Jack H. Dean

Suppression of T-helper cell function in mice following exposure to the carcinogen 7,12-dimethylbenz[a]anthracene and its restoration by interleukin-2

Previous studies in this laboratory have demonstrated that exposure of mice to the carcinogenic polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthrance (DMBA) results in suppression of cell-mediated immunity (CMI), specifically the ability to generate cytotoxic T cells. This is accompanied by an increased susceptibility to challenge with transplantable tumors. Our previous studies have demonstrated no appreciable change in the composition of splenic lymphocyte populations following exposure to DMBA, suggesting a modulation of lymphocyte function, probably at the level of the T lymphocyte. The purpose of this study was to examine the mechanism of DMBA-induced T lymphocyte dysfunction following DMBA exposure, and to determine whether CTL function could be reconstituted by the addition of untreated lymphocytes or lymphokines. Exposure to DMBA in vivo at doses of 50 and 100 micrograms/g and in vitro at doses of 20 and 40 microM suppressed the ability of splenic lymphocytes to generate cytotoxic T-lymphocytes (CTL). CTL-mediated lysis of allogeneic tumor target cells could be restored by the addition of 20% T cell-enriched naive lymphocytes and by 10% T-helper cell-enriched lymphocytes. DMBA suppressed splenocyte production of the lymphokine interleukin-2 (IL-2) in response to mitogenic or allogeneic stimulation by greater than 70% following in vitro exposure and greater than 45% following in vivo exposure. Although DMBA-exposed lymphocytes were impaired in their ability to produce IL-2, CTL responsiveness could be reconstituted by the addition of exogenous IL-2 (purified or recombinant DNA-produced). Complete restoration of CTL responsiveness by the addition of exogenous IL-2 suggests that the T-helper cell, rather than the T-cytotoxic cell, is the target for the DMBA-induced CMI lesion.

Immunotoxicology assessment in the pharmaceutical industry

Through several inter-laboratory evaluation studies, selected methods were optimized and evaluated using reference compounds in rodents, to determine their predictive value for detecting toxicity to the immune system. These provide the basis for the OECD, EPA and pending FDA Guidelines. To describe the status of immunotoxicity evaluation using these methods in the pharmaceutical industry, a survey was conducted, and is reported, of ongoing activities among the major pharmaceutical companies. The results describe which assays are performed, how compounds are selected for evaluation; and when, during the drug development process, an evaluation is performed. Finally, the strategy at Sanofi, for the evaluation and application of immunotoxicity methods during the preclinical development of new molecular entities (NMEs) is described. During the past 8 years, Sanofi has evaluated more than 27 NMEs from multiple therapeutic classes as well as four reference compounds (azathioprine, dexamethasone, cyclophosphamide and cyclosporin A). Our experience with multiple animal species (rat, dog and monkey) and immunotoxicity assays selected from the recommended tiers as well as the outcome from the evaluation of our NMEs and reference standards, is described. This experience has led us to believe that immunotoxicology parameters represent an important adjunct for the safety assessment of NMEs. In addition, these methods were easily integrated into the drug development process and yielded an unexpectedly low frequency of positive results. In summary, immunotoxicity can be evaluated on a case-by-case basis driven by pathology or clinical hematology findings, by the drugs indication, the chemical class or indication of the NME evaluated (for example, anti-viral agents), or systematically performed.

Evaluation of a lymph node proliferation assay for its ability to detect pharmaceuticals with potential to cause immune-mediated drug reactions.

Hypersensitivity reactions to systemically administered drugs cannot be predicted using available preclinical models. This research is a collaborative project to evaluate the ability of the Lymph Node Proliferation Assay (LNPA) to predict systemic hypersensitivity caused by pharmaceuticals. The assay design is a modification of the Local Lymph Node Assay with the major modification being injection of the test substance subcutaneously to achieve a known systemic exposure to the drug. Fourteen compounds were evaluated in the LNPA. These were two clinically negative drugs (Metformin, phenobarbital), an assay positive control (streptozotocin), eight human hypersensitivity positive drugs (sulfamethoxazole, procainamide, clonidine, ofloxacin, nevirapine, abacavir, lamotrigine, zomepirac), and 3 investigational drugs (CM40874, CM40954 and CM40420), one of which caused hypersensitivity in primates. Hypersensitivity-positive drugs were classified as such based on at least two of three independent data sources: U.S. FDA postmarketing database, drug labeling information, and clinical trial data. All drugs were tested in multiple laboratories for a total of 2–12 evaluations per compound. The pure drug substance was used for testing if it could be obtained commercially, otherwise the marketed drug formulation was used. Neither of the negative control drugs showed a positive reaction in the test system. Four of the eight hypersensitivity positive drugs showed a mixed or positive reaction. Two of the three investigational compounds gave a positive response. A smaller number of LNPAs were run concurrently using footpad injection and evaluation of the popliteal lymph node and gave generally comparable results. Additional development may increase the reproducibility of the assay and facilitate detection of drugs that require metabolic activation to become allergenic, or drugs for which there is dose-limiting toxicity. The data suggest that this method might be useful as a first-line screen to identify candidate drugs that are more likely to cause a high prevalence of human drug hypersensitivity.

Assessment of the immunotoxic potential of human pharmaceuticals:a workshop report

The assessment of the immunotoxic potential of human pharmaceuticals has drawn considerable attention worldwide in the past few years. In Europe, the Committee for Proprietary Medicinal Products released its immunotoxicity guidance documents. The Food and Drug Administrations Center for Drug Evaluation and Research in the United States and the Japanese Ministry of Health, Labor, and Welfare are in the process of finalizing similar guidance documents. This report summarizes the discussions on drug immunotoxicity assessment held at a November 2001 DIA workshop held in Noordwijk, The Netherlands. This workshop revealed that an important issue for company attendees was the timing of the immunotoxicity studies during the drug development process. The Committee for Proprietary Medicinal Products requires that immunotoxicity endpoints be monitored for all new compounds. Industry has interpreted this requirement, and the expectation that these endpoints be studied in a 28-day repeated dose study in rats, as obligatory immunotoxicity testing for all new compounds, regardless of the stage of development. The Committee for Proprietary Medicinal Products requirements, however, are applicable only at the stage of the marketing application. Workshop participants agreed that immunotoxicity screening should preferably be carried out prior or parallel to Phase 2 development. This will substantially reduce the number of compounds that require enhanced testing for immunotoxicity (approximately 80% of the compounds are dropped from development after Phase 1) and reduce the use of animals. Presentations and discussions at the workshop also demonstrated that there is a strong scientific basis for European requirements as included in the Revised Note for Guidance on Repeated Dose Toxicity Testing. For example, not evaluating the response to a T-cell-dependent antigen would have missed the immunotoxicological effects of several compounds. This is probably due to the dynamic nature of the immune system whereby immune effects are best demonstrated using an immune function assay, as opposed to reliance on an essentially static analysis such as histopathology.

Suppression of murine cytotoxic T-lymphocyte induction following exposure to 7,12-dimethylbenz[a]anthracene: Dysfunction of antigen recognition

Exposure of murine lymphocytes to the carcinogenic polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA) results in significant suppression of a variety of immunological parameters, including the induction of cytotoxic T-lymphocytes (CTL). This CTL suppression may be reversed in vitro by the addition of exogenous cellular activation products, including IL-2. The current study demonstrated that DMBA-induced CTL suppression occurs throughout the induction process, with the most pronounced effect occurring within 24-48 h of initiation. IL-2-mediated restoration of suppression is effective only within this critical window. These findings suggest an effect by DMBA on both the T-helper (Th) and CTL-precursor (CTLp) subsets. Alloantigen-specific CTL generated from murine thymocytes in the presence of DMBA and Th-derived factors (thus circumventing the Th) displayed an almost identical degree of CTL suppression to that of splenocytes, indicating a direct effect of DMBA on the CTLp. Antigen nonspecific, polyclonal CTL activation by the lectin leucoagglutinin or by IL-2 (LAK cells) was unaffected by chemical exposure, indicating that DMBA preferentially affects antigen-specific activation rather than the cytolytic process itself. Conversely, polyclonal CTL induced with monoclonal antibodies directed against the T-cell receptor-associated subunit CD3, in the presence of conditioned medium, was suppressed in a manner similar to antigen-mediated CTL. The CD3 receptor subunit acts as a transmembrane activation signal following binding of antigen to the specific receptor, further implicating dysfunction of antigen recognition or signal processing following DMBA exposure. This was confirmed by the observation that DMBA exposure prevents the anamnestic response of CTL memory cells following re-exposure to eliciting tumor antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphotoxin and interferon production by rosette-separated human peripheral blood leukocytes☆☆☆

Abstract Experiments were undertaken to investigate the abilities of human T and B lymphocytes to produce the lymphokines interferon (IF) and lymphotoxin (LT). Sheep erythrocyte rosette-forming cells (E-RFC) were separated from nonrosetting cells by sedimentation on Ficoll-Hypaque gradients. Unseparated peripheral blood mononuclear cells and T-cell-enriched and B-cell-enriched populations were assayed for lymphokine production and proliferative response after stimulation with the T-cell mitogen, phytohemagglutinin (PHA), or the B- and T-cell mitogen, staphylococcal phage lysate (SPL). PHA induced LT production and cell proliferation primarily in the E-RFC-enriched population. In contrast, SPL preferentially induced LT production in the E-RFC-depleted population. SPL stimulated cell proliferation and induced equivalent IF production in both populations. Thus, PHA and SPL have differential and contrasting effects in terms of the induction of LT production and cell proliferation in T-cell and B-cell populations. In the same experiments there was no population specificity in induction of IF production. Therefore, the potentials to produce LT and IF appear to be inherent in both cell populations with their in vitro expression being dependent upon the nature of the stimulus.

Toxicity to the Immune System: A Review

Immunotoxicology is defined as the study of events that lead to undesired effects as a result of interaction of foreign substances (e.g., xenobiotics) with the immune system. Toxic responses might arise when the immune system either (1) acts as a passive target of chemical insult, leading to a relatively broad-spectrum loss or potentiation of function; or (2) responds to the antigenic specificity of the chemical as part of a specific immune response. In the latter instance, a more limited population of antigen-specific immune cells is the initial target of the chemical interaction, with the potential for toxic responses to occur (e.g., in the skin or lungs), subsequent to the specific interaction between the chemical antigen (hapten) and host antibody or sensitized cells.

A Brief History of Immunotoxicology and a Review of the Pharmaceutical Guidelines

This article provides a brief history of the development of the field of immunotoxicology from one individual perspective and separates the discussion into five phases: the methods development and validation phase; the compound testing phase; the many meetings and organization of the specialty section phase; the mechanistic studies phase; and the guidelines phase. During the discussion of each phase, major highlights, accomplishments, contributors and key references are provided. The immunotoxicology guidelines for the testing of pharmaceutical chemical entities across the three major geographic axes are also presented and compared, along with some of the concerns raised by industry with these guidelines. The mandatory requirement of functional tests represents the major concern and difference between the Committee of Proprietary Medicinal Products (CPMP) and the new Food and Drug Administration (FDA) guidance. The scientific basis for the recommendation of the functional tests proposed in the guidance documents based on National Institute of Environmental Health and Safety/National Toxicology Program (NIEHS/NTP)-sponsored studies is described. Experience at Sanofi-Synthelabo, with the testing of 29 new chemical drug entities developed across a broad range of therapeutic classes using this testing paradigm and functional tests to define their immunotoxic potential, yielded a low number of compounds (6.8%) that produced any abnormal reaction. The two positive compounds might have been anticipated based on their pharmacology.

Immunotoxicity of Pharmaceuticals: Current Knowledge, Testing Strategies, Risk Evaluation & Consequences for Human Health

This paper provides an overview of the DIA workshop on immunotoxicity of pharmaceuticals held on October 2-4, 1996, in Montreux, Switzerland. The importance of immunotoxicity testing, and its current status, are discussed. The workshops agenda: current animal tests and clinical efforts for which preclinical immunotoxicity assays need to be developed, are highlighted. Conclusions from the meeting are provided.

ADOPTIVE CELL TRANSFER STUDIES TO EXAMINE THE ROLE OF T LYMPHOCYTES IN IMMUNITY TO TRYPANOSOMA MUSCULI

Previous studies in this laboratory utilizing monoclonal antibody-induced immunosuppression have demonstrated that the T-helper lymphocyte is primarily responsible for the T lymphocyte dependency of Trypanosoma musculi elimination from the bloodstream of mice, and that T-cytotoxic lymphocytes play a minimal role in this response. In the current study, these findings were extended by examining the effects of adoptive cell transfers on the course of infection with T. musculi using immune splenocytes enriched for T lymphocyte subpopulations. These studies demonstrated that adoptive transfer of immune splenic T lymphocytes resulted in a specific, dose-related enhancement of kinetics of trypanosome elimination. This effect was found to be due to the presence of L3T4+ T-helper cells in the immune splenocyte population. Adoptive transfer of Lyt-2+ T-cytotoxic cells or lymphokine-activated killer (LAK) cells was ineffective in altering the course of infection. In addition, it was found that immune B lymphocytes were equally capable of adoptively transferring immunity to T. musculi, suggesting that the primary role of the T-helper lymphocyte is to provide help in the induction of parasite-specific antibodies.