Effect of inhibitors of plant cell division on mammalian tumor cells in vitro.

We studied the activity of 14 compounds, all of which have been shown to interfere in plant cell division, in two animal tumor cell cultures, EL-4 and L1210. Four compounds [propham, chlorpropham, bensulide S-(O,O-diisopropylphosphorodithioate) ester of N-(2-mercaptoethyl)benzenesulfonamide), and siduron] had a 50% inhibitory dose less than 10(-4) M; six [2,3,5-triiodobenzoic acid, (2,4-dichlorophenoxy)acetic acid, bromacil, (2,4,5-trichlorophenoxy)acetic acid, naptalam, and (4-chloro-2-methylphenoxy)acetic acid] had a 50% inhibitory dose between 10(-4) and 10(-3) M, and the remaining four 2,3:4,6-di-O-isopropylidene-2-keto-L-gulonate, eptam, maleic hydrazide, and 4-(methylsulfonyl)-2,6-dinitro-N,N,-dipropylaniline] had a 50% inhibitory dose at higher than 10(-3) M. There was a significant correlation between the effect on the two cell lines as well as between the inhibition of cell proliferation and that of thymidine and leucine uptake. More detailed study of cell proliferation and leucine and thymidine uptake for bensulide and 2,3,5-triiodobenzoic acid revealed a dose-response pattern of inhibition starting shortly after exposure of the cells to the compounds. These results indicate that some inhibitors of plant cell division are capable of inhibiting the proliferation of animal tumor cells.

Autolymphocyte therapy--I. In vivo tumour-specific adoptive cellular therapy of murine melanoma and carcinoma using ex vivo activated memory T-lymphocytes.

Autolymphocyte therapy (ALT) is tumour-specific adoptive cellular therapy of neoplastic disease based upon non-specific ex vivo activation of autologous peripheral blood lymphocytes (PBL), using the supernatant derived from a previously prepared one-way mixed lymphocyte culture (MLC). To determine the requirement for tumour antigen during the activation process, splenocytes from C57BL/6J healthy syngeneic mice (HSM) and tumour-bearing mice (TBM) were activated ex vivo using a MLC-supernatant (MLCS). Ex vivo activation was performed both in the presence (HSM splenocytes) and absence (TBM splenocytes) of a 3M KC1 syngeneic tumour-antigen (STA) extract prepared from Lewis lung (3LL) carcinoma, B16 melanoma, or normal lung. Immunophenotyping of splenocytes pre- and post-activation by MLCS plus STA or MLCS only revealed expansion of activated CD44+ (memory) T-cells. Ex vivo tumour-specific cytotoxicity was demonstrated using MLCS-activated (TBM) or MLCS + STA-activated (HSM) splenocytes against 3LL or B16 target cells. CD44+ T-cells (ALT-cells) were then infused into synegeneic 3LL and B16 TBM. Significant antitumour activity was detected in 3LL and B16 TBM receiving cells from normal mice that were activated with MLCS in the presence of 3LL or B16 STA, respectively, and in 3LL and B16 TBM receiving splenocytes from 3LL-TBM and B16-TBM, respectively, activated by MLCS alone. Infusions of 3LL-derived or B16-derived ALT-cells into HSM provided specific immunity on tumour challenge. No antitumour activity was seen in 3LL and B16 TBM receiving fresh TBM splenocytes, ALT-cells derived from HSM which were activated ex vivo using MLCS without antigen, normal lung tissue as antigen, or using MLCS-activated splenocytes without STA derived from reciprocal TBM. Ex vivo depletion of CD44+ cells or Thy-1.2+ T-cells abrogated all antitumour activity in TBM and in HSM challenged with tumour. Depletion of NK-1.1+ natural killer (NK)-cells had no effect on antitumour efficacy. These data suggest that tumour-specific adoptive cellular therapy is possible using ex vivo activated HSM splenocytes with STA, or TBM splenocytes activated ex vivo without STA, and that these antitumour effects are dependent on CD44+ memory T-cells.

Adoptive transfer of ex vivo activated memory T-cells with or without cyclophosphamide for advanced metastatic melanoma: results in 36 patients.

Autolymphocyte therapy (ALT) is the infusion of autologous peripheral blood mononuclear cells (PBMC) activated ex vivo by a cytokine-rich supernatant (T3CS) generated from a previous autologous lymphocyte culture using low doses of the anti-CD3 mitogenic monoclonal antibody. The mechanism of action is enhancement of a recall response by CD45RO+ (memory) T-cells (ALT cells) to host tumour without dependence on exogenous interleukin (IL)-2. The existence of anti-tumour-specific T-cells in melanoma patients has been well described, and efforts to utilise them therapeutically have achieved modest tumour response rates. However, few long-term survival data have been reported. From 1986 to 1992, we treated 36 patients with disseminated melanoma using ALT alone (26 patients) or adoptive chemoimmunotherapy using ALT and cyclophosphamide (CY) (10 patients). Over this time period, the cell activation method evolved from using cytokine supernatants derived from a one-way allogeneic mixed lymphocyte culture (MLCS), to the current practice of utilising anti-CD3 and autologous cytokines (T3CS). There were 21 men and 15 women, average age 57 years, range 30-82. 27 had failed prior therapies and 9 had no prior therapy. A total of 161 infusion of ALT cells were given: 65 with cells activated in MLCS and 96 with T3CS. There were no grade 3 adverse events, and an approximate 20% incidence of grades 1 and 2 reactions to ALT-cell infusions. Transient cytopenias were seen in patients receiving CY. Sixty-one per cent (22/36) of patients received the planned six ALT-cell infusions, while 39% did not due to progressive disease. In 33 evaluable patients, there were four complete responses, four partial responses and 6 patients with stable disease (SD). Responding patients and those with SD had prolonged survival compared to historical controls when matched for number of organ systems involved. Ex vivo depletion of CD45RO+ T-cells revealed preferential lysis of autologous and HLA-A-matched melanoma targets that was dependent on these memory T-cells. These data suggest that adoptive cellular therapy using ex vivo activated memory T-cells with and without CY is active, has low toxicity, is tumour-specific and can result in clinical benefit in patients with disseminated melanoma.

Characterization of the inhibitory effects of the phytotoxic agent propachlor (alpha-chloro-N-isopropyl-acetanilide), on L1210 cell proliferation.

We recently reported that a variety of phytotoxic compounds are capable of inhibiting the proliferation of mammalian tumor cells. We now report that an additional herbicide, propachlor (alpha-chloro-N-isopropyl-acetanilide), has a strong inhibitory effect on the proliferation of L1210 mouse leukemia cells. When tested in vitro against L1210 cells, propachlor displayed an ID50 on cell proliferation of less than 3 x 10(-7) M. Propachlor also inhibited significantly the uptake of leucine, thymidine and uridine. Kinetic experiments indicate that the inhibitory effects on cell proliferation and precursor uptake are present after the first day of culture. Furthermore, the inhibitory effect of propachlor is largely reversible in that cells grown in propachlor and then washed free of the compound return to a nearly normal rate of proliferation. Finally, these effects of propachlor were dependent on cell density, with greater activity occurring at higher propachlor to cell ratios.

T8 chronic lymphocytic leukemia. A distinctive disorder related to T8 lymphocytosis.

A case of T chronic lymphocytic leukemia (CLL) was first suspected on histologic and cytochemical examination of a splenectomy specimen. Later the patient became frankly leukemic with an E+/T3+/T8+/T4- phenotype. From our case and other reported cases of T-CLL studied with monoclonal antisera, the authors conclude that there are significant clinical and morphologic differences between T4 CLL and T8 CLL. In contrast to T4 CLL, T8 CLL characteristically presents with relatively low lymphocyte counts, little organ involvement, and often only discreet bone marrow involvement, and has a long clinical course. The leukemic cells are often large and have abundant cytoplasm with azurophilic granules. Similar features in cases reported as T8 lymphocytosis with neutropenia lead us to consider both entities part of a spectrum of the same disorder.

Treatment of metastatic renal cell carcinoma with autolymphocyte therapy. Low toxicity outpatient approach to adoptive immunotherapy without use of in vivo interleukin-2.

Thirty-six patients with Stage IV renal cell carcinoma were treated with autolymphocyte therapy (ALT). This new form of adoptive immunotherapy is based on the infusion of relatively small numbers of autologous lymphocytes that are depleted of suppressor cells and immunized in vitro by a method designed for antigen-specific activation using a 3M KCl extract of autologous tumor and an autologous lymphokine mixture. Patients received six monthly infusions of immunized lymphocytes, all on an outpatient basis. The majority of patients experienced no toxicity. The few reactions that occurred were minor and self-limiting; none required any medical intervention or subsequent delay in therapy. Patients also received oral cimetidine to reduce in vivo suppressor cell function. Survival at twenty-four months is 36 percent. Median survival is fifteen months, a significant improvement over the natural history of this disease. A multi-site, randomized, controlled trial of ALT in renal cell carcinoma has been initiated to confirm that this treatment causes a significant prolongation of survival with virtually no toxicity in these patients.

Histiocytosis X. Langerhans' cell histiocytosis.

Histiocytosis X is a complex and poorly understood entity. Nevertheless, it would appear as if certain themes are found recurrently throughout the literature dealing with this disease and a review of them serves as a useful summary. 1. Problems with Nomenclature. To name or categorize a disease based on end-organ pathology is generally not clinically useful, but this is what we have done with histiocytosis X. It has caused substantial confusion among physicians and patients alike concerning diagnosis, prognosis, and treatment. Further attempts at improving the nosology of this disease will not be useful unless those new names also reflect scientific advances in our understanding of etiology, pathogenesis, and therapy. 2. Identification of the Langerhans' Cell as the Consistent Pathognomonic Cell in the Lesions of Histiocytosis X. Although the Langerhans' cell was identified more than a century ago, it has only recently been recognized as the cell that proliferates in this disease. Nevertheless, several important questions remain regarding the relationship of the Langerhans' cell to histiocytosis. Foremost among these questions is whether the Langerhans' cell is a truly normal Langerhans' cell, responding appropriately to immune system signals, or if it is an abnormal variant, possibly even neoplastic. 3. Recognition that Immune System Dysfunction Is a Critical Part of Histiocytosis X. The immune system is the focus of most recent clinical research. Results of these studies are obviously important with regard to both the biology and management of this disease. 4. Histiocytosis X Is an Extremely Heterogeneous Clinical Disorder. As mentioned before, the term histiocytosis X was originally intended by Lichtenstein to describe a pathologic, and not clinical, entity. It is rare to find two patients with this disease who are exactly alike. To make matters even more confusing, the disease includes both infants with disseminated fatal disease as well as middle-aged adults with solitary bony lesions. 5. The Disease Requires Improved Therapy, but it Is a Difficult Setting in which to Perform Clinical Studies. Improved therapy is required in patients with this disease, especially those with the disseminated form. But it will be difficult to develop improved therapy until definitive answers are provided to some of the basic questions of etiology and pathogenesis. Unfortunately, these clinical studies are not readily available because of the rare occurrence of this disease and its extreme clinical heterogeneity.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunotherapy of histiocytosis-X.

This article focuses on the interaction of histiocytosis-X and the immunotherapeutic agent, suppression. Studies with this and other agents have presented us with invaluable insights into the nature of histiocytosis-X.

Histiocytosis-X: an overview.

This article acts as an overview of this issue of Hematology/Oncology Clinics of North America. We discuss the several themes that recur in the research and therefore in this issue concerning histiocytosis-X.

Evaluating the role of therapy in histiocytosis-X. Clinical studies, staging, and scoring.

Histiocytosis-X is a difficult disease on which to perform clinical studies. It is a rare disease, with great clinical heterogeneity. The natural history is characterized by periods of spontaneous disease stabilization, remission, and exacerbation. Clinical care is given by several diverse types of physicians. Several suggestions are made for the optimal design and performance of clinical studies aimed at evaluating potentially new therapies, including the proper identification and use of controls. In addition, a staging system is described, and a comprehensive scoring system is given. This scoring system will allow accurate determination of disease activity, and permit analysis of the clinical effectiveness of any new treatment.

Autolymphocyte therapy: previous experience and future prospects.

Autolymphocyte therapy is outpatient medical treatment for selected patients, in which a relatively small amount of white blood cells is removed temporarily from the patients, modified in the laboratory through sophisticated biotechnology, and returned to the patient for the purpose of enhancing the immune system and contributing to improved health. We suggest that autolymphocyte therapy might be a useful approach in the prevention or treatment of several diseases. Previous work with autolymphocyte therapy in the treatment of metastatic renal cell carcinoma has demonstrated that this approach is minimally toxic and biologically active. Further clinical trials are justified and currently taking place.

Autolymphocyte therapy. II. Dependence of in vivo anti-tumor specificity and long-term immunity against murine melanoma and carcinoma on ex vivo activated donor memory T-cells.

Autolymphocyte therapy (ALT) is tumor-specific adoptive cellular therapy of neoplastic disease in human tumor-bearing hosts based upon nonspecific ex vivo activation of autologous peripheral blood lymphocytes. We have previously demonstrated that nonspecific ex vivo activation of splenocytes from murine tumor-bearing hosts using a mixed-lymphocyte culture supernatant without tumor antigen results in the expansion of the CD44+ (memory) T-cell subset and that depletion of these CD44+ T-cells results in the abrogation of all in vivo anti-tumor effects. To examine other means of generating anti-tumor-specific effectors, splenocytes of C57BL/6J healthy syngeneic mice and mice with B16 melanoma (B16 mice) or Lewis lung (3LL) carcinoma (3LL mice) were activated ex vivo using low doses of the mitogenic monoclonal antibody OKT3 and a mixture of previously prepared autologous cytokines (T3CS). Immunophenotypic studies of splenocytes pre- and postactivation by T3CS revealed preferential expansion of Thy-1.2+CD44+ (memory) T-cells. 3LL and B16 mice received ALT cells derived from splenocytes of 3LL mice, B16 mice, or healthy mice; fresh splenocytes from 3LL or B16 mice; or CD44-depleted ALT cells derived from splenocytes of 3LL or B16 mice. Significant anti-tumor activity as shown by a reduction in primary tumor size, decreased mean number of lung metastases (Day 21), and prolonged survival was demonstrated in 3LL mice that received 3LL-derived ALT cells and in B16 mice that received B16-derived ALT cells. To test long-term immunity, adoptive transfer of subtherapeutic numbers of ex vivo activated Thy-1.2+ memory T-cells were infused into healthy congenic B6.PL Thy-1a/CY (Thy-1.1+) mice. On rechallenge with 3LL or B16 tumor, long-term tumor-specific immunity was shown to be dependent on donor Thy-1.2+ memory T-cells. These data demonstrate that tumor-specific adoptive cellular therapy and long-term tumor immunity is possible using splenocytes from tumor-bearing mice that are activated ex vivo by T3CS and is dependent on the infusion of memory T-cells. These data are consistent with clinical results in patients with metastatic melanoma and renal cell carcinoma treated with T3CS-activated lymphocytes.

Problems in the investigational study and clinical use of cancer immunotherapy.

The last decade has witnessed a veritable explosion in the investigational study and clinical use of immunotherapy for the treatment of cancer. Although this is an exciting development, the promise of cancer immunotherapy has not yet been fulfilled. Why is there an apparent discrepancy between the theory of cancer immunotherapy and the actual results from clinical studies? Michael Osband and Susan Ross suggest that there are several basic problems with the clinical study and therapeutic use of immunotherapy that must be overcome before it can be considered a viable treatment for a broad range of tumors. The purpose of this article is to describe some of the more important of these problems.

Adoptively transferred ex vivo activated memory T cells with cyclophosphamide: effective tumor-specific chemoimmunotherapy of advanced metastatic murine melanoma and carcinoma.

Autolymphocyte therapy (ALT) is adoptive cellular therapy of neoplastic disease based upon ex vivo activation of autologous peripheral blood mononuclear cells by either the supernatant derived from a previously prepared one-way mixed lymphocyte culture (MLC) or using low doses of the mitogenic monoclonal antibody anti-CD3 and a mixture of previously prepared autologous cytokines (T3CS). We have previously demonstrated that nonspecific ex vivo activation of splenocytes from murine tumor-bearing hosts (TBH) using an MLC supernatant or T3CS without the use of tumor antigen results in the expansion of the CD44+ (memory) T-cell subset. These memory T cells or autolymphocytes (ALT cells) mediate in vivo specificity when infused into murine TBH. To determine if cyclophosphamide (CY) could enhance the effectiveness of these ALT cells, C57BL/6J TBH with B16 melanoma or Lewis lung (3LL) carcinoma were treated with adoptive chemoimmunotherapy (ACIT) consisting of ALT cells and varying doses of CY. ALT cells were derived from the splenocytes of C57BL/6J TBH with B16 melanoma or 3LL carcinoma and activated ex vivo with T3CS. 3LL or B16 TBH received ALT cells derived from 3LL TBH splenocytes, B16 TBH splenocytes, CY alone, or ACIT with ALT cells and CY. Significantly greater antitumor activity as demonstrated by a decreased number of lung metastases (Day 21) and cure of primary and metastatic disease (Day 100) was seen in B16 or 3LL TBH that received ACIT using CY with B16-derived and 3LL-derived ALT cells, respectively, rather than ALT cells alone, CY alone, or ACIT with CY and reciprocal tumor-derived ALT cells. TBH cured by ACIT using CY and ALT cells derived from syngeneic TBH with the identical tumor displayed tumor-specific immunity as they were able to reject a lethal challenge of their previous B16 or 3LL tumor but not reciprocal tumors. TBH cured by ACIT using CY and ALT cells derived from splenocytes of syngeneic TBH with reciprocal tumors were able to reject lethal challenges of both tumors. These data demonstrate that effective ACIT using CY combined with ex vivo T3CS-activated CD44+ memory T cells from TBH conveys long-term tumor-specific immunity.

Autolymphocyte therapy. III. Effective adjuvant adoptive cellular therapy with in vivo anti-tumor specificity against murine melanoma and carcinoma using ex-vivo-activated memory T-lymphocytes.

Autolymphocyte therapy (ALT) is adoptive cellular therapy of neoplastic disease based upon ex vivo activation of lymphocytes by either the supernatant derived from a previously prepared one-way mixed lymphocyte culture (MLC) or using low doses of the mitogenic monoclonal antibody OKT3 and a mixture of previously prepared cytokines (T3CS). We have previously demonstrated that nonspecific ex vivo activation of splenocytes from murine tumor-bearing hosts (TBH) using an MLC-supernatant or T3CS without the use of tumor antigen results in the expansion of the CD44+ (memory) T-cell subset. These CD44+ T-cells are the principal mediators of anti-tumor specificity in the ALT-cell population in advanced metastatic murine tumors and are able to protect against tumor challenge in healthy syngeneic mice (HSM). To determine if ALT is effective in an adjuvant setting, C57BL/6J splenocytes from HSM and TBH with B16 melanoma or Lewis lung (3LL) carcinoma were activated ex vivo using T3CS. Mice were implanted with either B16 melanoma or 3LL carcinoma and then underwent surgical excision of tumor. Tumor-excised mice (TEM) then received small numbers (10(6)) of ALT-cells derived from 3LL-TBH or B16-TBH splenocytes, HSM-derived ALT-cells, fresh splenocytes derived from 3LL-TBH or B16-TBH, or CD44-depleted ALT-cells. Significant anti-tumor activity as shown by prolonged survival (Day 100), cure of disease, and rejection of a local and systemic tumor rechallenge was demonstrated in 3LL-TEM that received B16-derived ALT-cells.(ABSTRACT TRUNCATED AT 250 WORDS)