Tim R. Mosmann

Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays

A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.

THE EXPANDING UNIVERSE OF T-CELL SUBSETS : TH1, TH2 AND MORE

Since their discovery nearly ten years ago, T helper 1 (Th1) and Th2 subsets have been implicated in the regulation of many immune responses. In this article, Tim Mosmann and Subash Sad discuss the increasing number of T-cell subsets defined by cytokine patterns; the differentiation pathways of CD4+ and CD8+ T cells; the contribution of other cell types to these patterns; and the cytokine interactions during infection and pregnancy.

Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon?

Pregnant females are susceptible to intracellular pathogens and are biased towards humoral rather than cell-mediated immunity. Since TH1 cytokines compromise pregnancy and TH2 cytokines are produced at the maternal-fetal interface, we hypothesize that these TH2 cytokines inhibit TH1 responses, improving fetal survival but impairing responses against some pathogens.

Heterogeneity of Cytokine Secretion Patterns and Functions of Helper T Cells

Publisher Summary Because cytokine functions are complex and overlapping, the development of precise, monospecific bioassays, and enzyme-linked immunosorbent assays (ELISAs) was essential, before a clear picture of T cell cytokine synthesis could be obtained. In vitro , some mast cell lines synthesize IL-4 and other cytokines of the Th2 pattern in response to cross-linking of surface IgE. Although functional evidence using mixed cell populations suggested different types of Th cells, the lack of discriminatory cell-surface markers has hampered efforts to define the different subtypes. The development of in vitro T cell clones led to the description of four types of helper T cell clones, and later, two types of Th clones (Th1 and Th2) were defined on the basis of different patterns of cytokine secretion. These patterns have been confirmed in several panels of Th clones and this is currently the most clear-cut criterion for separation of mouse Th subtypes. As the two types differ in the synthesis of many cytokines, and the cytokines have a major role in the regulation of immune responses, the two types of Th cells have markedly different functions. When injected simultaneously with antigen into the footpads of naive mice, Th1 clones cause an antigen-specific and major histocompatibility complex (MHC)-restricted inflammatory reaction that peaks at about 24 hours. Th2 clones do not produce a swelling reaction under these conditions. One of the major functions of helper T cells is to provide signals for activation, proliferation, and differentiation to B cells that have encountered an antigen. Several strategies have been employed for studying the functions of mouse Th clones in micro and for analyzing the roles of specific Th products in these functions. In spite of the clear dichotomy of mouse Th1 and Th2 clones, and the accumulating evidence for their involvement in normal immune responses, human T cells do not appear to segregate into two clear subsets. Many human T cell clones secrete both Th1and Th2 cytokines.

Two types of mouse helper T-cell clone: Implications for immune regulation

Lymphocytes mediate many important functions of the immune system, such as antibody production, cytotoxicity, and immediate and delayed type hypersensitivity (DTH). In 1968, two major divisions of lymphocytes were recognized: B cells (derived from bone marrow) that produce antibody, and T cells (thymus-dependent) responsible for DTH, cytotoxicity, and regulation of many B- and T-cell functions. The discovery of subset-specific cell surface antigens subsequently allowed division of T cells into two classes: mainly responsible for helper and DTH functions, T cells bearing Lyt-1 and L3T4 antigens, and T cells bearing the Lyt-2 antigen responsible for the majority of cytotoxic and suppressor functions. Evidence from studies with normal cell populations and T-cell clones has since suggested that the Lyt-1(+)L3T4(+)Lyt-2(-) helper T cell population can be divided into at least two types. In this article, Tim Mosmann and Robert Coffman review evidence for this subdivision obtained with T-cell clones grown in vitro and discuss the implications of differences in function and lymphokine synthesis between the two types of cloned helper T cell.

Cytokine-induced differentiation of precursor mouse CD8^+ T cells into cytotoxic CD8^+ T cells secreting Th1 or Th2 cytokines

Alloantigen-stimulated CD8+ mouse spleen cells, either spontaneously or in the presence of IL-12 or IFN gamma plus anti-IL-4, differentiate into CD8+ T cells secreting a Th1-like cytokine pattern (IL-2 and IFN gamma). IL-4 induced differentiation into CD8+ T cells secreting Th2 cytokines (IL-4, IL-5, IL-6, and IL-10), whereas anti-IFN gamma suppressed the development of CD8+ cells secreting IFN gamma. Clones of IL-4- or IFN gamma-producing CD8+ T cells were relatively stable, as IL-4 or IFN gamma did not cause interconversion of committed CD8+ T cells. Both CD8+ subsets were cytotoxic, failed to provide cognate help for B cell antibody production, and remained CD4-, CD8 alpha+ CD8 beta+. We propose the names TC1 and TC2 for cytotoxic CD8+ T cells secreting Th1-like and Th2-like cytokines, respectively.

Properties and functions of interleukin-10.

Publisher Summary This chapter focuses on properties and functions of interleukin-10. IL10 inhibits several macrophage functions, including some microbicidal properties and presentation of antigen to TH 1 cells. In contrast, IL10 has generally enhancing or stimulatory functions on B cells and mast cells. Because IL10 is produced by macrophages and other cell types in addition to the T cells from which it was originally identified, it is clear that IL10, in common with several other cytokines, has a much more complex role in the immune system than could be inferred from the original activity. IL10 inhibits the synthesis of several cytokines that are normally secreted by human and mouse monocytes/macrophages in response to activation by lipopolysaccharide. Several functions of IL10 are centered on inhibition of macrophage activation and function. TH1-like responses are in general inhibited by IL10, which appears to be a consistent part of strong TH2 responses against a variety of pathogens and in several other disease states. The early results regarding IL10 production and IL10 interventions in vivo suggest that IL10 can be deleterious to situations in which a delayed type hypersensitivity response is required, for example many intracellular pathogen infections.

Functional diversity of T lymphocytes due to secretion of different cytokine patterns.

Different helper T cell subsets secrete different patterns of cytokines when stimulated by antigen. The TH1 and TH2 subsets differ in the secretion of at least eight cytokines, and three or more other cytokine secretion patterns also exist among both mouse and human T cell clones. Several properties of strong immune responses suggest that at least the TH1 and TH2 phenotypes can be present in vivo. As cytokines are major determinants of the functions of the T cells that produce them, these patterns lead to different properties of the T cell subsets. TH1 cells mediate several functions connected with cytotoxicity and local inflammatory reactions, and so these T cells are particularly effective at combating viruses and intracellular bacteria and parasites. TH2 cells are much more effective at stimulating B cells to produce antibody, and so should be more effective against free‐living bacteria, and in inducing protective humoral immunity. Antibody and delayed inflammatory reactions are often mutually exclusive during immune responses, and this can be at least partially explained by cross‐inhibition of TH1 and TH2 cells. A newly discovered cytokine, IL10, has been implicated as one of the cross‐regulatory cytokines, as this TH2 product inhibits cytokine synthesis by TH1 cells.—Street, N. E.; Mosmann, T. R. Functional diversity of T lymphocytes due to secretion of different cytokine patterns. FASEB J. 5: 171–177; 1991.

Role of T‐Cell Derived Cytokines in the Downregulation of Immune Responses in Parasitic and Retroviral Infection

Parasitic infection is frequently accompanied by a downregulation in host cell-mediated immunity. Recent studies suggest that this modulation of helper T cells and effector cell function can at least in part be attributed to the action of a set of inhibitory cytokines produced by T lymphocytes as well as by a number of other cell types. The best characterized of these inhibitory lymphokines are IL-4, IL-10 and TGF-beta. Interestingly, both IL-4 and IL-10 are produced by the Th2 but not the Th1 subset of CD4+ helper cells. The former subset dominates in many situations of chronic or exacerbated parasitic infection and is thought to suppress Th1 function as a consequence of the cross-regulatory activity of these two cytokines. The latter hypothesis is supported by recent experiments demonstrating that mAb-mediated neutralization of IL-10 reverses suppressed IFN-gamma responses and/or disease susceptibility in mice with parasitic infections. In vivo neutralization of TGF-beta has also been reported to increase host resistance to parasite challenge. In addition to suppressing T-cell differentiation, function or proliferation, IL-4, IL-10 and TGF-beta each inhibit the ability of IFN-gamma to activate macrophages for killing of both intracellular and extracellular parasites. Moreover, the three cytokines are able to synergize with each other in downregulating these parasiticidal effects. Interestingly, each of the cytokines inhibits the production of reactive nitrogen oxides, an effector mechanism previously demonstrated to play a major role in parasite killing by activated macrophages. In the case of IL-10, this suppression of nitrogen oxide production appears to result from an inhibition of TNF-alpha synthesis leading to defective macrophage stimulation. While distant from parasites in their biology and phylogeny, some retroviruses also appear to induce an over-production in downregulatory cytokines which is closely associated with the onset of immunodeficiency. Thus, in an animal model involving infection of mice with LP-BM5 MuLV and in human HIV infection, Th2 (IL-10 and/or IL-4) cytokine synthesis is increased while Th1 (IFN-gamma and/or IL-2) cytokine production is suppressed. These observations suggest that cytokine-mediated cross-regulation may play a role in the pathogenesis of acquired immune deficiency disease, contributing both to the progression of retroviral infection and the increase in susceptibility to opportunistic infections and malignancy. Observations of similar cytokine cross-regulatory activities in organisms as diverse as helminths, protozoa and retroviruses predict that comparable mechanisms may operate in a wide variety of infectious diseases.

Diversity of cytokine synthesis and function of mouse CD4+ T cells

The immune response is capable of invoking a variety of difTerent effector mechanisms, each of which is particularly effective against a certain set of pathogens. The regulation of the type of effector mechanisms chosen during an immune response is of critical importance to the host, and thus it is not surprising that this aspect of the immune system is subject to precise and complex regulation. Over the past few years, it has become clear that subsets of T cells secreting distinct patterns of cytokines have a major role in the regulation of the effector functions induced against a particular infectious agent. Two major patterns of cytokine synthesis were initially recognized, and these two patterns appear to correlate with the induction of delayed-type hypersensitivity (DTH) and help for antibody synthesis, thus providing a possible explanation for the separate and often reciprocal regulation of these two responses. Recent information suggests that additional cytokine secretion phenotypes exist, and that there is extensive regulation of the differentiation and effector function of the various cell types.