in FDG uptake within malignant nodes was observed early after vaccine administration in a patient who achieved a CR (fig 1-3-4-5), while ranges of tracer uptake variations were less important with a continuous increase of FGD uptake, size and number of involved loci in non responding patients (fig 2: follow up of intensity of FDG uptake by 6 tumoral nodes in a non responding pt). In conclusion, FDG-PET is a non invasive imaging procedure which besides being useful for the staging and the evaluation of treatment efficacy in FL, might be of crucial interest to detect cell responses induced by immunotherapies. [Display omitted]

Melanoma-Reactive T Cells in the Bone Marrow of Melanoma Patients: Association with Disease Stage and Disease Duration

Using IFN-gamma enzyme-linked immunospot, we investigated reactivity of T cells from bone marrow and peripheral blood to melanoma lysate-pulsed autologous dendritic cells in 40 melanoma patients. Melanoma-reactive T cells were present in the bone marrow of seven patients and in peripheral blood of four patients. In the bone marrow, melanoma-reactive T cells were present in 6 of 21 stage IV patients and in 1 of 10 stage III patients, whereas none were detected in stage I to II patients (0 of 9). The occurrence of tumor-reactive bone marrow T cells in melanoma patients was associated with advanced disease stage, disease duration and tumor load, and independent of treatment. These findings provide new insights into the generation of T-cell responses in melanoma patients.

Poor ex vivo induction of T-cell responses to idiotype or tumor cell lysate-pulsed autologous dendritic cells in advanced pre-treated multiple myeloma

This study evaluated the feasibility of using dendritic cells (DCs) to generate, ex vivo, anti-tumor cytotoxic T lymphocytes (CTL) in patients with stage III multiple myeloma (MM). Nucleated cells from eight patients who had received chemotherapy (three of whom had undergone autologous hemopoeitic stem cell transplantation) were collected by apheresis. Their monocytes were enriched using counter-current centrifugation, differentiated into DCs which were further co-cultured with autologous CD8 lymphocytes to induce CTL. The DCs were pulsed either with the idiotypic paraprotein (regarded as a tumor-specific antigen) or with autologous MM cell lysate before co-culture. Specific T-cell responses were measured in IFNγ enzyme-linked immunospot and chromium release assays of autologous plasmocyte targets. A slight increase in IFNγ secretion by T-cells was observed for two patients (DCs pulsed with idiotypic paraprotein for one, MM cell lysate for the other). No or weak specific lysis of plasmocyte targets was observed in the chromium release assays. In conclusion, the T-cell response to pulsed DCs was very weak or absent. There are clinical and technical reasons that could explain, in part, this lack of response.

Pancreatic cancer cell lysate-pulsed autologous dendritic cells induce T-lymphocyte response

Abstract Introduction: Dendritic cells (DC) are potent APC playing an important role in T-cell mediated immune reactions. Tumor cell lysates have the possible advantage in stimulation of cytotoxic T-cells (CTL) by presenting a broad variety of antigens. Methods: DC (generated using GM-CSF/IL-4) were pulsed with tumor cell lysate (TU-LY) of a newly established autologous pancreatic cancer cell line (DD-TU). In controls the immunogenic protein KLH was added. Antigen-pulsed DC were. Autologous lymphocytes were weekly stimulated with lysate-pulsed DC (activated with TNF-alpha/prostaglandin E2), cultured with IL-2/IL-7. IL-127/IFN-gamma secretion was evaluated (ELISA) and correlated with expression of the activation marker CD69 (FACS). After 4 stimulations, functional cytotoxicity tests of CTL with DD-TU as targets were performed with and without anti-HLA class I mAB. Results: CD54, MHC class II, CD80 and CD86 expression revealed autologous DC generation. Stimulation of CTL with TU-LY resulted in expression of CD86, CD83 and MHC class II molecules and CTL proliferation. DC/TU-LY stimulation induced syntheses of IL-12 and IFN-gamma (P Conclusions: This first description of a complete autologous DC-tumor lysate system in pancreatic cancer revealed a CTL/Th1-like cytokine profile showing significant autologous tumor cell lysis and provides solid ground for the development of future DC-based immunotherapeutic vaccination strategies in pancreatic cancer.

In vitro dendritic cell-induced T cell responses to B cell chronic lymphocytic leukaemia enhanced by IL-15 and dendritic cell-B-CLL electrofusion hybrids.

HLA class II-restricted proliferative and cytotoxic T cell (CTL) responses to B cell chronic lymphocytic leukaemia (B-CLL) can be generated using autologous dendritic cells (DCs) pulsed with tumour cell lysate. In this study a number of different approaches were used to optimize further the in vitro system. First, the effects of a variety of maturation agents were studied. The addition of TNF-alpha, polyriboinosinic polyribocytidylic acid (Poly(I:C)) and LPS to autologous DCs resulted in the emergence of only a small percentage of CD83+ DCs, IFN-alpha having no demonstrable effect. Only the addition of Poly(I:C) to DCs resulted in modestly increased specific cytotoxicity to B-CLL targets, IFN-alpha and LPS having no effect. Secondly, T cells were pretreated with IL-15, prior to culturing with lysate-pulsed autologous DCs. A significant increase in T cell activation (P = 0.038), IFN-gamma secretion (P = 0.030) and specific cytotoxicity to B-CLL targets (P = 0.006) was demonstrated compared to untreated T cells. Thirdly, monocyte derived DCs electrofused with B-CLL B cells were compared with lysate-pulsed DCs. T cells stimulated by fused DCs generated higher levels of specific cytotoxicity to autologous B-CLL B cell targets than those stimulated by lysate pulsed DCs (P = 0.013). Blocking studies demonstrated inhibition of this cytotoxicity by both anti-CD4 (P = 0.062) and anti-CD8 monoclonal antibodies (P = 0.018), suggesting the generation of both HLA class I- and HLA class II-restricted CTL responses. In summary, in vitro B-CLL-specific T cell responses can be enhanced further by preincubating T cells with IL-15 and using autologous fused DC-B-CLL hybrids instead of autologous lysate-pulsed DCs. These preliminary data require confirmation with larger numbers of patients. Such an approach, however, may eventually provide effective immunotherapy for treatment of B-CLL.

Induction of tumour-specific CD8(+) cytotoxic T lymphocytes by tumour lysate-pulsed autologous dendritic cells in patients with uterine serous papillary cancer.

Uterine serous papillary carcinoma is a highly aggressive variant of endometrial cancer histologically similar to high grade ovarian cancer. Unlike ovarian cancer, however, it is a chemoresistant disease from onset, with responses to combined cisplatinum-based chemotherapy in the order of 20% and an extremely poor prognosis. In this study, we demonstrate that tumour lysate-pulsed autologous dendritic cells can elicit a specific CD8+ cytotoxic T lymphocyte response against autologous tumour target cells in three patients with uterine serous papillary cancer. CTL from patients 1 and 2 expressed strong cytolytic activity against autologous tumour cells, did not lyse autologous lymphoblasts or autologous EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Patient 3 CD8+ T cells expressed a modest but reproducible cytotoxicity against autologous tumour cells only at the time of the first priming. Further priming attempts with PBL collected from patient 3 after tumour progression in the lumboaortic lymph nodes were unsuccesful. Cytotoxicity against autologous tumour cells could be significantly inhibited by anti-HLA class I (W6/32) and anti-LFA-1 MAbs. Highly cytotoxic CD8+ T cells from patients 1 and 2 showed a heterogeneous CD56 expression while CD56 was not expressed by non-cytotoxic CD8+ T cells from patient 3. Using two colour flow cytometric analysis of intracellular cytokine expression at the single cell level, a striking dominance of IFN-γ expressors was detectable in CTL populations of patients 1 and 2 while in patient 3 a dominant population of CD8+ T cells expressing IL-4 and IL-10 was consistently detected. Taken together, these data demonstrate that tumour lysate-pulsed DC can be an effective tool in inducing uterine serous papillary cancer-specific CD8+ CTL able to kill autologous tumour cells in vitro. However, high levels of tumour specific tolerance in some patients may impose a significant barrier to therapeutic vaccination. These results may have important implications for the treatment in the adjuvant setting of uterine serous papillary cancer patients with active or adoptive immunotherapy.British Journal of Cancer (2002) 86, 151–157. DOI: 10.1038/sj/bjc/6600026 www.bjcancer.com© 2002 The Cancer Research Campaign