Independent regulation of cutaneous lymphocyte-associated antigen expression and cytokine synthesis phenotype during human CD4+ memory T cell differentiation

Although considerable attention has been paid to the development of cytokine synthesis heterogeneity during memory T cell differentiation, little information is available on how this function is coregulated with homing receptor expression. The development of skin-homing, CD4+ memory T cells in the human provides an excellent model for such investigation, since 1) the skin supports both Th1- and Th2-predominant responses in different settings, and 2) the skin-homing capability of human memory T cells correlates with and appears to depend on expression of the skin-selective homing receptor cutaneous lymphocyte-associated Ag (CLA). In this study, we used multiparameter FACS analysis to examine expression of CLA vs IFN-gamma, IL-4, and IL-2 synthesis capabilities among fresh peripheral blood CD4+ memory T cells, and Th1 vs Th2 memory T cells generated in vitro from purified CD4+ naive precursors by cyclic activation in polarizing culture conditions. Among normal peripheral blood T cells, CLA expression was essentially identical among the IFN-gamma- vs IL-4-producing CD4+ memory subsets, clearly indicating the existence of in vivo mechanisms capable of producing both Th1 vs Th2 skin-homing T cells. In vitro differentiation of naive CD4+ T cells confirmed the independent regulation of CLA and all three cytokines examined, regulation that allowed differential production of IFN-gamma-, IL-4-, and IL-2-producing, CLA+ memory subsets. These studies also 1) demonstrated differences in regulatory factor activity depending on the differentiation status of the responding cell, and 2) revealed CLA expression to be much more rapidly reversible on established memory cells than cytokine synthesis capabilities.     

T cell cytokine responses in persons with tuberculosis and human immunodeficiency virus infection

Tuberculosis causes more extensive and life-threatening disease in patients with HIV infection than in immunocompetent persons. To investigate the hypothesis that these severe manifestations of tuberculosis may be due to alterations in cytokine production, we evaluated cytokine patterns in HIV-infected tuberculosis patients. Upon stimulation with Mycobacterium tuberculosis in vitro, PBMC from HIV-infected tuberculosis patients had reduced proliferative and type 1 responses, compared with HIV-seronegative tuberculosis patients. The reduction in proliferative responses was independent of the CD4 cell count, but the reduced type 1 response was a direct result of CD4 cell depletion. There was no enhancement of type 2 cytokine production in HIV-infected patients, although production of IL-10 was prominent in all tuberculosis patients. In HIV-infected tuberculosis patients, M. tuberculosis-induced proliferative responses were significantly enhanced by neutralizing antibodies to IL-10 but not by antibodies to IL-4 or by recombinant IL-12. The M. tuberculosis-induced type 1 response was augmented both by antibodies to IL-10 and by recombinant IL-12. Tuberculosis in the context of HIV infection is characterized by diminished type 1 responses, probably induced by immunosuppressive cytokines produced by macrophages/monocytes, rather than by type 2 cells.     

Aberrant responses of human lymphocytic neoplasms to cytokine regulation

Studies in this laboratory have recently focused on two hemic neoplasms: B cell chronic lymphocytic leukemia (B-CLL) and a T cell disorder, Sézary syndrome. These tumors do not have consistent cytogenetic or molecular genetic alterations, and so we have concentrated on their response to and production of various regulatory cytokines. Although B-CLL cells show variable proliferative responses when exposed to transforming growth factor beta (TGF beta), these cells have consistently shown resistance to the pro-apoptotic effects of this cytokine. Also, interleukin 4 (IL4), IL5, and interferon-gamma (IFN gamma) all show a consistently increased protective effect against apoptosis in B-CLL cells as compared to normal human B cells. Thus, a defect in apoptosis appears to be an important factor in the pathogenesis of CLL. By contrast, the neoplastic T cells of Sézary syndrome show a consistent resistance to the antiproliferative effects of TGF beta, suggesting that aberrant proliferation is more important than apoptosis in this disorder. In both neoplasms, we have shown that the defective responses to cytokines are in some instances related to alterations in receptor expression, but this has not been true in all circumstances, and other stages in the signaling pathways are being investigated. As we define more precisely the specific defects that contribute to the clonal expansion of these neoplasms, the findings may ultimately lead to improved clinical control of these disorders.     

Differential activation of T cells by natural antigen peptide analogues: influence on autoimmune and alloimmune in vivo T cell responses

Recent studies using synthetic altered peptide ligands (Analogues) have led to the fine dissection of TCR-mediated T cell functions elicited by Ag recognition. Certain Analogues behave as full agonists of the antigenic peptide while others are partial agonists in that they only trigger selected T cell functions. Additionally, peptide Analogues can behave as antagonists by inhibiting functions of T cell clones when coincubated with the wild-type peptide. In fetal thymic organ cultures, synthetic altered peptide ligands can impact T cell repertoire selection. However, the influence of naturally occurring peptide Analogues on T cell immunity in vivo remains hypothetical. We previously reported that, in B10.A mice, immunogenicity and tolerogenicity of the self-MHC class I peptide, Ld 61-80, were influenced by the presentation of a cross-reactive self-peptide, Kk 61-80. Here, we show that Kk 61-80 self-peptide represents a partial agonist of Ld 61-80 in that it induced the proliferation but not the lymphokine production of Ld 61-80-primed T cells. Next, we showed that presentation of Kk 61-80 Analogue peptide mediated T cell tolerance toward Ld 61-80 self-peptide. Alternatively, when Ld protein represented an alloantigen displayed on transplanted cells, immunization with Kk 61-80 Analogue sensitized recipient mice to Ld 61-80 peptide, thus inducing potent immune responses to donor cells. These results show that the presentation of natural Analogue peptides may represent an essential component of T cell responses involved in autoimmunity and transplant rejection.     

Differential regulation of cytokine production by nitric oxide

Nitric oxide (NO) has recently been identified as a potent and pleiotropic intracellular mediator produced by and acting on many cells of the body. Although considerable attention has been devoted to the regulation of NO by inflammatory cytokines, and also to the role of NO as an important effector molecule in immune function, there is very little information on the role of this mediator in modulating T-cell-dependent cytokine production. In this study we show that physiological levels of NO (either produced by activated macrophages or by the addition of exogenous NO donors) can selectively down-regulate interleukin-3 (IL-3) production by spleen cells from contact-sensitized mice, while leaving IL-2 activity unaffected. Thus NO may have an important role as an immunomodulatory as well as effector molecule in the immune system.     

Differential effects of neuropeptides on cytokine production by mouse helper T cell subsets

DNA damage caused by tamoxifen and its derivatives was examined by estimating the conversion of supercoiled pUC18 plasmid DNA to linear form by means of agarose gel electrophoresis. N-Desmethyltamoxifen induced DNA cleavage and its effect was enhanced by the addition of reducing agents such as dithiothreitol, NADPH and 2-mercaptoethanol. 4-Hydroxytamoxifen itself had little effect, but the cleavage was slightly enhanced by the addition of reducing agents. DNA damage was higher with alpha-hydroxytoremifene than with alpha-hydroxytamoxifen, which had a prominent effect only at high concentration. The cleavage by alpha-hydroxy derivatives were not enhanced by reducing agents. No damage was induced by tamoxifen, toremifene, 3-hydroxytamoxifen or N-desmethyltoremifene. The DNA cleavage by N-desmethyltamoxifen was inhibited by the addition of EDTA, mannitol, sodium azide, methionine, catalase and superoxide dismutase. The formation of 8-hydroxy-2'-deoxyguanosine was also examined with calf thymus DNA in vitro. A slight increase of its level was found with 4-hydroxytamoxifen in the presence of dithiothreitol and also with N-desmethyltamoxifen in the presence of NADPH, but alpha-hydroxytoremifene and alpha-hydroxytamoxifen were ineffective. These experimental data suggest that among metabolites of tamoxifen, N-desmethyltamoxifen and probably also 4-hydroxytamoxifen cause oxidative DNA damage in which redox cycling is involved. The DNA damage by alpha-hydroxytoremifene appears to involve a different mechanism from that by N-desmethyltamoxifen. Tamoxifen and toremifene are possibly metabolized to the forms contributing to DNA damage.     

Detection of mosquito saliva-specific IgE and IgG4 antibodies by immunoblotting

IgE and IgG subclass antibodies against Aedes communis mosquito saliva were studied by immunoblotting in 12 adults with immediate and/or delayed skin reactions to mosquito bites. Four antigenic proteins, with molecular weights of 22, 30, 36, and 64 kd, were found in the mosquito saliva. Almost all subjects (11 of 12) had anti-mosquito saliva-specific IgE antibodies directed against the 36 kd protein. The IgG antibody response appeared to be restricted mostly to IgG4 (11 of 12) and IgG1 (8 of 11) subclasses against the same 36 kd antigen. Ten of the 12 subjects had both IgE and IgG4 antibodies to the 36 kd protein. No anti-mosquito antibodies were found in pooled sera of five infants never exposed to mosquito bites. These results show that most persons with immediate skin reactivity to A. communis mosquito bites have both IgE and IgG4 antibodies that recognize the 36 kd antigen present in the mosquito saliva, suggesting that anti-saliva antibodies may play a role in the pathogenesis of mosquito bite reactions.     

Differential regulation of early phase and late phase responses in human neutrophils by cAMP

The elevation of intracellular levels of cyclic AMP by forskolin stimulation of adenylate cyclase regulates early and late phase neutrophil responses differentially. Early phase neutrophil responses as measured by shape change in response to chemotactic factors, transmigration across a polycarbonate membrane and priming were unaffected by forskolin-induced elevation of intracellular cAMP. Late phase neutrophil responses such as release of superoxide anions, activation of phospholipase A2 and platelet activating factor (PAF) synthesis were inhibited by increasing intracellular cAMP through the addition of 10 microM forskolin for 10 min prior to stimulation. N-Formyl-methionyl-leucyl-phenylalanine-stimulated arachidonic acid release fell from 9.3% (untreated cells) to 4.6% in forskolin-treated cells. PAF generation was also inhibited from 430 pg/10(6) cells in untreated cells to background levels in forskolin-treated cells (110 pg/10(6) cells). Also, the reduction of cytochrome c by superoxide anions fell from 4.2 nmol/10(6) cells in the absence of forskolin to 2.0 nmol/10(6) cells following forskolin treatment. These results indicate that in neutrophils the elevation of cAMP acts differentially on cellular responses, not affecting early activation events, but markedly inhibiting late events such as the release of inflammatory mediators.     

Correlation of antigen specific IgG and IgG(T) responses with Anoplocephala perfoliata infection intensity in the horse

There is increasing interest in the application of serological methods to macro-parasite infections to indicate infection intensity, which in turn is related to pathogenicity. Colic is the single most important cause of mortality in horses and there is evidence that a proportion of colic cases are associated with infection with the intestinal cestode Anoplocephala perfoliata. In order to develop better tools to investigate this association, the correlation between antigen-specific equine IgG and IgG(T) and infection intensity of A. perfoliata was investigated. Affinity purification of a 12/13 kDa protein doublet from crude excretory/secretory (E/S) products, and its use in enzyme linked immunosorbent assays (ELISA) is described. Its use in the immunodiagnosis of equine cestodosis and the correlation of anti-12/13 kDa IgG and IgG(T) with parasite burden is investigated using sera from 94 horses of known tapeworm infection intensity. The anti-12/ 13 kDa IgG and IgG(T) ELISAs gave correlation coefficients with infection intensity of 0.56 and 0.63 respectively. Linear regression analysis also indicated that anti-12/ 13 kDa IgG(T) was the best predictor of infection intensity. The decay of anti-12/13 kDa IgG(T) in horses following the elimination of A. perfoliata is demonstrated for four horses. Specificity of the anti-12/13 kDa IgG(T) ELISA is investigated with sera from 33 A. perfoliata negative horses with other helminth infections. Immunoblotting studies demonstrate no cross-reactivity between A. perfoliata 12/13 kDa antigen and the protein antigens of other helminths. It is concluded that assay of anti-12/13 kDa IgG(T) provides a useful tool for the assessment of A perfoliata infection intensity for clinical diagnosis and for epidemiological studies.     

Differential regulation of in vitro cytokine production by human blood cells in response to methylmethacrylate

The effect of methylmethacrylate (MMA) on human whole blood cultures (WBC) obtained from healthy donors was investigated. Lymphocyte transformation and cytokine production, that is, interleukin 6 (IL-6), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha), were used to evaluate the immunological activities of MMA. Primary cytotoxicity testing of MMA in Jurkat cells showed that this compound decreased the cell proliferation to 50% at a concentration of >60 mmol/L. Similarly, MMA significantly decreased lymphocyte transformation in either phytohemagglutinin (PHA) or Staphylococcus aureus protein A (SpA) activated WBC at 100 mmol/L. In contrast to activated WBC, MMA had no observed effect on resting blood cells. Cytokine expression in WBC seemed differentially modulated by MMA. There was a tendency for IL-6 production in both resting and PHA-stimulated WBC to be upregulated, while IL-6 induced in SpA stimulated cultures was downregulated. TNF-alpha was slightly increased by MMA in resting WBC at early incubation periods, and it was slightly downregulated in response to PHA or SpA activation. Suppression of IFN-gamma secretion was observed in WBC with or without PHA or SpA stimulation. The overall results demonstrated that MMA at physiological levels could influence the cytokine production in normal human blood cells in vitro. Alterations of cytokine production patterns by MMA indicate that this compound has multiple regulatory effects on immune reactions in normal human blood.     

Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR

During lymphocyte development, cell-fate decisions are determined by a myriad of signals produced by the micro- environment of the thymus and the bone marrow. These yet to be fully defined developmental cues regulate stage-specific gene expression, and the extraordinarily well-characterized stages of T and B cell development have provided attractive model systems for studying regulation of cellular differentiation. In particular, studies on the contribution of both antigen receptors and cytokine receptors to lymphoid development have illuminated essential signalling pathways in early T and B cells. Here, we review investigations supporting an obligatory role for the IL-7 receptor pathway in early T cell development. IL-7 is produced by both thymus and bone marrow stromal cells, and its potential contribution to survival, differentiation and proliferation of pro-T cells is discussed. We also address the contribution of the pre-T cell receptor (pre-TCR) to differentiation past the pro-T cell stage, and recent advances in deciphering the composition and function of the pre-TCR complex are discussed. Finally, we suggest future directions in this field that may serve to reveal whether and how signals initiated by the cytokine receptors and pre-TCR may intersect, and to define which down-stream molecular events are regulated by these receptors.     

Glucocorticoids inhibit human antigen-specific and enhance total IgE and IgG4 production due to differential effects on T and B cells in vitro

Although anti-inflammatory properties of glucocorticoids (GC) are well documented, their activity in allergic diseases is still controversial. Recently, it has been reported that GC can increase, both in vivo and in vitro, the polyclonal production of total IgE. In this study we investigated the effects of GC on the antigen (Ag)-specific IgE response in a human in vitro system with peripheral blood mononuclear cells or B cells of bee venom-sensitized individuals that allows the production of bee venom phospholipase A2 (PLA)-specific IgE and IgG4 antibodies (Ab). PLA-specific Ab were induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti-CD2 and soluble CD40 ligand (sCD40L) in the presence of interleukin (IL)-4. Indeed, dexamethasone and prednisolone enhanced the formation of total IgE and IgG4 in PBMC, while the production of PLA-specific IgE and IgG4 Ab was selectively inhibited in a dose-dependent manner. The suppressive effect of GC was mediated during Ag-specific stimulation and T cell-B cell interaction. This was due to GC suppressing specific T cell proliferation and cytokine production, whereas neither allergen-specific nor total IgE and IgG4 production by sCD40L/IL-4-stimulated pure B cells was affected. In contrast to GC, cyclosporine A inhibited both total and PLA-specific IgE and IgG4 secretion in peripheral blood mononuclear cells and B cell cultures. Further experiments showed that increase in nonspecific total isotype response resulted from inhibition of IL-4 uptake by cells other than B cells and sufficient availability of IL-4 to B cells for isotype switch and synthesis. Furthermore, demonstration of opposite regulatory effects of GC on specific and total isotype formation in vitro, including the inhibition of allergy-relevant Ag-specific IgE response, may contribute to a better understanding of apparently controversial observations, and explain why most allergic patients benefit from GC therapy.     

CD4+ T cells from IRF-1-deficient mice exhibit altered patterns of cytokine expression and cell subset homeostasis

The cyclic hexapeptide CWLDVC (TBC 772) is an antagonist of alpha4 integrins and a potent inhibitor of lymphocyte interactions with fibronectin, vascular cell adhesion molecule-1, and muscosal vascular addressin cell adhesion molecule-1 (MAdCAM-1). As such, peptide TBC 772 effectively inhibits the activation of freshly isolated human T lymphocytes stimulated with purified vascular cell adhesion molecule-1 coimmobilized with anti-CD3 mAb. The influence of peptide binding on distinct sites of the alpha4beta1 complex was determined by flow cytometry and cellular adhesion assays employing a panel of mAbs. Binding of the alpha4-specific mAb L25 and the beta1-specific mAb 33B6 was not altered by the peptide; however, binding of mAb 19H8, which is specific for a combinatorial epitope of alpha4beta1, was dramatically inhibited. Treatment of lymphocytes with the peptide caused an increase in a ligand-induced epitope on beta1 integrin defined by mAb 15/7. In T cell activation studies using coimmobilized anti-CD3 mAb and the anti-integrin mAbs, the peptide had broader inhibitory activity, suppressing costimulation induced by all the integrin mAbs. The peptide was not generally toxic and was integrin selective in its suppressive activity, as coactivation by ligation of CD3 in conjunction with CD28 or CD26 was not affected. These results suggest that the antagonist peptide CWLDVC can effectively neutralize integrin coactivation systems by a mechanism independent of competitive binding.     

Natural killer cell depletion fails to influence initial CD4 T cell commitment in vivo in exogenous antigen-stimulated cytokine and antibody responses

The role played by NK- and NK1.1-expressing T cells in CD4 T cell activation and induction of immune responses in vivo is controversial. These effector cells of the innate immune response are hypothesized to play a pivotal role in shaping initial T cell activation, with some groups reporting that classical NK cells are required for optimal Th1-like T cell activation, and others supporting a role for NK1.1+ alphabeta T cells in Th2 generation. Here, we examine the impact of in vivo NK cell depletion on the development of exogenous Ag-specific cytokine and Ab responses using a murine model of human immediate hypersensitivity. OVA-specific immune responses were induced in 1) C57Bl/6 bg/bg and bg/+ mice, 2) BALB/c mice pretreated with anti-asialoGM1 or control Ab, and 3) C57Bl/6 mice depleted of NK1.1-expressing cells by in vivo administration of anti-NK1.1 mAb PK136. Depletion efficacy was assessed by functional assays and flow cytometric analysis. Each of these approaches indicated that depletion of NK cells and NK1.1+ CD4+ T cells fails to alter the Th1:Th2 balance of Ag-driven cytokine synthesis, as indicated by OVA-stimulated cytokine synthesis in primary bulk culture. Similarly, the kinetics and intensity of effector responses such as OVA-specific IgG2a and IgE synthesis were neither increased nor decreased in any of the three models examined. The results argue that NK cells and peripheral NK1.1+ T cells do not play an essential role in shaping the induction of Ag-specific immune responses to soluble exogenous Ags, the most common class of inhalant allergen.     

Single-cell cytokine analysis of gamma delta T cell responses to nonpeptide mycobacterial antigens

TCR gamma delta T cells are considered important in the rapid immune response to intracellular infection. We investigated the early response of peripheral blood gamma delta T cells to the nonpeptide Ag isopentenyl pyrophosphate and to its synthetic analogue ethyl pyrophosphate. In healthy donors, an increase in the number of gamma delta T cells was detected as soon as 4 days after stimulation with the nonpeptide Ags. Single-cell analysis of cytokine production was performed by intracellular staining of IFN-gamma and IL-4. gamma delta T cells were found to rapidly expand and produce IFN-gamma in response to nonpeptide Ags. Furthermore, IL-12 augmented the IFN-gamma response. In contrast, gamma delta T cells from the majority of HIV+ donors did not expand or express IFN-gamma in response to nonpeptide Ags, even in the presence of IL-12. These findings indicate a role for nonpeptide-reactive gamma delta T cells in effective cell-mediated immunity for intracellular pathogens.     

Steroid hormone regulation of cytokine secretion by proteolipid protein-specific CD4+ T cell clones isolated from multiple sclerosis patients and normal control subjects

Steroid hormones have long been known to modulate immune function, and recent studies indicate that one of the means by which they do so involves effects on the secretion of immunoregulatory cytokines. Our laboratory has found recently that estradiol (E2) selectively modifies cytokine secretion in proteolipid protein (PLP)-specific, CD4+ T cell clones isolated from patients with the demyelinating disease, multiple sclerosis, and from normal control subjects. The data suggest that E2 may play a role in regulating the balance between pro- and antiinflammatory conditions, especially at concentrations typical of pregnancy. To determine whether other pregnancy-associated steroid hormones are capable of similar activity, we expanded our testing to include estrone (E1), estriol (E3), progesterone, and dexamethasone. The results indicate that E1 and E3 enhance secretion of Ag- or anti-CD3-stimulated IL-10 and IFN-gamma in dose-dependent fashion, almost identical to that of E2. The effect on IL-10 was more potent than occurred with IFN-gamma. In addition, E1 and E3, like E2, had a biphasic effect on TNF-alphabeta secretion, with low concentrations stimulatory, and high doses inhibitory. None of the estrogens influenced IL-4 or TGF-beta secretion. Progesterone enhanced secretion of IL-4, without affecting any other tested cytokine. Finally, dexamethasone induced TGF-beta secretion, but inhibited IFN-gamma and TNF-alphabeta. This differential effect of steroid hormones on the secretion of cytokines by CD4+ human T cell clones is consistent with the possibility that, collectively, they promote antiinflammatory conditions at high concentrations typical of pregnancy.     

Positive and negative regulation of human T cell activation mediated by the CTLA-4/CD28 ligand CD80

CD28 and CTLA-4 are related receptors that differentially regulate T cell activation. Despite the fact that they bind the same ligands, CD28 is a classical costimulator enhancing proliferation whereas CTLA-4 appears to perform negative regulatory functions. In this study, we have utilized the natural ligand for CD28 and CTLA-4 (CD80) to determine under what circumstances positive and negative effects are operative. We show here that the stimulation of purified human T cells with phorbol ester and ionomycin is inhibited in the presence of Chinese hamster ovary (CHO) cells expressing CD80. This inhibition is reversed by blocking with both anti-CD80 or Fab fragments of anti-CTLA-4 but also requires CD28 engagement. Furthermore, we show that the inhibitory function of CD80 requires elevated intracellular calcium since inhibition was observed only in the presence of ionomycin. In the absence of intracellular calcium elevation, CTLA-4 was not expressed at the cell surface, and CD80 acted positively as a costimulator of T cells, via CD28. These results demonstrate that the natural ligand CD80 can either costimulate or inhibit T cell responses depending on the conditions of T cell stimulation.