Effector functions in the immune system are carried out by a variety of cell types, and as our understanding of the complexity of the system expands, the number of recognized subdivisions of cell types also continues to increase. B lymphocytes, producing antibody, were initially distinguished from T lymphocytes, which provide help for B cells (1, 2). The T-cell population was further divided when surface markers allowed separation of helper cells from cytotoxic cells (3). Although there were persistent reports of heterogeneity in the helper T-cell compartment (reviewed below), only relatively recently were distinct types of helper cells resolved. In this review we describe the differences between two types of cloned helper T cells, defined primarily by differences in the pattern of lymphokines ynthesized, and we also discuss the different functions of the two types of cells and their lymphokines. Patterns of lymphokine synthesis are convenient and explicit markers to describe T-cell subclass differences, and evidence increases that many of the functions of helper T cells are predicted by the functions of the lymphokines that they synthesize after activation by antigen and presenting cells. The separation of many mouse helper T-cell clones into these two distinct types is now well established, but their origin in normal T-cell populations is still not clear. Further divisions of helper T cells may have to be recognized before a complete picture of helper T-cell function can be obtained.

TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties.

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.

Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins.

A cytokine synthesis inhibitory factor (CSIF) is secreted by Th2 clones in response to Con A or antigen stimulation, but is absent in supernatants from Con A-induced Th1 clones. CSIF can inhibit the production of IL-2, IL-3, lymphotoxin (LT)/TNF, IFN-gamma, and granulocyte-macrophage CSF (GM-CSF) by Th1 cells responding to antigen and APC, but Th2 cytokine synthesis is not significantly affected. Transforming growth factor beta (TGF-beta) also inhibits IFN-gamma production, although less effectively than CSIF, whereas IL-2 and IL-4 partially antagonize the activity of CSIF. CSIF inhibition of cytokine synthesis is not complete, since early cytokine synthesis (before 8 h) is not significantly affected, whereas later synthesis is strongly inhibited. In the presence of CSIF, IFN-gamma mRNA levels are reduced slightly at 8, and strongly at 12 h after stimulation. Inhibition of cytokine expression by CSIF is not due to a general reduction in Th1 cell viability, since actin mRNA levels were not reduced, and proliferation of antigen-stimulated cells in response to IL-2, was unaffected. Biochemical characterization, mAbs, and recombinant or purified cytokines showed that CSIF is distinct from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IFN-gamma, GM-CSF, TGF-beta, TNF, LT, and P40. The potential role of CSIF in crossregulation of Th1 and Th2 responses is discussed.

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Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones.

Mucins — large extracellular proteins that are heavily glycosylated with complex oligosaccharides — establish a selective molecular barrier at the epithelial surface and engage in morphogenetic signal transduction. Alterations in mucin expression or glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion and immune surveillance. Mucins are used as diagnostic markers in cancer, and are under investigation as therapeutic targets for cancer.

Mucins in cancer: Protection and control of the cell surface

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.

Heterogeneity of cytokine secretion patterns and functions of helper T cells.

Sixteen metastatic breast cancer patients were immunized with a low dose (5 micrograms) of a 16 amino acid MUC1 peptide (GVTSAPDTRPAPGSTA) conjugated to KLH (BP16-KLH) plus DETOX adjuvant and evaluated for antibody titers against MUC1 peptide and KLH and for cytotoxic lymphocyte (CTL) activity using class 1 HLA-matched MUC1-positive tumor targets. All patients generated strong anti-KLH IgG responses. Only 3 patients developed an anti-MUC1 IgG response, which was weak in magnitude. As it is controversial whether human cancer patients generate class-1-restricted CTL against MUC1, we examined anti-MUC1 CTL activity of PBLs following 4 immunizations with BP16-KLH. The generation of MUC1-specific CTLs required only a 6-day in vitro stimulation of patients' T-cells with synthetic MUC1-peptide-pulsed autologous APCs. The assay for CTL activity was a 4 hour 51Cr release from labeled adenocarcinoma target cells. Eleven of the 16 immunized patients were tested for CTL activity using class-1-matched adenocarcinoma target cell lines. Evidence for class-1-restricted killing of MUC1-expressing tumor cell lines was obtained in 7 of these 11 patients.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/%28SICI%291097-0215%2819980610%2976%3A6%3C817%3A%3AAID-IJC9%3E3.0.CO%3B2-0

Anti-MUC1 class I restricted CTLs in metastatic breast cancer patients immunized with a synthetic MUC1 peptide

Sialyl Tn (sTn) is a mucin-associated carbohydrate antigen expressed in most types of human adenocarcinoma. Defining the configuration of tumor cell surface sTn recognized by antibodies is important for understanding the basis for the cancer cell specificity of sTn-reactive mAbs, for the development of more effective mAbs, and for designing cancer vaccines against sTn. In this study, we compared the immunogenicity of synthetic single sTn disaccharide epitopes and clusters [sTn(C)] of 3 sTn epitopes covalently linked via serine to keyhole limpet hemocyanin [KLH; sTn-KLH and sTn(C)-KLH, respectively]. The cell surface sTn configurations were analyzed with the use of sera from mice immunized with these neoglycoproteins and a panel of sTn-reactive mAb. Sera from mice immunized with sTn-KLH reacted in direct and inhibition assays with sTn-human serum albumin (HSA) but only weakly with sTn(C)-HSA, whereas sera from mice immunized with sTn(C)-KLH reacted with sTn(C)-HSA but not with sTn-HSA. Both anti-sTn and anti-sTn(C) sera reacted with ovine submaxillary mucin (a natural source of sTn) and with sTn-positive human tumor cell line LS-C but not with sTn-negative LS-B cells. With regard to the sTn-reactive mAbs, B72.3 reacted exclusively with clustered sTn, whereas mAb B195.3R11 reacted preferentially with unclustered sTn. Results with mAbs TKH2, B239.1, and CC49 were less clear, although all reacted more strongly with clustered sTn than with unclustered sTn. These results suggest that sTn is recognized at the tumor cell surface in at least two quite distinct configurations, as clustered and nonclustered arrays.

https://cancerres.aacrjournals.org/content/canres/55/15/3364.full.pdf

Immune sera and monoclonal antibodies define two configurations for the sialyl Tn tumor antigen.

.Many functions have been proposed for MUC1. The extensive ex-pression of MUC1 from mid-gestation throughout adulthood in sim-ple secretory epithelial tissues, and the elevated level of expressionfound in carcinomas and metastatic lesions suggest important func-tions in epithelial morphogenesis and tumor progression (reviewed inRef. 6). MUC1 could function at several levels: (1) by steric hin-drance owing to its large glycosylated extracellular domain, (2) byremodeling the cytoskeletal network

The biological role of mucins in cellular interactions and immune regulation: prospects for cancer

https://www.cell.com/molecular-medicine/pdf/S1357-4310(98)01322-7.pdf

The biological role of mucins in cellular interactions and immune regulation: prospects for cancer immunotherapy

Human mucin 1 (MUC1) is an epithelial mucin glycoprotein that is overexpressed in 90% of all adenocarcinomas including breast, lung, pancreas, prostate, stomach, colon, and ovary. MUC1 is a target for immune intervention, because, in patients with solid adenocarcinomas, low-level cellular and humoral immune responses to MUC1 have been observed, which are not sufficiently strong to eradicate the growing tumor. The hypothesis for this study is that enhancing MUC1-specific immunity will result in antitumor immunity. To test this, the authors have developed a clinically relevant breast cancer model that demonstrates peripheral and central tolerance to MUC1 and develops spontaneous tumors of the mammary gland. In these mice, the authors tested a vaccine formulation comprised of liposomal-MUC1 lipopeptide and human recombinant interleukin-2. Results indicate that when compared with untreated mice, immunized mice develop T cells that express intracellular IFN-gamma, are reactive with MHC class I H-2Db/MUC1 tetramer, and are cytotoxic against MUC1-expressing tumor cells in vitro. The presence of MUC1-specific CTL did not translate into a clinical response as measured by time of tumor onset, tumor burden, and survival. The authors demonstrate that some of the immune-evasion mechanisms used by the tumor cells include downregulation of MHC-class I molecule, expression of TGF-beta2, and decrease in IFN-gamma -expressing effector T cells as tumors progress. Finally, utilizing an injectable breast cancer model, the authors show that targeting a single tumor antigen may not be an effective antitumor treatment, but that immunization with dendritic cells fed with whole tumor lysate is effective in breaking tolerance and protecting mice from subsequent tumor challenge. A physiologically relevant spontaneous breast cancer model has been developed to test improved immunotherapeutic approaches.

B. Michael Longenecker

Papers

Anti-MUC1 class I restricted CTLs in metastatic breast cancer patients immunized with a synthetic MUC1 peptide

Immune sera and monoclonal antibodies define two configurations for the sialyl Tn tumor antigen.

The biological role of mucins in cellular interactions and immune regulation: prospects for cancer

The biological role of mucins in cellular interactions and immune regulation: prospects for cancer immunotherapy

Mucin 1-specific immunotherapy in a mouse model of spontaneous breast cancer.