Induction of anti-tumor CD8 T cell responses by experimental ECP-induced human dendritic antigen presenting cells.

Extracorporeal photochemotherapy (ECP), or photopheresis, is distinguished by the specificity of the clinically potent immunologic reactions it initiates or regulates. The selectivity of ECP-induced immunoprotection for the malignant clone in cutaneous T cell lymphoma (CTCL), and for the pathogenic clones in allograft rejection and graft-versus-host disease (GVHD), has suggested a central mechanistic role for dendritic antigen presenting cells (DC). Discovery of ECP's induction of monocyte-derived DC, via monocyte signaling by ECP-plate activated platelets, and the absolute dependency of experimental ECP on such induced DC, supports that premise. Herein, we show that ECP-induced DC are capable of stimulating CD8 T cell responses to tumor antigens with which they are loaded. They internalize an antigen-specific melanoma-associated protein then present it onto a class I major histocompatibility, which then stimulates expansion of anti-tumor CD8 T cell populations. We conclude that ECP-induced DC prominently contribute to its initiation of anti-tumor immunity and raise the possibility that the therapy may be applicable to the immunotherapeutic management of a broader spectrum of cancers.

Integrin-driven monocyte to dendritic cell conversion in modified extracorporeal photochemotherapy.

Due to clinical efficacy and safety profile, extracorporeal photochemotherapy (ECP) is a commonly used cell treatment for patients with cutaneous T cell lymphoma (CTCL) and graft-versus-host disease (GVHD). The capacity of ECP to induce dendritic antigen-presenting cell (DC)-mediated selective immunization or immunosuppression suggests a novel mechanism involving pivotal cell signalling processes that have yet to be clearly identified as related to this procedure. In this study we employ two model systems of ECP to dissect the role of integrin signalling and adsorbed plasma proteins in monocyte-to-DC differentiation. We demonstrate that monocytes that were passed through protein-modified ECP plates adhered transiently to plasma proteins, including fibronectin, adsorbed to the plastic ECP plate and activated signalling pathways that initiate monocyte-to-DC conversion. Plasma protein adsorption facilitated 54·2 ± 4·7% differentiation, while fibronectin supported 29·8 ± 7·2% differentiation, as detected by DC phenotypic expression of membrane CD80 and CD86, as well as CD36, human leucocyte antigen D-related (HLA-DR) and cytoplasmic CD83. Further, we demonstrate the ability of fibronectin and other plasma proteins to act through cell adhesion via the ubiquitous arginine-glycine-aspartic (RGD) motif to drive monocyte-to-DC differentiation, with high-density RGD substrates supporting 54·1 ± 5·8% differentiation via αVß3 and α5ß1integrin signalling. Our results demonstrate that plasma protein binding integrins and plasma proteins operate through specific binding domains to induce monocyte-to-DC differentiation in ECP, providing a mechanism that can be harnessed to enhance ECP efficacy.

The balance between immunity and tolerance: the role of Langerhans cells.

Langerhans cells are immature skin-homing dendritic cells that furnish the epidermis with an immune surveillance system, and translate information between the internal and external milieu. Dendritic cells, in particular Langerhans cells, are gaining prominence as one of the potential principal players orchestrating the decision between immunity and tolerance. Langerhans cells capture aberrant self-antigen and pathogen-derived antigen for display to the efferent immune response. Recent evidence suggests redundancy in the antigen-presenting function of Langerhans cells, with dermal dendritic subsets capable of fulfilling an analogous role. There is mounting evidence that Langerhans cells can cross-prime T cells to recognize antigens. Langerhans cells are proposed to stimulate T regulatory cells, and are implicated in the pathogenesis of cutaneous T cell lymphoma.The phenotype of Langerhans cells, which may be tolerogenic or immunogenic, appears to depend on their state of maturity, inciting immunogen and cytokine environment, offering the potential for manipulation in immunotherapy.

Cutaneous T cell lymphoma: translating immunobiology into therapeutic opportunities.

Cutaneous T cell lymphoma (CTCL) has always served as a proving ground where conceptual advances in immunology can be tested and the results translated into clinical practice. From the earliest studies that used sheep red blood cells to identify the malignant cell as a T lymphocyte to molecular demonstration of the clonalilty of the disease, basic science techniques have provided sign posts that allow us to understand the clinical features seen in the patients. We continue to apply this paradigm to develop new insights into the role of the immune system in CTCL with the goal of using this knowledge to enhance the therapeutic options available to the patient. This article will review the studies that have led to our current understanding of the immunobiology of CTCL and the new therapeutic approaches that are being tested in this disease.

Cutaneous T cell lymphoma: the helping hand of dendritic cells.

Since its introduction 25 years ago, cutaneous T cell lymphoma has become the preferred designation for clonal malignancies of those CD4 thymus-derived lymphocytes ("cutaneous T cells") that preferentially migrate to skin. The varied cutaneous clinical presentations, dependent on the specific features of the dominant subclones of the malignant lymphocytes, historically led to confusing descriptive terms (mycosis fungoides, Sézary syndrome, lymphoma cutis, leukemia cutis, reticulum cell sarcoma of the skin). Recognition that all of these clinical presentations are cancers of a single type of cell has permitted their unification under the single, clarified heading cutaneous T cell lymphoma, or CTCL. As a neoplastic amplification of the skin-homing T cells from which it is derived, CTCL's distinctive features can be explained. The triad of skin localization, remarkable avoidance of bone marrow, often even in the context of extremely high leukemic counts, and infiltration of perifollicular T cell zones of the lymph nodes and spleen reflect the migratory pathway and homing patterns of cutaneous T cells. The usually retained levels of serum immunoglobulins and the resulting capacity to defend against encapsulated bacteria, often even in advanced CTCL, are manifestations of the helper function of the malignant T cells-that is, their functional capacity to stimulate B lymphocytes to produce immunoglobulin in a polyclonal manner. In contrast, the often-extreme normal T cell deficits in advanced CTCL, equivalent to those of late-stage AIDS, probably resulting from the production of suppressive cytokines such as IL-10, cause susceptibility to a broad range of opportunistic infections, the most common direct cause of death. Pautrier microabscesses, the pathognomonic feature of epidermotropic early CTCL, hold the clues to the pathogenesis of the cancer. These intraepidermal collections of stimulated and proliferating malignant cells, adherent to the dendrites of intraepidermal dendritic antigen-presenting cells (Langerhans' cells [LCs]), indicate a dynamic communication between the two cell types. Since CTCL cells are derived from CD4 T cells, which normally receive signaling from dendritic cells (DCs) via presentation of antigenic peptides as part of class II major histocompatibility complexes to antigen-specific T cell receptors (TCRs), it seems likely that CTCL is a clonal proliferation of T cells responding to specific antigenic stimulation from LCs. This is supported by our recent finding that CTCL cells proliferate in vitro in response to TCR stimulation by autologous DCs, which have previously ingested and processed antigens from apoptotic autologous CTCL cells. In short, CTCL may be a malignancy of T cells stimulated to proliferate against its own tumor antigens. The most intriguing possibility is that a yet-unidentified transforming retrovirus, harbored by LCs, simultaneously attracts, stimulates, and transforms a single clone of antigen-specific cutaneous T cells. Longstanding disease-free remissions have been induced by transimmunization (via a photopheresis apparatus). This treatment, introduced more than a decade ago by our group and the first and still the only FDA-approved selective anticancer immunotherapy, has been performed more than 200,000 times worldwide on advanced CTCL, as well as in reversal/prevention of heart transplant rejection and treatment of graft-versus-host disease and selected autoimmune disorders. Transimmunization induces clinically relevant suppression, and occasionally elimination, of pathogenic T cell clones. The common denominator between these diverse groups of responding patients is the presence of clonally distinctive TCRs on the disease-causing malignant or autoaggressive T cell clones. In CTCL at least one source of tumor-specific antigens is derived from the clone-specific (idiotypic) segments of the TCR protein chains. In the photopheresis apparatus, two synergistic phenomena are initiated: induction of apoptosis of the CTCL cells and mass conversion of blood monocytes to DCs. The young DCs then ingest the apoptotic CTCL cells, process and present the CTCL antigens to responding anti-CTCL cytotoxic T cells, and stimulate clinically important CTCL suppression. Now that it is better understood, transimmunization may have much broader applications in other types of cancer as well.

The clonotypic T cell receptor is a source of tumor-associated antigens in cutaneous T cell lymphoma.

To develop cancer vaccines for the treatment of cutaneous T cell lymphoma (CTCL), immunogenic peptides were identified by two approaches. First, through the use of "reverse immunology" the peptide sequence of the idiotypic region of the beta chain of the T cell receptor (TCR) was determined and a series of overlapping peptides synthesized and tested for CD8 T cell recognition. In two patients, the idiotypic CDR3 region provided immunogenic epitopes that were recognized in a class I-restricted fashion by autologous CD8 T cell lines. In a second strategy, peptides were isolated directly from class I MHC molecules on the CTCL surface and sequenced. A peptide with partial homology to sequences contained in the conserved variable portion of the clonotypic TCR beta chain was recognized as immunogenic by autologous CD8 T cells. Therefore, both approaches demonstrated that the clonotypic TCR in CTCL is a source of immunogenic tumor epitopes. To confirm that recognition of TCR-derived sequences provides immunoprotection against tumor growth, a murine model of T cell lymphoma was studied. The immunogenicity of a thymoma, which lacks cell surface TCR expression, was enhanced by transfection of the beta chain of the TCR. The studies reviewed in this paper demonstrate that the TCR can serve as one source for immunogenic tumor peptides in T cell lymphoma in vitro and in vivo. Presentation of TCR epitopes on dendritic cells that express high levels of MHC, costimulatory, and adhesion molecules may provide an effective means for immunization against T cell malignancy.

Reversible resistance to apoptosis in cutaneous T cell lymphoma.

Mycosis fungoides and its leukemic variant, Sézary syndrome, represent the most common forms of cutaneous T cell lymphomas (CTCL). These disorders are clonal neoplasms characterized by the progressive accumulation of cells that resemble activated/memory CD4+ T cells. Unlike their normal counterparts, these malignant lymphocytes have prolonged life spans and are resistant to dying following treatment with most chemotherapeutic agents. This suggests that CTCL undergo abnormal programmed cell death; however, data regarding apoptotic defects in CTCL are limited. Regulation of apoptosis in lymphocytes that regularly undergo clonal expansion is necessarily complex and will be reviewed here. Clonally expanded lymphocytes rely primarily on Fas-mediated pathways to initiate apoptosis. Factors leading to the resistance of apoptosis in CTCL and new therapeutic approaches for reversing this resistance will be discussed, including the important role that the Fas death pathway may play in the pathogenesis and treatment of CTCL.

Induction of human tumor-loaded dendritic cells.

A preferred anti-cancer vaccine would be tumor-specific, simple to rapidly construct and safe to administer. It would permit immunization against a spectrum of the tumor's distinctive antigens, without requiring their prior identification. Toward these goals, we describe a modification of standard extracorporeal photopheresis (ECP) which initiates, within a single day, both monocyte-to-dendritic cell (DC) differentiation and malignant cell apoptosis. The transition of monocytes to immature DCs was identified by the expression of cytoplasmic CD83 and membrane CD36 in the absence of membrane CD14 staining, as well as induction of membrane CD83 expression. Differentiating DCs were avidly phagocytic and engulfed apoptotic malignant T cells. Differentiating DCs were capable of stimulating significant proliferation of normal alloreactive lymphocyte responders, indicting increased expression of membrane MHC class II molecules. This approach provides a clinically practical means of developing tumor-loaded cells that have initiated the transition to DCs without the requirement of exogenous cytokines, excessive cellular manipulation or isolation. Construction of DC vaccines using this methodology can be generalized to other diseases and may offer a novel approach for improved cancer immunotherapy.

P-glycoprotein expression and multidrug resistance in cutaneous T-cell lymphoma.

Advanced-stage cutaneous T-cell lymphoma (CTCL) is generally resistant to standard chemotherapy. Because P-glycoprotein (P-gp) has been detected in other types of resistant solid tumors, leukemias, and lymphomas, we analyzed P-gp expression in CTCL. Twenty-seven patients with CTCL and circulating Sezary cells in the peripheral blood as observed on a peripheral smear treated at the Yale Photopheresis Center between 1987 and 1993 were identified. Twenty-five of these patients had skin biopsies evaluated for expression of P-gp using JSB-1 (Accurate Chemical), MRK-16 (gift of T. Tsuruo), and UIC-2 (gift of E. Metchner). P-gp expression was considered present if immunoreactivity was noted with two of the three antibodies. Eighteen of 25 patients (72%) evaluated exhibited expression. The patients were treated with various combinations of drugs consisting of topical and systemic steroids electron beam therapy, psoralens in combination with UV light A (PUVA), systemic chemotherapy, and photopheresis before testing the tissue for P-gp expression. Treatment with systemic chemotherapy in P-gp-positive patients produced responses in 3 and no responses in 11 patients (4 were lost to follow-up). Seven patients did not express P-gp: One patient responded to treatment, five did not respond, and one patient was lost to follow-up. These results demonstrate that P-gp is frequently expressed in CTCL. P-gp expression in our study was not a useful predictor of drug resistance.

Cutaneous T-cell lymphoma: pathogenesis and treatment.

Cutaneous T-cell lymphoma (CTCL) is a malignancy of a distinctive subset of T-helper cells designated "cutaneous T cells" because of their central role in the normal functioning of the skin immune system. Guided by selective adhesion molecules, activated/memory T cells of the skin immune system normally circulate among the skin, lymph nodes, and peripheral blood. Thus, a better understanding of the skin immune system, which normally functions to provide immunosurveillance against cutaneous pathogens and other insults, has led to a better understanding of the clinical spectrum, pathogenesis, staging, and management of CTCL. This article describes the major subtypes of CTCL and provides an update on the pathogenesis and treatment of this lymphoma.

Experience with total skin electron beam therapy in combination with extracorporeal photopheresis in the management of patients with erythrodermic (T4) mycosis fungoides.

OBJECTIVE: We compared the prognosis of patients with erythrodermic mycosis fungoides (MF) administered total skin electron beam radiation (TSEB) plus neoadjuvant, concurrent, and adjuvant extracorporeal photopheresis (ECP) with the prognosis of patients administered only TSEB. Outcomes of clinical interest include disease-free survival (DFS), progression-free survival (PFS), overall survival (OS), and cause-specific survival (CSS). METHODS: This study was a retrospective nonrandomized series. Between 1974 and 1997, a total of 44 patients with erythrodermic MF from the Department of Therapeutic Radiology, Yale University School of Medicine, and the Department of Radiation Oncology, Cancer Care Ontario, Hamilton, Ontario, were collected and analyzed as a group (Hamilton = 15, Yale = 29). These patients received TSEB consisting of 32 to 40 Gy via 4 to 6 MeV. Twenty-one patients at Yale also received ECP treatment 2 days per month for a median of 6 months. Median age was 68 years (range, 29-82 years) at the commencement of TSEB, and 66% were male. Seventy-three percent of patients had received other therapies before TSEB, including 75 courses that failed to control disease (n = 15 systemic therapy, 16 biologicals, and 44 topical therapies). At TSEB, 59% had hematologic involvement (B1), 30% were stage IVA (N3), and 13% were IVB (M1). Median follow-up was 2.2 years (range, 0.3-13.9 years) subsequent to TSEB and 3.7 years from diagnosis (range, 0.8-16.8 years). RESULTS: All patients responded to TSEB within 2 months of completion, with a cutaneous complete response rate of 73%. For the 32 complete responders the 3-year DFS was 63%. It was 49% for those 17 patients who received only TSEB compared with 81% for those 15 patients who received TSEB + ECP. Cox regression analysis demonstrated that ECP was associated with prolonged remission (DFS multivariate P =.024, adjusting for B1 and stage). The 2-year PFS, CSS, and OS for the TSEB group were 36%, 69%, and 63%, respectively, compared with 66%, 100%, and 88% for the TSEB + ECP cohort. Cox regression demonstrated that ECP was associated with CSS (multivariate P =.048, adjusting for B1 and stage). For those who progressed, a total of 49 subsequent courses of therapy were administered (n = 20 chemotherapy, 10 biologicals, and 19 topical therapies). Thirteen patients died from MF-related causes, and 8 died from other causes. Acute and chronic toxicities were consistent with those previously reported. CONCLUSION: ECP given concurrently with, or immediately after, TSEB (32-40 Gy) significantly improves both PFS and CSS for patients with erythrodermic MF compared with TSEB without the addition of ECP.

Integrins alpha4beta7 and alphaEbeta7 are expressed on epidermotropic T cells in cutaneous T cell lymphoma and spongiotic dermatitis.

Integrin alpha4beta7 has been associated with tissue-specific homing of malignant and inflammatory lymphocytes to gastrointestinal mucosa, whereas integrin alphaEbeta7 has been associated with intraepithelial lymphocytes in both the gut and the skin. This prompted us to examine the expression of alpha4beta7 on skin-infiltrating lymphocytes in 12 cases of patch/plaque stage cutaneous T cell lymphoma (CTCL) and in 4 cases of spongiotic dermatitis, which also display intraepidermal T cell accumulation. alpha4beta7 was found to be expressed on 64.8+/-7.4% of intraepidermal and 39.1+/-5.0% of intradermal T lymphocytes in CTCL. There was a significant positive correlation (r=0.58) between the degree of epidermotropism and the percentage of intraepidermal T cells expressing alpha4beta7. Similar findings were observed in spongiotic dermatitis, indicating that this result is not unique to malignant T cells. We evaluated staining of T cells in the same specimens for presence of alphaEbeta7 and observed a strong correlation between the expression of both beta7 integrins in each specimen. Staining with antibodies directed against the known ligands of alpha4beta7 was also performed on skin biopsies from CTCL patients. There was significantly increased dermal microvascular endothelial expression of vascular cell adhesion molecule-1 in lesional compared with nonlesional skin, and in nonlesional skin compared with skin of normal control subjects. Dermal and epidermal expression of the CS-1 domain of fibronectin was present but not increased in lesional biopsies compared with nonlesional or normal controls, whereas expression of mucosal addressin cell adhesion molecule-1 was not detectable in any skin biopsy specimens. In summary, alpha4beta7, like alphaEbeta7, is expressed at high levels on epidermotropic T cells and may interact with endothelial cell vascular cell adhesion molecule-1 as part of stepwise recruitment of lymphocytes from the blood to the epidermis.

Photoactivated 8-methoxypsoralen treatment causes a peptide-dependent increase in antigen display by transformed lymphocytes.

Ex vivo exposure of malignant human T cells to photoactivated 8-methoxypsoralen (8-MOPa), followed by their i.v. return, appears to vaccinate patients against tumor-associated antigens of cutaneous T cell lymphoma in a procedure termed photopheresis. The molecular basis of this Food and Drug Administration-approved therapy, administered in 100 centers worldwide, is unclear. Most of the attention to the mechanism of action of the drug has focused on its capacity to form covalent cross-links with pyrimidine bases of DNA, thereby inhibiting cellular proliferation. Because immunologic factors appear to be important in the clinical response and could potentially serve as a model for immunotherapy of other malignancies, we explored the possibility that 8-MOP-treated cells display increased quantities of antigenic peptides at their cell surface. In this work, human B-lymphoblastoid tissue culture lines were exposed to 8-MOPa and expression of cell surface class I major histocompatibility complex proteins assessed, since CD8 T cells recognize antigenic moieties in the context of class I molecules. A peak 200-300% increase in MHC class I expression in 8-MOPa-treated cells occurred at 20 hr. 8-MOPa was far more effective in inducing this increase in class I MHC than other modalities, including mitomycin C, gamma-irradiation, ultraviolet B or heat or cold shock. This increase in surface class I MHC molecules appears to be driven by the degradation of cytoplasmic proteins into small peptides, followed by the transport of these peptides to MHC class I molecules in the endoplasmic reticulum. The data suggest that 8-MOPa treatment may augment the immunogenicity of tumor and/or antigen-presenting cells by enhancing processing and transport of class I MHC antigenic peptides.

Tumor-specific peptides in cutaneous T-cell lymphoma: association with class I major histocompatibility complex and possible derivation from the clonotypic T-cell receptor.

We wished to identify and characterize tumor-associated class I peptides which could potentially serve as immunogens for an immunoprotective CD8 response in cutaneous T-cell lymphoma (CTCL). Candidate idiotypic peptides were identified from the third complementarity determining region (CDR3) of the clonotypic T-cell receptor (TCR) expressed on malignant T cells and native class I peptides were identified from CTCL cells. Idiotypic peptides were designed by sequencing of patients' CDR3 and identifying 9 amino acid peptides that could be accommodated in the peptide-binding motif of the class I alleles. Three candidate idiotypic peptides were synthesized and tested by measuring release of tumor necrosis factor-alpha (TNF-alpha) from autologous CD8 cells. Native peptides were acid-eluted from class I molecules on CTCL lymphocytes, fractionated, tested in the TNF-alpha assay and sequenced. Two unique idiotypic peptides were specifically recognized by autologous CD8 cells from CTCL patients. In addition, a native peptide eluted from class I molecules of CTCL tumor cells was identified, in the protein data base, as a novel molecule with partial sequence homology to the conserved portion of the patient's TCR. This homology was used to construct an extended native peptide sequence that was immunogenic for CD8 cells from both CTCL patients. Our results demonstrate that peptides derived from the TCR can be used as tumor-specific immunogens that are recognized by CD8 cells. Moreover, novel class I peptides isolated from the tumor cell also serve as immunogens. These peptides might form the basis of an anti-tumor vaccine for immunotherapy of CTCL.

Literature as an introduction to psychiatric ethics.

Responding to requests for an earlier introduction to psychiatric ethics, the authors added "An Introduction to Psychiatric Ethics Through Literature" seminar to their institution's first-year curriculum. The authors' primary objective was to increase the sensitivity of beginning psychiatric residents to ethical dilemmas. The 11-session seminar was co-led by a psychiatrist and a literary scholar. At each session, the group discussed a short story selected by the seminar leaders, with the discussion centering on ethical considerations raised by the story and their relevance to the practice of psychiatry. The residents gave the seminar high ratings for its increasing their ethical sensitivity and the stimulating content of the stories.