Joseph W. Fay

Flt3-Ligand and Granulocyte Colony-Stimulating Factor Mobilize Distinct Human Dendritic Cell Subsets In Vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c− plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c− IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c− precursors (>7-fold). Freshly sorted CD11c+ but not CD11c− cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c− cells can be induced to secrete high levels of IFN-α, in response to influenza virus. CD11c+ and CD11c− cells can mature in vitro with GM-CSF + TNF-α or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c− subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.

A Modular Analysis Framework for Blood Genomics Studies: Application to Systemic Lupus Erythematosus

The analysis of patient blood transcriptional profiles offers a means to investigate the immunological mechanisms relevant to human diseases on a genome-wide scale. In addition, such studies provide a basis for the discovery of clinically relevant biomarker signatures. We designed a strategy for microarray analysis that is based on the identification of transcriptional modules formed by genes coordinately expressed in multiple disease data sets. Mapping changes in gene expression at the module level generated disease-specific transcriptional fingerprints that provide a stable framework for the visualization and functional interpretation of microarray data. These transcriptional modules were used as a basis for the selection of biomarkers and the development of a multivariate transcriptional indicator of disease progression in patients with systemic lupus erythematosus. Thus, this work describes the implementation and application of a methodology designed to support systems-scale analysis of the human immune system in translational research settings.

Dendritic cell subsets in health and disease

Summary:  The dendritic cell (DC) system of antigen‐presenting cells controls immunity and tolerance. DCs initiate and regulate immune responses in a manner that depends on signals they receive from microbes and their cellular environment. They allow the immune system to make qualitatively distinct responses against different microbial infections. DCs are composed of subsets that express different microbial receptors and express different surface molecules and cytokines. Our studies lead us to propose that interstitial (dermal) DCs preferentially activate humoral immunity, whereas Langerhans cells preferentially induce cellular immunity. Alterations of the DC system result in diseases such as autoimmunity, allergy, and cancer. Conversely, DCs can be exploited for vaccination, and novel vaccines that directly target DCs in vivo are being designed.

Disabling Immune Tolerance by Programmed Death-1 Blockade With Pidilizumab After Autologous Hematopoietic Stem-Cell Transplantation for Diffuse Large B-Cell Lymphoma: Results of an International Phase II Trial

PURPOSE The Programmed Death-1 (PD-1) immune checkpoint pathway may be usurped by tumors, including diffuse large B-cell lymphoma (DLBCL), to evade immune surveillance. The reconstituting immune landscape after autologous hematopoietic stem-cell transplantation (AHSCT) may be particularly favorable for breaking immune tolerance through PD-1 blockade. PATIENTS AND METHODS We conducted an international phase II study of pidilizumab, an anti-PD-1 monoclonal antibody, in patients with DLBCL undergoing AHSCT, with correlative studies of lymphocyte subsets. Patients received three doses of pidilizumab beginning 1 to 3 months after AHSCT. RESULTS Sixty-six eligible patients were treated. Toxicity was mild. At 16 months after the first treatment, progression-free survival (PFS) was 0.72 (90% CI, 0.60 to 0.82), meeting the primary end point. Among the 24 high-risk patients who remained positive on positron emission tomography after salvage chemotherapy, the 16-month PFS was 0.70 (90% CI, 0.51 to 0.82). Among the 35 patients with measurable disease after AHSCT, the overall response rate after pidilizumab treatment was 51%. Treatment was associated with increases in circulating lymphocyte subsets including PD-L1E-bearing lymphocytes, suggesting an on-target in vivo effect of pidilizumab. CONCLUSION This is the first demonstration of clinical activity of PD-1 blockade in DLBCL. Given these results, PD-1 blockade after AHSCT using pidilizumab may represent a promising therapeutic strategy in this disease.

Dendritic cells loaded with killed allogeneic melanoma cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity.

Dendritic cells (DCs) loaded with killed allogeneic tumors can cross-prime tumor-specific naive CD8+ T cells in vitro, thereby providing an option to overcome human leukocyte antigen restriction inherent to loading DC vaccines with peptides. We have vaccinated 20 patients with stage IV melanoma with autologous monocyte-derived DCs loaded with killed allogeneic Colo829 melanoma cell line. DCs were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (granulocyte macrophage-colony stimulating factor) and interleukin (IL-4) and activated by additional culture with tumor necrosis factor and CD40 ligand. A total of 8 vaccines were administered at monthly intervals. The first patient was accrued December 2002 and the last November 2003. Fourteen patients were alive at 12 months, 9 patients were alive at 24 months, and 8 patients are alive as of January 2006. The estimated median overall survival is 22.5 months with a range of 2 to 35.5 months. Vaccinations were safe and tolerable. They induced, in 2 patients who failed previous therapy, durable objective clinical responses, 1 complete regression (CR) and 1 partial regression (PR) lasting 18 and 23 months, respectively. Three out of 13 analyzed patients showed T-cell immunity to melanoma antigen recognized by autologous T cells (MART-1) tissue differentiation antigen. Two of 3 patients showed improved immune function after vaccinations demonstrated by improved secretion of interferon (IFN)-γ or T-cell proliferation in response to MART-1 derived peptides. In one of these patients, vaccination led to elicitation of CD8+ T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. Thus, the present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma.

Taming cancer by inducing immunity via dendritic cells

Summary: Immunotherapy seeks to mobilize a patients immune system for therapeutic benefit. It can be passive, i.e. transfer of immune effector cells (T cells) or proteins (antibodies), or active, i.e. vaccination. In cancer, passive immunotherapy can lead to some objective clinical responses, thus demonstrating that the immune system can reject tumors. However, passive immunotherapy is not expected to yield long‐lived memory T cells that might control tumor outgrowth. Active immunotherapy with dendritic cell (DC)‐based vaccines has the potential to induce both tumor‐specific effector and memory T cells. Early clinical trials testing vaccination with ex vivo‐generated DCs pulsed with tumor antigens provide a proof‐of‐principle that therapeutic immunity can be elicited. Yet, there is a need to improve their efficacy. The next generation of DC vaccines is expected to generate large numbers of high‐avidity effector CD8+ T cells and to overcome regulatory T cells. Therapeutic vaccination protocols will combine improved ex vivo DC vaccines with therapies that offset the suppressive environment established by tumors.

Circulating tumor antigen-specific regulatory T cells in patients with metastatic melanoma

Although it is accepted that regulatory T cells (T regs) contribute to cancer progression, most studies in the field consider nonantigen-specific suppression. Here, we show the presence of tumor antigen-specific CD4+ T regs in the blood of patients with metastatic melanoma. These CD4+ T regs recognize a broad range of tumor antigens, including gp100 and TRP1 (melanoma tissue differentiation antigens), NY-ESO-1 (cancer/testis antigen) and survivin (inhibitor of apoptosis protein (IAP) family antigen). These tumor antigen-specific T regs proliferate in peripheral blood mononuclear cells (PBMC) cultures in response to specific 15-mer peptides, produce preferentially IL-10 and express high levels of FoxP3. They suppress autologous CD4+CD25− T cell responses in a cell contact-dependent manner and thus share properties of both naturally occurring regulatory T cells and type 1 regulatory T cells. Such tumor antigen-specific T regs were not detected in healthy individuals. These tumor antigen-specific T regs might thus represent another target for immunotherapy of metastatic melanoma.

Treatment of resistant malignant lymphoma with cyclophosphamide, total body irradiation, and transplantation of cryopreserved autologous marrow.

Twenty-seven patients with malignant lymphoma in whom primary chemotherapy had failed and the prognosis was poor were treated with cyclophosphamide, total body irradiation, and transplantation of cryopreserved autologous marrow. The median time to recovery of more than 500 neutrophils per microliter and more than 10,000 platelets per microliter was 18 and 24 days, respectively. Complete remission was achieved in 15 patients (56 per cent), five of whom were in continuous remission at this writing 19 to 71 months after transplantation without further therapy and one of whom was alive in a subsequent remission at 20 months. Fifteen patients died of lymphoma, three of interstitial pneumonitis, two of sepsis, and one of congestive heart failure. This experience shows that intensive therapy and autologous-marrow transplantation can produce prolonged remissions in patients with malignant lymphoma in whom conventional chemotherapy has failed.

Harnessing human dendritic cell subsets for medicine.

Summary:  Immunity results from a complex interplay between the antigen‐non‐specific innate immune system and the antigen‐specific adaptive immune system. The cells and molecules of the innate system employ non‐clonal recognition receptors including lectins, Toll‐like receptors, NOD‐like receptors, and helicases. B and T lymphocytes of the adaptive immune system employ clonal receptors recognizing antigens or their derived peptides in a highly specific manner. An essential link between innate and adaptive immunity is provided by dendritic cells (DCs). DCs can induce such contrasting states as immunity and tolerance. The recent years have brought a wealth of information on the biology of DCs revealing the complexity of this cell system. Indeed, DC plasticity and subsets are prominent determinants of the type and quality of elicited immune responses. In this article, we summarize our recent studies aimed at a better understanding of the DC system to unravel the pathophysiology of human diseases and design novel human vaccines.

Immune and clinical outcomes in patients with stage IV melanoma vaccinated with peptide-pulsed dendritic cells derived from CD34+ progenitors and activated with type I interferon.

Twenty-two HLA A*0201+ patients with stage IV melanoma were enrolled in a phase 1 safety and feasibility trial using a composite dendritic cell (DC) vaccine generated by culturing CD34+ hematopoietic progenitors and activated with IFN-α. The DC vaccine was loaded with peptides derived from four melanoma tissue differentiation antigens (MART-1, tyrosinase, MAGE-3, and gp100) and influenza matrix peptide (Flu-MP). Twenty patients were evaluable, 14 of whom received vaccination with peptide-pulsed DCs without keyhole limpet hemocyanin (KLH) and 6 of whom received vaccination with KLH-loaded DCs. Patients were vaccinated until disease progression or until they had received eight vaccinations. None of the analyzed patients showed the expansion of melanoma-peptide-specific circulating effector memory T cells that secrete IFN-γ in direct ELISPOT. Melanoma-peptide-specific recall memory CD8+ T cells able to secrete IFN-γ and to proliferate could be detected in six of the seven analyzed patients. There were no objective clinical responses. The estimated median overall survival was 12 months (range 2-38), and the median event-free survival was 4 months (range 1-12). There was no statistically significant survival advantage in patients who received KLH-loaded vaccines. As of March 2005, four patients remained alive, 26+, 28+, 28+, and 36+ months. Three of them had received KLH-loaded vaccines and all of them had had additional therapy. Overall, these results suggest that IFN-α-activated CD34-DCs are safe but elicit only limited immune responses, underscoring the need to test different DC maturation factors.