Characterization of the DNA copy-number genome in the blood of cutaneous T-cell lymphoma patients.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous non-Hodgkin's lymphoma that may variably involve the skin, lymph nodes, and peripheral blood. Malignant burden ranges from cutaneous patches and plaques with little evidence of blood involvement to erythroderma often in association with frank leukemia, as in Sézary syndrome. Toward a better understanding of the pathogenesis of this CD4+ T-cell malignancy, we conducted a high-resolution genomic analysis combining DNA (23 samples) and mRNA (12 samples) data of peripheral blood isolates from CTCL patients across a spectrum of stages. Strikingly, even patients with limited involvement, e.g., normal CD4 counts, contained significant copy-number alterations. Defining genomic characteristics of CTCL blood involvement included gains on 8q and 17q, and deletions on 17p and chromosome 10. A consensus analysis of 108 leukemic CTCL samples demonstrated global similarities among patients with varied blood involvement, narrowing 38 of 62 loci. Toward an annotated framework for in vitro testing, we also characterized genomic alterations in five CTCL cell lines (HH, HUT78, PNO, SeAx, and Sez4), revealing intact core features of leukemic CTCL. Together, these studies produce the most comprehensive view of the leukemic CTCL genome to date, with implications for pathogenesis, molecular classification, and potential future therapeutic developments.

Development of immunogenic tumor-loaded dendritic cells through physical perturbation and apoptotic cell loading.

To improve understanding of the forces that drive monocytes to transition into dendritic cells (Liyanage et al., 2002), we developed an experimental system that converts monocytes to DC by passage of leukocytes through a 400 microm silica bead column. The results demonstrate that overnight culture of column-treated monocytes causes a phenotypic conversion that is characteristically displayed by immature DC. These phenotypic changes were enhanced when the DC were loaded with apoptotic cells, leading to increased expression of the DC maturation-associated markers CD83, CD80 and the chemokine receptor CCR7. The DC demonstrated potent induction of allogeneic T cell proliferation and the capacity to activate autologous CD8(+) T cells. The CD8 T cells expressed augmented levels of perforin, IFN-gamma and TNF-alpha and mediated CTCL cell apoptosis. These studies demonstrate that physical contact with silica beads combined with loading of apoptotic tumor cells induces synchronized, rapid conversion of human monocytes to DC, which can efficiently stimulate CD8(+) T cells. These results may aid in the development of more efficient DC vaccines that can be loaded with the universe of antigens available in apoptotic tumor cells in a rapid, clinically practical fashion.

Improved generation of anti-tumor immunity by antigen dose limitation.

BACKGROUND: The malignant cells of cutaneous T cell lymphoma (CTCL) display immunogenic peptides derived from the clonal T cell receptor (TCR) providing an attractive model for refinement of anti-tumor immunization methodology. To produce a clinically meaningful anti-tumor response, induction of cytotoxic anti-CTCL cells must be maximized while suppressive T regulatory cells (Treg) should be minimized. We have demonstrated that engulfment of apoptotic CTCL cells by dendritic cells (DC) can lead to either CD8 anti-CTCL responses or immunosuppressive Treg induction. Treg generation is favored when the number of apoptotic cells available for ingestion is high. METHODS: In this study, we sought to determine whether the balance between immunity and immunosuppression could be shifted towards a CD8 anti-CTCL response by lowering the ratio of apoptotic CTCL cells available for DC ingestion. CTCL cell apoptosis was produced by engagement of the TCR by anti-CD3 antibody affixed to magnetic beads. RESULTS: The physical perturbation inherent in passage through a separation column induced monocytes to differentiate into DC, demonstrated by increased expression of class II and CD86 and decreased expression of the monocyte marker CD14. The immature DC internalized and processed apoptotic CTCL cells and could potentially present the tumor-derived peptides in the context of MHC class I and II. As the number of apoptotic cells increased, there was a dose-dependent increase in the expression of Treg markers CTLA-4, CD25, and FoxP3, with a ratio of apoptotic cell/DC loading of > 10:1 corresponding to the greatest Treg induction. These inducible phenotypic Treg also functionally inhibited CD8-mediated perforin expression in vitro. At lower levels of apoptotic cell/DC loading of < 5:1, there was an expansion of the CD8 T cell compartment with increased perforin expression and increased CTCL cell death, indicating anti-tumor activity. CONCLUSION: These findings demonstrate that the ratio of apoptotic cells supplied to DC is an important determinant of whether CD8 anti-tumor immunity or immunosuppression is generated.

Transimmunization for cutaneous T cell lymphoma: a Phase I study.

Extracorporeal photochemotherapy (ECP) is a widely used immunotherapy for cutaneous T cell lymphoma (CTCL). It involves four sequential steps: conversion of blood monocytes into dendritic antigen presenting cells (DC) by repetitive adherence and disadherence to plastic surface; reinfusion of the new DC; presumed in vivo loading of the new DC with apoptotic malignant leukocytes; and expansion of the anti-tumor CD8 T cell pool. To assess the safety of a methodology designed to increase ex vivo contact between the apoptotic malignant cells and new DC prior to reinfusion, a single-center, open-label Phase I clinical study of a revised procedure--referred to as "Transimmunization"--was conducted in CTCL patients. Twenty-seven subjects were treated monthly for 3 to 5 months, alone or in combination with electron beam therapy. For those receiving Transimmunization alone, there was an overall diminution in infiltrative lesions in eleven (55%) of twenty patients. In the twelve leukemic CTCL patients, there was a significant mean reduction of 50.1% in the circulating malignant cells, as determined with family-specific anti-T cell receptor Vbeta monoclonal antibodies (P

Langerhans cells: mediators of immunity and tolerance.

Langerhans cells provide the epidermis with a surveillance network that samples the external environment influencing the decision between immunity and tolerance. Langerhans cells are immature dendritic cells acquiring antigens from foreign invaders as well as damaged native tissue for display to the immune response. The current paradigm suggests that the state of maturity of Langerhans cells, defined by the display of molecules that provoke immune responses (histocompatibility, co-stimulators, adhesion and homing receptors), determines whether emigration of the Langerhans cell to lymph nodes signals immunity or tolerance. Other factors such as type of immunogen ingested, environmental danger signals and the level of cell death may also play a role in tipping the balance towards immunity or immunosuppression. As modulators of the immune response, Langerhans cells play a role in cutaneous autoimmunity in lupus and in cancers that have an affinity for the epidermis such as cutaneous T cell lymphoma.

Increased expression of CTLA-4 in malignant T-cells from patients with mycosis fungoides -- cutaneous T cell lymphoma.

Mycosis fungoides (MF) is a low-grade lymphoma of cluster of differentiation (CD)4+, CD45RO+, cutaneous leukocyte antigen (CLA)+ T cells that homes to the skin. To understand the functional abnormalities in this disease, we study the regulation of cytotoxic T-lymphocyte antigen (CTLA)-4 in peripheral blood mononuclear cells (PBMCs) from patients with MF. CTLA-4 is a costimulatory molecule for T cells that functions in immunoregulation. Unlike the expression of CD28, which is expressed constitutively on T cells, CTLA-4 expression is highly regulated. In the analysis of PBMCs in MF, we found that CTLA-4 is stimulated by phorbol myristate acetate/A23187 to a greater level when compared to normals. This defect was seen in the dominant clones of T cells. The increased CTLA-4 expression was significant between normal and MF, with a correlation between higher expression of CTLA-4 and a higher grade of MF. In a patient whose disease progressed, the CTLA-4 level increased. The abnormal level of CTLA-4 was confirmed at both the transcription and translation levels. Although MF is associated with a Th2 bias, Th1 cytokines IL-2 and IFN-gamma enhanced CTLA-4 expression, while IL-4 did not. These findings reveal an abnormal regulation of CTLA-4 expression in MF and show that PBMCs from patients with MF have properties that are divergent from those of normal T cells.

Rapid construction of a dendritic cell vaccine through physical perturbation and apoptotic malignant T cell loading.

We have demonstrated that adherence and release of monocytes from a plastic surface drives their differentiation into immature dendritic cells (DC,) that can mature further during overnight incubation in the presence of apoptotic malignant T cells. Based on these results, we sought to develop a clinically, practical, rapid means for producing DC loaded with malignant cells. A leukapheresis harvest containing the clonal, leukemic expansion of malignant CD4+ T cells was obtained from the blood of patients with cutaneous T cell lymphoma (CTCL). CTCL cells were purified with a CD3-magnetic bead column where CD3 engagement rendered the malignant T cells apoptotic. The monocyte fraction was simultaneously activated by column passage, re-added to the apoptotic CTCL cells and co-cultured overnight. CTCL cell apoptosis, DC differentiation and apoptotic malignant T cell ingestion were measured by immunostaining. The results demonstrate that as monocytes passed through the column matrix, they became activated and differentiated into semi-mature DC expressing significantly increased levels of class II, CD83 and CD86 (markers associated with maturing DC) and reduced expression of the monocyte markers CD14 and CD36. Apoptotic malignant T cells were avidly engulfed by the phagocytic transitioning DC. The addition of supportive cytokines further enhanced the number of DC that contained apoptotic malignant T cells. Functional studies confirmed that column passaged DC increased class II expression as shown by significantly enhanced stimulation in mixed leukocyte culture compared to control monocytes. In addition, DC loaded with apoptotic CTCL cells stimulated an increase in the percentage and absolute number of CD8 T cells compared to co-cultivation with non-loaded DC. After CD8 T cells were stimulated by DC loaded with malignant cells, they mediated increased apoptosis of residual CTCL cells and TNF-alpha secretion indicating development of enhanced cytolytic function. We report a simple one-step procedure where maturing DC containing apoptotic malignant T cells can be prepared rapidly for potential use in vaccine immunotherapy. Ready access to both the DC and apoptotic cells provided by this system will allow extension to other malignancies through the addition of a variety of apoptotic tumor cells and maturation stimuli.

Cutaneous T-cell lymphoma: malignant proliferation of T-regulatory cells.

Studies in an in vitro model of cutaneous T-cell lymphoma (CTCL) demonstrated that CTCL cell proliferation is stimulated by direct contact with autologous, immature dendritic cells (DCs), suggesting that CD4(+) CTCL cell division is driven by antigens presented by DC major histocompatibility complex (MHC) class 2. We now report that the T-cell receptor (TCR) of the CD4(+) CTCL cells is triggered after interaction with DCs loaded with apoptotic CTCL cells, as shown by reduced membrane expression of CD3 and the TCR, up-regulation of cytotoxic T lymphocyte antigen-4 (CTLA-4), and calcium mobilization. CTCL cells adopt a T-regulatory (Treg) phenotype expressing CD25/CTLA-4 and FoxP3 and secreting interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). Treg CTCL cells suppress normal T-cell antigen-driven secretion of IL-2 and interferon-gamma (IFN-gamma). Blocking DC MHC class 2 expression or transport inhibited CTCL cell adoption of a Treg phenotype. Allogeneic CTCL cells or normal CD4 T cells served as sources of apoptotic material for CTCL cell conversion to a Treg phenotype. Conversion of CTCL cells to Treg cells may explain the anergic, immunosuppressive nature of the malignancy.

The life cycle of cutaneous T cell lymphoma reveals opportunities for targeted drug therapy.

The diverse clinical presentations of cutaneous T cell lymphoma (CTCL) have been unified by immunologic characterization of the malignant T cells as an expansion of clonal, CD4+ inducer T cells with affinity for epidermal association with Langerhans cells (LC), an immature member of the dendritic cell (DC) family. Features of the life cycle of CTCL have recently been elucidated through development of an in vitro cell culture system. In this system, the proliferation and survival of the CTCL cells is tied to an association with immature monocyte-derived DC. Growth of the CTCL cells requires direct contact with the DC and both cell types survive in the presence of supportive cytokines for 3 months. Separation of the CTCL cells and the DC, or DC maturation truncates the synergy between the two cell populations and results in rapid death of both cell types. The CTCL cells perpetuate DC immaturity and survival through secretion of interleukin 10 (IL10) and transforming growth factor-beta (TGF-beta). The immature DC are aggressively phagocytic and can engulf apoptotic CTCL cells that have exhausted their proliferative potential and present peptides derived from the apoptotic material in class II MHC molecules to the T cell receptor (TCR) of the CD4+ CTCL cell. CTCL cells are induced to become T-regulatory (Treg) cells when their TCR is triggered by DC class II presentation of peptides derived from apoptotic material. Treg CTCL cells suppress immune responses and secrete IL10 and TGF-beta, cytokines that perpetuate DC immaturity, providing continued opportunity for DC stimulation of CTCL cell growth. Understanding the CTCL cell life cycle unveils a variety of potential targets that can be exploited for therapeutic intervention.

Current concepts of the immunobiology and immunotherapy of cutaneous T cell lymphoma: insights gained through cross-talk between the clinic and the bench.

An understanding of the immunologic features of cutaneous T cell lymphoma (CTCL) has led to insights into the life cycle of the malignancy. The identification of the T cell lineage of the neoplastic CTCL cells has allowed unification of diverse clinical presentations under a single entity. The CD4 inducer T cell phenotype of the malignant cells has provided an understanding of the patient's ability to resist infection with certain bacteria. The clonality of the tumor cells, beyond its diagnostic implications, has made them a valuable resource for studying both normal and neoplastic T cell biology. The recently identified immunosuppressive features of the malignant T cells and their dependency for survival on an interaction with immature dendritic cells have explained previously cryptic clinical observations and identified new targets for immunotherapy. Future insights gained both from the bedside and the bench will provide not only an understanding of the immunobiology of the malignancy but also open new avenues for therapeutic intervention.

Transimmunization, a novel approach for tumor immunotherapy.

This review describes our experience with the development of a novel form of immunotherapy that may represent the first practical and effective means of performing tumor-loaded dendritic cell (DC) immunotherapy. We have modified the highly successful extracorporeal photopheresis (ECP) treatment that has been used in the therapy of cutaneous T cell lymphoma (CTCL). autoimmune disease, transplantation rejection episodes and graft-versus-host disease to enhance its efficacy by the addition of an overnight incubation period. This adaption of ECP is termed "transimmunization (TI)" since the new therapy permits transfer of tumor antigens that have been previously poorly recognized to potent antigen presenting cells where the tumor epitopes can be displayed in the full context of major histocompatibility, co-stimulatory and adhesion molecules. The TI modification of ECP is a practical and safe means of rapidly inducing DC differentiation from peripheral blood monocytes in the presence of apoptotic tumor cells. Uptake of the apoptotic CTCL cells by the immature DC, in the presence of inflammatory cytokines, further drives their maturation into potent antigen presenting cells. Reinfusion of these tumor-loaded DC, that have access to the full spectrum of tumor antigens, has the potential to invoke an anti-tumor immune response in the recipient. Standard ECP has been a very useful form of immunotherapy and a modification of this approach that can enhance its ellicacy and utility should broaden its application to a larger variety of disorders including potentially the treatment of solid tumors and the modulation of the immune response in graft-versus-leukemia and graft-versus-host transplantation regimens. An understanding of the mechanism of ECP and TI will provide the physician with the ability to more finely tune the desired immune response and thereby, provide an enhanced immunotherapy for malignancy and other disorders of immunocompetence.

The growth of cutaneous T-cell lymphoma is stimulated by immature dendritic cells.

In the initial stage of cutaneous T-cell lymphoma (CTCL), proliferating CTCL cells are concentrated in the epidermis in close association with an immature dendritic cell (DC), the Langerhans cell. Because long-term in vitro culture of CTCL cells has proven difficult, the in vivo association with the major antigen-presenting cell (APC) of the epidermis has been postulated to play a role in directly stimulating the clonal T-cell proliferation. We report that CTCL cells can be reproducibly grown in culture for 3 months when cocultured with immature DCs. CTCL cells retain the phenotype and genotype of the initial malignant clone, whereas the APCs are a mixture of immature and mature DCs. CTCL cell and DC survival was dependent on direct membrane contact. Growth was inhibited by antibodies that bound to the T-cell receptor (TCR) or interfered with the interaction of CD40 with its ligand on the CTCL cell. Addition of antibody to CD3 or the clonotypic TCR caused rapid CTCL cell apoptosis followed by engulfment by avidly phagocytic immature DCs and subsequent DC maturation. The opportunity to study CTCL cells and immature DCs for prolonged periods will facilitate studies of tumor cell biology and will allow investigation of the intriguing hypothesis that CTCL cell growth is driven through TCR recognition of class II-presented self-peptides. In addition, the culture of CTCL cells will permit evaluation of therapies in vitro before clinical intervention, thereby improving safety and efficacy.