Immuno-suppressive lymphocyte factors. I. Purification of inhibitor of DNA synthesis to homogeneity.

Inhibitor of DNA synthesis (IDS) is a T-lymphocyte factor, whose role in immunnoregulation might be to nonspecifically suppress the immune system especially in situations where very high, prolonged tolerogenic doses of antigens are present. We have purified IDS-contained supernates of stimulated lymphocytes to homogeneity, through isoelectric focusing and Sephadex gel chromatography. IDS has an isoelectric point of 2.73-2.75 and in its monomeric form has a mol wt of 20,000 but exists in the supernate usually as an aggregated tetrameric form. Di- and trimeric forms are also seen. All forms are biologically active. Purity was confirmed by SDS gel electrophoresis and the binding of dansyl chloride to terminal or free amino groups of proteins and peptides. We have, further confirmed that pure IDS is not cytotoxic and is probably a glycoprotein whose activity depends on an intact carbohydrate moiety.

Regulatory substances produced by lymphocytes. VI. Cell cycle specificity of inhibitor of DNA synthesis action in L cells.

IDS inhibits DNA synthesis and mitosis of L cells only when present during the late G1 phase of the cell cycle, as shown with L cells synchronized by a variety of methods. This corresponds well with earlier findings that IDS inhibits DNA synthesis in mitogen-stimulated lymphocytes when present between 16 and 24 h after adding mitogen. In both cell types, the inhibition produced by IDS appears to be totally the result of elevation of cAMP level. Thus, inhibitors of cAMP phosphodiesterase work synergistically with IDS, and activators of cAMP phosphodiesterase overcome the inhibition by IDS. This paper shows that IDS raises cAMP levels in L cells only within a narrow interval of the cell cycle, around 6-8 h after mitosis. This cell cycle specificity, which may be related to appearance of receptors for IDS only at discrete times, may be important in limiting IDS action to suppression, as elevated cAMP levels have a variety of other effects during other phases of the cell cycle.

Blockade of T lymphocyte costimulation with cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (CTLA4Ig) reverses the cellular pathology of psoriatic plaques, including the activation of keratinocytes, dendritic cells, and endothelial cells.

Efficient T cell activation is dependent on the intimate contact between antigen-presenting cells (APCs) and T cells. The engagement of the B7 family of molecules on APCs with CD28 and CD152 (cytotoxic T lymphocyte-associated antigen 4 [CTLA-4]) receptors on T cells delivers costimulatory signal(s) important in T cell activation. We investigated the dependence of pathologic cellular activation in psoriatic plaques on B7-mediated T cell costimulation. Patients with psoriasis vulgaris received four intravenous infusions of the soluble chimeric protein CTLA4Ig (BMS-188667) in a 26-wk, phase I, open label dose escalation study. Clinical improvement was associated with reduced cellular activation of lesional T cells, keratinocytes, dendritic cells (DCs), and vascular endothelium. Expression of CD40, CD54, and major histocompatibility complex (MHC) class II HLA-DR antigens by lesional keratinocytes was markedly reduced in serial biopsy specimens. Concurrent reductions in B7-1 (CD80), B7-2 (CD86), CD40, MHC class II, CD83, DC-lysosomal-associated membrane glycoprotein (DC-LAMP), and CD11c expression were detected on lesional DCs, which also decreased in number within lesional biopsies. Skin explant experiments suggested that these alterations in activated or mature DCs were not the result of direct toxicity of CTLA4Ig for DCs. Decreased lesional vascular ectasia and tortuosity were also observed and were accompanied by reduced presence of E-selectin, P-selectin, and CD54 on vascular endothelium. This study highlights the critical and proximal role of T cell activation through the B7-CD28/CD152 costimulatory pathway in maintaining the pathology of psoriasis, including the newly recognized accumulation of mature DCs in the epidermis.

Cytokine inhibition of DNA synthesis: effect on cyclic adenosine monophosphate in lymphocytes.

A greater than twofold increase in intracellular adenosine 3',5'-monophosphate (cyclic AMP) inhibited DNA synthesis, in stimulated rat lymphocytes. A two- to fourfold rise of intracellular cyclic AMP, starting at 16 hours, was produced by purified inhibitor od DNA synthesis added to such cells either at 0 or 16 hours, in close association with the initiation of DNA synthesis.

CTLA4Ig-mediated blockade of T-cell costimulation in patients with psoriasis vulgaris.

Engagement of the B7 family of molecules on antigen-presenting cells with their T cell-associated ligands, CD28 and CD152 (cytotoxic T lymphocyte-associated antigen-4 [CTLA-4]), provides a pivotal costimulatory signal in T-cell activation. We investigated the role of the CD28/CD152 pathway in psoriasis in a 26-week, phase I, open-label dose-escalation study. The importance of this pathway in the generation of humoral immune responses to T cell-dependent neoantigens, bacteriophage phiX174 and keyhole limpet hemocyanin, was also evaluated. Forty-three patients with stable psoriasis vulgaris received 4 infusions of the soluble chimeric protein CTLA4Ig (BMS-188667). Forty-six percent of all study patients achieved a 50% or greater sustained improvement in clinical disease activity, with progressively greater effects observed in the highest-dosing cohorts. Improvement in these patients was associated with quantitative reduction in epidermal hyperplasia, which correlated with quantitative reduction in skin-infiltrating T cells. No markedly increased rate of intralesional T-cell apoptosis was identified, suggesting that the decreased number of lesional T cells was probably likely attributable to an inhibition of T-cell proliferation, T-cell recruitment, and/or apoptosis of antigen-specific T cells at extralesional sites. Altered antibody responses to T cell-dependent neoantigens were observed, but immunologic tolerance to these antigens was not demonstrated. This study illustrates the importance of the CD28/CD152 pathway in the pathogenesis of psoriasis and suggests a potential therapeutic use for this novel immunomodulatory approach in an array of T cell-mediated diseases.

Carcinoembryonic antigen-induced release of a suppressor factor from normal human lymphocytes in vitro.

Although it is generally accepted that tumor-bearing patients may be immunosuppressed, the mechanism for this effect is unclear. Therefore, we tested the hypothesis that a tumor-associated macromolecule, carcinoembryonic antigen (CEA), could itself suppress lymphocyte function, as quantitated by uptake of [3H]thymidine by lymphocytes stimulated with the plant lectin, phytohemagglutinin. Normal human peripheral blood mononuclear cells, after exposure to CEA for 48 hr, subsequently released a factor in vitro which markedly inhibited phytohemagglutinin-stimulated lymphocytes. In further experiments, this factor release was confirmed to be initiated by CEA and not by a contaminant, and to be induced over a broad range of CEA concentrations (0.2 to 100 ng/ml). Suppression could not be accounted for by factor-associated cytotoxicity toward indicator cells, nor was it secondary to a mixed-lymphocyte reaction, nor could CEA alone (without factor) modulate proliferation. In studies to characterize the factor, its molecular weight was greater than 10,000, its activity was partially denatured by heat and proteases, and the isoelectric point was 3.4. Polyacrylamide gel electrophoresis of an "active" fraction revealed protein bands with molecular weights of 52,000, 77,000, and 171,000. Knowledge of immunomodulatory molecules present in cancer patients may suggest new modalities for therapy.

Association of the human type C retrovirus with a subset of adult T-cell cancers.

To determine whether the human T-cell lymphoma-leukemia virus (HTLV) is associated with particular cancers, patient sera were surveyed for HTLV-specific antibodies. An association was seen with aggressive cancers of mature T-cells, specifically Japanese adult T-cell leukemia (ATL) and T-cell lymphosarcoma cell leukemia (TLCL), a similar cancer of Caribbean blacks. Ninety to 100% of these patients possessed HTLV-specific antibody. Forty-seven and 20% of relatives of ATL and TLCL patients, respectively, and 12 and 4% of healthy donors from ATL and TLCL endemic areas were also antibody positive. Visceral organ involvement, hypercalcemia, and skin manifestation, features of ATL and TLCL, were often seen in other antibody-positive patients. Childhood cancers, most cutaneous T-cell and all non-T-cell leukemias and lymphomas, myeloid leukemias, Hodgkin's disease, and solid tumors were not associated with HTLV. Healthy United States donors and European patients with non-malignant diseases were antibody negative. HTLV is thus associated with a subtype of adult T-cell leukemia-lymphoma, clustered in viral endemic areas, with apparent racial and geographic predilection.

Pivotal phase III trial of two dose levels of denileukin diftitox for the treatment of cutaneous T-cell lymphoma.

PURPOSE: The objective of this phase III study was to determine the efficacy, safety, and pharmacokinetics of denileukin diftitox (DAB389IL-2, Ontak [Ligand Pharmaceuticals Inc, San Diego, CA]) in patients with stage Ib to IVa cutaneous T-cell lymphoma (CTCL) who have previously received other therapeutic interventions. PATIENTS AND METHODS: Patients with biopsy-proven CTCL that expressed CD25 on > or = 20% of lymphocytes were assigned to one of two dose levels (9 or 18 microg/kg/d) of denileukin diftitox administered 5 consecutive days every 3 weeks for up to 8 cycles. Patients were monitored for toxicity and clinical efficacy, the latter assessed by changes in disease burden and quality of life measurements. Antibody levels of antidenileukin diftitox and anti-interleukin-2 and serum concentrations of denileukin diftitox were also measured. RESULTS: Overall, 30% of the 71 patients with CTCL treated with denileukin diftitox had an objective response (20% partial response; 10% complete response). The response rate and duration of response based on the time of the first dose of study drug for all responders (median of 6.9 months with a range of 2.7 to more than 46.1 months) were not statistically different between the two doses. Adverse events consisted of flu-like symptoms (fever/chills, nausea/vomiting, and myalgias/arthralgias), acute infusion-related events (hypotension, dyspnea, chest pain, and back pain), and a vascular leak syndrome (hypotension, hypoalbuminemia, edema). In addition, 61% of the patients experienced transient elevations of hepatic transaminase levels with 17% grade 3 or 4. Hypoalbuminemia occurred in 79%, including 15% with grade 3 or 4 changes. Tolerability at 9 and 18 microg/kg/d was similar, and there was no evidence of cumulative toxicity. CONCLUSION: Denileukin diftitox has been shown to be a useful and important agent in the treatment of patients whose CTCL is persistent or recurrent despite other therapeutic interventions.

Immunosuppressive lymphocyte factors: characterization of the IDS-producing cell in the experimental model of antigenic competition.

Inhibitor of DNA synthesis, is a soluble, protein lymphocyte factor which nonspecifically suppresses in vitro lymphocyte responses to antigens or mitogens. It is secreted in large amounts in vivo in some experimentally induced immunological paralysis. Here, we have defined the cell secreting IDS in one experimental model of non-specific immune-suppression, ie., that of antigenic competition. Lymphocytes of rats injected with a large dose of ovalbumin intravenously, show no immunologic response to the same or other antigens or mitogens 24 hr later. At this time, spleen cells of these rats secrete large amounts of the inhibitor into culture supernatants. However spleen cell supernatants of T-depleted rats do not contain the activity. Further, maximal inhibitor concentrations are obtained in the first 2 days of culture when more than 65% of cultured cells are large blasts actively synthesizing protein. As the number of actively metabolizing blast cells decrease in subsequent days of culture inhibitor concentration falls. Finally thymocytes of rats pretreated with hydrocortisone acetate, to deplete thymus cortex cells are unable to secrete inhibitor in culture. These findings reveal that the cells producing inhibitory DNA synthesis in an animal made tolerant with a supra-optimal dose of antigen is an active blast transformed T cell (present in the spleen and thymus). In the thymus the cell making inhibitor appears to reside in the thymus cortex. Previous experiments have confirmed that an identical cell causes nonspecific immune-suppression in vitro. We suggest that this cell produces in vivo tolerance in antigenic competition through the release of inhibitor to DNA synthesis.

Darier's disease: a partially immunodeficient state.

Darier's disease (Keratosis follicularis) a dominantly inherited keratinizing disorder of the skin, is associated with the development of severe, progressive viral and bacterial skin infections. We investigated the possibility that an inadequacy of the immune system might be responsible for this tendency. Seven of our 8 patients with Darier's disease showed complete anergy to common skin test antigens and their peripheral blood lymphocytes failed to produce the lymphokine, leukocyte inhibitory factor (LIF) in vitro when stimulated with the same antigens. One Darier's patient and 6 controls showed positivity to at least one skin test antigen and produced lymphokine in vitro to the appropriate antigen. All patients had normal leukocyte and differential counts and normal numbers of circulating T and B cells. All 8 patients with Darier's disease demonstrated no proliferative response to optimal doses of the T cell mitogen Con A while showing normal responses to the T cell stimulant PHA and the T cell dependent B cell stimulant PWM. This previously unreported finding suggests a subtle abnormality of T cells in Darier's and might be a marker for these patients. Serum from 2 patients with Darier's disease did not suppress the in vitro immunologic activity of lymphocytes from normals. Finally, 13-Cis-retinoic acid in dosages adequate to clear their skin disease did not alter the in vivo or in vitro immunologic functions in 3 Darier's patients, suggesting that the immune dysfunction is not a secondary phenomenon.

Differentiation of human T lymphocytes: II. Phenotypic difference in skin and blood malignant T-cells in cutaneous T-cell lymphoma.

The cell surface antigen phenotype of circulating and skin malignant T-cells in patients with cutaneous T-cell lymphoma were studied. The mature T-cell antigen phenotype of the malignant T-cells was identical for circulating and skin malignant T-cells. In contrast, skin malignant T-cells expressed the immature human T-cell antigen Thy-1, surface membrane transferrin receptors, and Ia-like determinants while circulating malignant T-cells did not express these antigens. Skin infiltrating T-cells in benign dermatoses also expressed Thy-1 and Ia-like determinants. These observations support the notion that the skin is a major site of extrathymic T-cell differentiation and may promote phenotypic changes in circulating T-cells that home to skin.

Anti-V region antibodies as "almost clonotypic" reagents for the study of cutaneous T cell lymphomas and leukemias.

Despite recent advances in the understanding of normal T lymphocyte immunobiology, there has been little progress in characterizing the non-HTLV cutaneous T-cell lymphomas and leukemias (CTCL) Mycosis Fungoides and Sezary syndrome. The two major impediments to in vitro studies of these malignancies have been the contamination of CTCL cells with normal T cells and the inability to induce a vigorous proliferative response or establish long-term cultures with standard T-cell mitogens. The ideal reagent for identifying CTCL cells in a given patient would be tumor specific. Although a monoclonal antibody to the clonotypic antigen receptor on CTCL cells would approach this ideal, it is not currently feasible to generate such antibodies for each CTCL patient. As a compromise, we chose to test an "almost clonotypic" reagent by examining whether monoclonal antibodies directed at the variable (V) region of the T-cell antigen receptor could be applied to CTCL. We identified three Sezary patients, who by standard T-cell phenotype and Southern blot analysis for clonality had a virtually pure peripheral blood population of leukemic cells (PBL). We then screened the PBL of these patients with a panel of seven commercially available monoclonal anti-V region antibodies and found one patients' cells reacted greater than 99% with alpha V beta 5. The other patients' cells were non-reactive. In addition, we utilized a solid-phase system to cross-link V beta 5 on the one CTCL patients' PBL cells, and found that they proliferated vigorously in the presence of 10 units of IL-2 and IL-4. Parallel cultures have been maintained for one month by restimulation twice a week. These findings suggest that anti-V region antibodies should prove useful for investigating the immunobiology of CTCL.

Possible role of Epstein-Barr virus infection in cutaneous T-cell lymphomas.

Although cutaneous T-cell lymphoma (CTCL) is a neoplastic helper T-cell disorder of unknown etiology, prolonged antigenic stimulation has been postulated to contribute to the development of this disease. Because Epstein-Barr Virus (EBV) infection has been associated with several different lymphomas, the sera of 21 CTCL patients were examined for antibodies to EBV antigens. By using complement immunofluorescence (CIF) techniques, 13 of 21 CTCL patients had detectable antibodies to Epstein-Barr Nuclear Antigens (EBNA), whereas only five of 20 control psoriatic patients were CIF positive. When immunoblot analysis was employed, all 21 of the CTCL patients had antibodies to the EBV antigens, EBNA, whereas only 12 of the control patients had detectable antibodies to these antigens. In addition, three of 21 CTCL patients had antibodies to the EBV-associated antigen, rheumatoid arthritis nuclear antigen (RANA), as determined by double immunodiffusion, whereas none of the control sera contained anti-RANA antibodies. These results indicate that antibodies against EBV antigens are found with a higher frequency and concentration in patients with CTCL when compared to controls and suggest that EBV products might serve as a possible stimulus for the development of this malignant disease.

Establishment of a human cutaneous T-cell lymphoma in C.B-17 SCID mice.

Investigation into the immunobiology of cutaneous T-cell lymphomas (CTCL) would be facilitated by the development of a suitable experimental system. The recent use of mice with severe combined immune deficiency (SCID) as a vehicle to study the human immune system prompted us to try to establish CTCL in SCID mice. We found that a CD4+ lymphocytic infiltrate characteristic of CTCL was maintained within patient skin grafts in place on natural killer cell depleted SCID mice for the month of observation. CTCL cells were not found outside the human skin graft. This chimeric model using SCID mice and patient lesional skin should provide a useful tool to characterize CTCL/skin microenvironmental interactions and to test new therapeutic approaches.

Chronic mucocutaneous candidiasis. Immunologic studies of three generations of a single family.

A family consisting of eight members in three generations (age 10 months to 53 years) affected with chronic mucocutaneous candidiasis was studied along with three unaffected relatives. Dermatophytosis, loss of teeth and recurrent viral infections were present in some members. Results of tests for endocrinologic, muscle or liver disease, thymoma, iron deficiency, antitissue antibodies and malabsorption were normal in all patients. Antibody function and levels, B cell counts, serum complement, leukocyte enzymes, chemotaxis, phagocytosis and adherence were normal in all members. Plasma inhibitors to lymphocyte transformation and leukocyte inhibitory factor were not found. No unique HLA haplotype or antigen segregated in this family. Evaluation of cell-mediated immunity revealed total cutaneous anergy in three of eight whereas four of the other five had negative lymphocyte transformation and skin tests to Candida but responded normally to other antigens. Leukocyte inhibitory factor was not produced to Candida antigen in all four patients tested. T cell counts were within normal limits in all. Extensive evaluation of all limbs of the immune system in this family revealed a defect in cell-mediated immunity to Candida that appeared to be inherited as a dominant characteristic.

Proliferation cell nuclear antigen and soluble interleukin 2 receptor levels in cutaneous T cell lymphoma: correlation with advanced clinical diseases.

Cutaneous T-cell lymphoma and leukemias (CTCL) are malignant clonal proliferation of T lymphocytes which have a predilection to home to and proliferate in skin. There are no clinical and laboratory parameters which consistently correlate with stage of disease, which varies from patch, plaque, tumor, or erythroderma. Soluble IL-2 receptor (sIL2-R) levels are elevated both in benign and malignant diseases involving immune activation. Proliferation cell nuclear antigen (PCNA) is a marker of the G1 and G/S phases of cell cycle and can be used to quantitate proliferation. We studied 43 skin biopsies of CTCL in various clinical stages for the presence of PCNA via immunoperoxidase techniques to establish a relationship between PCNA and the stage of disease. In addition, sIL2-R levels were determined in 14 patients. PCNA reactivity was detected in the nuclei of infiltrating cells in a total of 25 patients (58%). According to clinical stage there were 2/12 patch (12%), 9/17 plaque (53%), 4/4 tumor (100%) and 9/10 erythrodermic (90%) stage patients with PCNA positive cells. Thus PCNA positivity correlated with advanced clinical stage. sIL2-R levels were elevated in 14 of 14 patients and the degree of elevation correlated with advanced clinical stage of disease and with increased numbers of PCNA positive cells. Immunohistochemical studies for PCNA and serum sIL2-R levels can be used as laboratory parameters to correlate with clinical stage of disease and enhance prognostication in CTCL.

Lymphocyte abnormalities in the BB rat.

The BB rat has a marked T cell lymphocytopenia, with a near absence of peripheral "helper" T cells recognized by monoclonal antibody W3/25 (W3/25+ T cells). The lymphocytes of the BB rat's spleen and thymus were examined for the presence of W3/25+ T cells, which were found to be absent in the spleen but present in normal amounts in the thymus. Concanavalin A (Con A) responsiveness was absent in the BB's peripheral blood and spleen but present in the thymus. Thus, in these three lymphoid compartments, Con A responsiveness directly correlated with the presence or absence of W3/25+ T cells. These lymphocyte abnormalities in the BB rat are notably different from lymphocyte changes present in human type I diabetes.

Oxiconazole nitrate: pharmacology, efficacy, and safety of a new imidazole antifungal agent.

Oxiconazole nitrate (1%) cream became available in the United States in 1989 for the once-daily treatment of tinea pedis, tinea cruris, and tinea corporis. It has also proved valuable in the once-daily treatment of tinea (pityriasis) versicolor. In vitro oxiconazole is highly effective against many dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, and Epidermophyton floccosum. After application to the skin, oxiconazole is rapidly absorbed into the stratum corneum, maximum concentrations often being attained within 100 minutes. Fungicidal concentrations are maintained in the epidermis, upper corium, and deeper corium for at least five hours, and levels exceeding the minimum inhibitory concentrations of susceptible fungi are present in the corneum, epidermis, upper corium, and the hair follicle for over 16 hours. Applied once daily for four weeks in the treatment of tinea pedis or for two weeks in the treatment of tinea corporis, tinea cruris, and tinea versicolor, 1% oxiconazole cream has produced mycologic and clinical cures in at least 80% of patients. In plantar-type tinea pedis caused primarily by T rubrum, once-daily oxiconazole cream resulted in a mycologic cure in 76% of patients. The efficacy of once-daily and twice-daily regimens is similar. In comparative clinical trials of various types of dermatophytoses, oxiconazole was shown to be as effective as or more effective than miconazole, clotrimazole, and tolnaftate creams, and as effective as econazole and bifonazole creams. Tolerability of oxiconazole and the other antifungal creams was similar; in irritation studies oxiconazole was better tolerated than econazole. Oxiconazole cream exerts no detectable systemic effect since only a negligible amount is absorbed from the skin. Once-daily use of oxiconazole cream could be valuable in patients with a history of noncompliance with multiple-daily regimens of other topical antifungal agents.

Alopecia areata and Down syndrome.

The skin of 214 institutionalized patients with the Down syndrome was carefully examined. There were 19 cases of alopecia areata and four cases of vitiligo. Since persons with the Down syndrome are predisposed to immunological deficiency in thymus-dependent (T-cell) function, findings from the skin examinations suggest that immunologic factors might contribute to the increased incidence of vitiligo and alopecia areata seen in the Down syndrome. Syringoma was also common and affected female patients twice as frequently as male patients.

Leiomyosarcoma of the skin and subcutaneous tissue.

Leiomyosarcomas arising in the skin and subcutaneous tissue are rare tumors, and diagnosis usually is made by microscopic examination of the lesion. These lesions typically appear as a single, smooth nodule in the skin but may also occur as multiple nodules with varying surface changes. After local excision, these lesions recur in a large proportion of patients. However, metastatic spread that leads to death seems to occur only in patients with the subcutaneous variety of the tumor. Three patients had varied clinical appearances of leiomyosarcomas. In one case, which was available for long-term evaluation, no recurrence was noted six years after surgical excision of a single lesion.