Human T-helper Research Articles

Human T-helper clones induce IgG production in a subclass-specific fashion

The mechanisms governing the induction of IgG subclasses by T-helper cells in humans were investigated. As preliminary bulk-culture experiments had indicated that a direct B cell contact with viable T cells was an essential requirement for optimal IgG subclass production, 256 CD4 + human T cell clones were preactivated with PHA and cultured in direct contact with autologous B cells. These clones induced IgG production in a strikingly subclass-specific fashion. Moreover, the distribution of subclass-specific helper clones was very similar to the IgG subclass profile observed in serum and peripheral lymphoid tissue plasma cells (IgG1 ≈ 60%, IgG2 ≈ 30%, IgG3 ≈ 5–10%, IgG4 ⩽ 5%) and unlike that observed in resting B cells (which is IgG1 ≈ 40% and IgG2 ≈ 50%). It would, therefore, seem that a predominance of T cells capable of delivering IgG 1 -specific, as opposed to IgG2-specific, help is an essential factor for the preferential induction of IgG1 antibodies during B cell proliferation and differentiation. There was no relationship between IL2, IL4, IL6, and IFN-γ secreted by the T-helper clones and their IgG subclass induction patterns. In addition, only a few supernatants were able to reproduce the helper effects of the clones themselves. Therefore, direct contact of B cells with helper clones is crucial for IgG-subclass production in humans.

Receptor-mediated immunomodulation by corticotropin-releasing factor

In both normal and adrenalectomized rats, exogenous Corticotropin-releasing factor (CRF) suppresses immune function, and stress-induced immunosuppression can be partially reversed with either CRF antibody or CRF antagonist, suggesting a role for CRF in immunomodulation. We now report binding of CRF to human monocyte-macrophages and T-helper lymphocytes but not to T-suppresser or B lymphocytes. Bound CRF was displaced by synthetic CRF as well as CRF antagonist. CRF binding at these sites was accompanied by increases in the concentration of cAMP (but not cGMP) in the cells, with minimal and maximal effective CRF doses of 10 −13 and 10 −6 M for the monocyte-macrophage and 10 −12 M and 10 −8 M for the T-helper cell. Production of cAMP in response to CRF was effectively inhibited by CRF antagonist in both cell types. Moreover, rat splenocyte proliferation induced by interleukin 2 (IL-2; 110 IU) was blocked by CRF, half-maximally at a CRF dose of 2.2 × 10 −10 M and completely at 3.5 × 10 −9 M. Finally, when CRF was added together with a 50-fold molar excess of CRF antagonist the IL-2 effect was fully restored. This demonstration of specific, physiologically relevant CRF receptors on two key immunocytes, the monocyte-macrophage and the T-helper lymphocyte, along with in vitro immunosuppression concomitant with CRF binding reinforces the growing body of evidence for a prominent role for CRF in immunomodulation.

Purification and characterization of cyclosporine and FK-506 binding proteins from a human T-helper cell line

Cytosolic proteins that specifically bind cyclosporine A and FK-506 were isolated and purified from the JURKAT human T-helper cell line. These binding proteins were purified by affinity, molecular weight exclusion and weak cation exchange column chromatography. Radiolabeled cyclosporine A specifically bound to a approximately 17 kDa molecule which is cyclophilin and also bound to a approximately 50 kDa protein(s). Radiolabeled FK-506 did not bind to the approximately 17 kDa molecular weight protein, but specifically bound to soluble approximately 10 kDa and approximately 50 kDa proteins.

Interference of cephalosporins with immune response: Effects of cefonicid on human T-helper cells

To determine the immunosuppressive effect(s) of cephalosporin cefonicid (CEFO) on human T-helper cells (Th), we exposed human peripheral blood mononuclear cells (PBMC) to various concentrations of CEFO during in vitro stimulation with a panel of T-lymphocyte stimulators that activate different Th/antigen presenting cell (APC) pathways. We evaluated the proliferation and IL-2 production induced by influenza virus (FLU), allogeneic lymphocytes (ALLO), xenogeneic mouse splenocytes (XENO) or phytohemagglutinin (PHA). The proliferative responses to FLU and XENO were much more depressed by CEFO than those to ALLO or PHA. After 7 days of culture with the highest dose of CEFO tested (200 mg/l) the stimulation index (stimulated/unstimulated culture) was near to 0 in FLU and XENO treated cultures, indicating that the response against these antigens was completely abrogated. The responses to ALLO and PHA were also impaired, but not abrogated (stimulation index >1). Since Flu and XENO utilize the CD4+Th/self-APC pathway our data suggested that this pathway was extremely sensitive to CEFO-induced inhibition both when the response requires memory Th cells (FLU) and virgin Th cell (XENO). The incubation with CEFO (200 mg/l) reduced the IL-2 production by XENO, FLU and ALLO to less than 20% of control cultures, while paradoxically increases to 120% the production by PHA.

Activation of human CD4+ and CD8+ cells bySarcocystis gigantea lectin

As recently reported, Sarcocystis gigantea lectin (SGL) is a powerful mitogen and a polyclonal activator (Syn. S. ovifelis) of human peripheral B-cells. In the present study we investigated the reactivity of human T-helper (CD4) and T-suppressor (CD8) cells to SGL. Mononuclear cells (MNCs) from five newborns and six adults were examined cytofluorometrically for the expression of cell-surface differentiation and activation antigens using a set of seven monoclonal antibodies. In all, 96% of cord-blood and 81% of adult CD4 cells expressed receptors for interleukin-2 (Tac+) after 64 and 164 h microgram/ml). The percentages of neonatal and adult Tac+ CD8 cells amounted to 67% and 59%, respectively. Major histocompatibility complex (MHC) class II antigens identified using the monoclonal anti-human leukocyte antigen (HLA)-DR antibody L243 were expressed on 30% (CD4) and 44% (CD8) of adult T-cells. Neonatal HLA-DR+ T-lymphocytes were not detectable. In parallel, functional tests were performed to examine cell proliferation and MNC antibody production.

Lack of leu‐3a epitope on t‐helper (cd4) lymphocytes

Human T-helper cells express membrane-bound CD4 antigen whose many epitopes are recognized by different monoclonal antibodies. The epitope recognized by Leu-3a and similar clones has been shown to be the location for human immunodeficiency virus (HIV) receptor. We have found a unique blood donor whose CD4+ T-helper lymphocytes were lacking Leu-3a epitope. CD4+ T-helper cells lacking Leu-3a epitope might be resistant to HIV infection.

Selective inhibition of interleukin 2 but not of interferon-γ, tumour necrosis factor-α or granulocyte/macrophage colony stimulating factor secretion by human helper T cell clones after antigen-driven tolerisation

Alloreactive human T-helper cell clones chronically exposed to specific antigen can become tolerised, i.e. lose their autocrine proliferative capacity and no longer undergo antigen-driven clonal expansion. To study parameters of possible clonal anergy in such cells, lymphokine secretion before and after tolerisation was measured. 10/10 CD4+ clones were found to secrete interleukin 2 (IL-2), interferon gamma, tumour necrosis factor alpha and granulocyte/macrophages colony stimulating factors. Tolerised clones which had lost autocrine proliferative capacity and had lost the ability to secret IL-2 nevertheless retained the ability to secrete the other lymphokines. These results thus show a highly selective lesion in the noncoordinate regulation of lymphokine secretion by tolerised cells.

HIV infection of primate lymphocytes and conservation of the CD4 receptor

The CD4 T-lymphocyte differentiation antigen is an essential component of the cell surface receptor for human immunodeficiency viruses (HIVs) causing AIDS (acquired immune deficiency syndrome) (refs 1-3). Peripheral blood lymphocytes of apes, New World and Old World monkeys express cell surface antigens homologous to CD4 of human T-helper lymphocytes. The cells of several of these species can be infected in short term culture with diverse strains of the type-1 or type-2 human immunodeficiency viruses (HIV-1 and HIV-2). HIV-1 is the prototype AIDS virus, and HIV-2 is the second type of AIDS virus, prevalent in West Africa. Infection of the primate cells correlates with evolutionary conservation on CD4 of one particular epitope cluster, and is inhibited by treatment of the cells with monoclonal antibodies to this epitope. The capacity of HIV to replicate in simian cells may provide a means for evaluating antiviral drugs and vaccines.

Human T-helper lymphocytes in myasthenia gravis recognize the nicotinic receptor alpha subunit.

Myasthenia gravis is a human disease caused by an autoimmune response against the nicotinic acetylcholine receptor (AcChoR). Since the molecular structure of AcChoR is well known, myasthenia gravis is an excellent system for studying the recognition of a complex membrane antigen in the human immune system. Human T-helper (TH) cell lines reactive to the AcChoR were isolated from four myasthenic patients by selection with native AcChoR from Torpedo californica. The selected TH cells could efficiently recognize native and fully denatured AcChoR. The vast majority of the TH-stimulating AcChoR epitopes were located on the denatured alpha subunit of AcChoR. Antibody competition experiments using a panel of rat anti-AcChoR monoclonal antibodies showed that 39-45% of the autoantibodies present in the sera of these same patients bound to the conformation-sensitive "main immunogenic region" (MIR), also located on the alpha subunit. However, AcChoR-induced stimulation of the T cells could not be inhibited with up to 20-fold molar excess of different rat anti-MIR monoclonal antibodies. These results suggest that the Torpedo AcChoR alpha subunit contains conformation-insensitive epitopes that play a role in the autosensitization of TH cells and that seem to be physically separated from the MIR. The specificity of the TH cell response may contribute to directing the B-cell response to other alpha-subunit determinants, such as the MIR itself.

Purification of the 33,000-dalton ligand binding-protein constituent of the lymphoblast substance P receptor.

Substance P (SP) acts as an immunoregulator by binding to specific functional cell surface receptors on a subpopulation of human T-helper lymphocytes. Receptors with similar properties have also been characterized on the human IM-9 B-lymphoblast cell line. Four distinct proteins of molecular weight (MW) 33,000, 58,000, 78,000, and 116,000 can be specifically affinity labeled using [125I]-SP Bolton-Hunter reagent and disuccinymidyl suberate (DSS). Peptide-mapping studies of these individually purified affinity labeled proteins have shown that the 33,000 MW membrane protein is present in the higher molecular weight cross-linked proteins. In the present studies, the 33,000 MW affinity-labeled protein was purified using semipreparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by high-performance hydroxylapatite chromatography. Starting with 800 mg of affinity-labeled protein, the final yield was 39.0 micrograms of 33,000 MW affinity-labeled protein. Based on an estimate of 53 mg receptor per 800 mg membrane protein, this represents an overall yield of greater than 70%. Peptide mapping was done by digesting 20 micrograms of the receptor protein with bovine trypsin. The proteolytic fragments were separated by reverse-phase high-performance liquid chromatography, and the amino acid content of 13 distinct peptides was determined. With this procedure, sufficient receptor can now be purified so that partial amino acid sequences can be obtained for further structural studies.

The delta- and epsilon-chains of the human T3/T-cell receptor complex are distinct polypeptides.

The T3/T-cell receptor complex on the surface of human thymus-derived lymphocytes consists of four glycoproteins: the alpha-chain of relative molecular mass (Mr) 40,000-50,000 (40-50K), the beta-chain (37-45K); the gamma-chain (25K) and the delta-chain (20K). The T3 alpha- and beta-chains have been identified as clonotypic T-cell receptors, but functionally the T3/T-cell receptor chains seem to form a single complex: monoclonal antibodies directed at the 20K T3 components are mitogenic for normal human T lymphocytes and, at higher concentrations, anti-clonotypic and anti-20K reagents block T-cell function. Recently, Zanders et al. showed that incubation of human T-helper clones with high concentrations of antigen abolishes antigen-specific proliferation and induces disappearance of T3 from the cell surface. Thus, the T3/T-cell receptor complex consists of two variable subunits, the T3 alpha- and beta-chains, which interact with antigen and the monomorphic 20K/25K T3 chains. Recently, the existence of a fifth polypeptide chain, the unglycosylated T3 epsilon-chain, has been postulated. Here we confirm that a 20K epsilon-chain does exist. The T3 epsilon-chain differs from the T3 delta-chain in primary structure as judged by N-terminal amino acid sequencing, peptide mapping and immunoblotting with anti-T3-delta and anti-T3-epsilon antibodies. Treatment with endoglycosidase F revealed two nonglycosylated T3 delta polypeptide backbone chains (16K and 14K) with identical amino termini. Together with previous pulse-chase experiments this observation suggests that the 14K T3 polypeptide is derived from the 16K T3 precursor by proteolytic processing near the C-terminus of the molecule.

Long-term human T-helper lines producing specific helper factor reactive to influenza virus.

Our understanding of the mechanism of T-cell help in antibody production in the mouse has been improved by the production of long-term lines of T cells, either using T-cell growth factor (TCGF or IL-2) or by creating T-cell hybridomas. Such cells have been shown to produce non-antigen-specific1,2 or antigen-specific helper factors3 with genetically restricted or unrestricted activity1–3. However, the factors are still not well characterized. We have examined the role of human T-helper cells and their soluble factors in inducing antibody production, initially in a xenogeneic system, using mouse B cells4. Using a human in vitro secondary antibody response system for influenza virus5 we have investigated the effect of helper factors on human B cells. We report here the production of long-term cultures of antigen-specific human helper cells. Both the cells and the high-potency antigen-specific helper factor (HF) which they produce act on human B cells in a genetically restricted manner. Preliminary mapping of the restriction indicates associations, but not identity, with HLA-DR.

Modified Ridley lenses.

Background: Radiation and systemic chemotherapy are standard treatment strategies for advanced or metastatic head and neck cancer. However, little is known about the implications and changes in the tumor microenvironment, including the T-helper (TH)₁/TH₂ balance in response to these treatment regimens. The aim of the current study was to unravel the effects of chemotherapeutic drugs and radiation on cytokine changes. Materials and Methods: In this study, the effect of radiation and chemotherapeutic treatment (5-fluorouracil and cisplatin) on eight cell lines was determined. Before and after exposure, cytokine levels in culture supernatants of cell lines were evaluated using the Bio-Plex Assay (Bio-Rad) and the Human TH₁/TH₂ Cytometric Bead Array (Becton Dickinson). Results were correlated with parallel measurements for cellular proliferation assessed by cytotoxicity assay. Results: Seven out of eight cell lines of primary tumors or metastases demonstrated an enhanced level of the cytokines interleukin (IL)-1β, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α), after sub-lethal radiation doses. Under treatment with low concentrations of 5-fluorouracil and cisplatin, all examined cell lines showed an increasing secretion of the cytokines IL-6 and G-CSF. In contrast, sub-lethal doses of both cytostatic drugs revealed a dose-dependent decrease in secretion IL-1β. Regarding GM-CSF and TNF-α, we demonstrated an increase in secretion by the primary tumors under low doses of 5-fluorouracil and cisplatin, whereas the metastases showed a sharp drop of GM-CSF and TNF-α secretion. Chemotherapeutic treatment led to no changes of the IL-8 cytokine profile. Conclusion: The results suggest complex cytokine changes of the tumor microenvironment and more aberrant expression profiles under treatment with radiation and the chemotherapeutic drugs 5-fluorouracil and cisplatin.

Contraception

Background: Radiation and systemic chemotherapy are standard treatment strategies for advanced or metastatic head and neck cancer. However, little is known about the implications and changes in the tumor microenvironment, including the T-helper (TH)₁/TH₂ balance in response to these treatment regimens. The aim of the current study was to unravel the effects of chemotherapeutic drugs and radiation on cytokine changes. Materials and Methods: In this study, the effect of radiation and chemotherapeutic treatment (5-fluorouracil and cisplatin) on eight cell lines was determined. Before and after exposure, cytokine levels in culture supernatants of cell lines were evaluated using the Bio-Plex Assay (Bio-Rad) and the Human TH₁/TH₂ Cytometric Bead Array (Becton Dickinson). Results were correlated with parallel measurements for cellular proliferation assessed by cytotoxicity assay. Results: Seven out of eight cell lines of primary tumors or metastases demonstrated an enhanced level of the cytokines interleukin (IL)-1β, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α), after sub-lethal radiation doses. Under treatment with low concentrations of 5-fluorouracil and cisplatin, all examined cell lines showed an increasing secretion of the cytokines IL-6 and G-CSF. In contrast, sub-lethal doses of both cytostatic drugs revealed a dose-dependent decrease in secretion IL-1β. Regarding GM-CSF and TNF-α, we demonstrated an increase in secretion by the primary tumors under low doses of 5-fluorouracil and cisplatin, whereas the metastases showed a sharp drop of GM-CSF and TNF-α secretion. Chemotherapeutic treatment led to no changes of the IL-8 cytokine profile. Conclusion: The results suggest complex cytokine changes of the tumor microenvironment and more aberrant expression profiles under treatment with radiation and the chemotherapeutic drugs 5-fluorouracil and cisplatin.