Quantitative measurement of T-lymphocyte activation by an enzyme-linked immunosorbent assay (ELISA) detecting interleukin-2 receptor expression

A monoclonal antibody prepared against the murine interleukin-2 receptor (IL-2R) was employed to develop an ELISA method for measuring the immunological activation of T-cells. The assay detects an increase in IL-2R expression on activated lymphocytes. Stimulated splenic lymphocytes displayed markedly higher IL-2R expression compared to unstimulated controls. A significant increase in IL-2R expression on lymphocytes was detected in mitogen-stimulated responses, in a one-way mixed leukocyte reaction (MLR) and in the antigen-specific responses to conalbumin and purified protein derivative (PPD) in vitro. At a constant cell number, the level of IL-2R expression was found to be dependent on the dose of the stimulant. A comparative study of the kinetics of activation of splenic lymphocytes in response to mitogen, antigen and allogeneic cells as measured by the IL-2R ELISA and the conventional tritiated thymidine (3HTdR) uptake assay revealed remarkable similarity. For both assays, the mitogenic response was detected within 12 h and peaked at 72 h, the MLR was detectable within 2-3 days and peaked at day 6, and the specific antigenic response was detected within 2 days and peaked on day 4-5. Hydroxyurea, an inhibitor of DNA synthesis, had no effect on early IL-2R expression by mitogen-stimulated splenic lymphocytes, however, only 20% of maximum IL-2R expression could be detected at later stages of incubation. In contrast, cycloheximide, an inhibitor of protein synthesis, completely abrogated IL-2R expression and proliferation of stimulated lymphocytes.     

Kinetics of cytokine gene expression in human CD4+ and CD8+ T-lymphocyte subsets using quantitative real-time PCR

The time kinetics of five cytokines [interleukin-2 (IL-2), IL-5, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha)] and one cytotoxic effector protein (granzyme B) was analysed by real-time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T-cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell-shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD-activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4-8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48-72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen-specific T lymphocytes. It might be recommended that cytokine gene expression (IL-2, IL-5 and IFN-gamma) should be measured after 4-8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48-72 h of activation.     

Soluble interleukin 2 receptor release, interleukin 2 production, and interleukin 2 receptor expression in activated T-lymphocytes in vitro.

Following activation in vitro, peripheral blood mononuclear cells (PBMC) express cell-associated interleukin-2 receptors (IL2R). The present study was undertaken to define the proportion of T lymphocyte subsets that express the IL2R (CD25 antigen) upon different mitogenic stimulation. Double immunofluorescence staining with different fluorochromes, fluorescein isothiocyanate and phycoethyrin, was applied for identification of IL2R positive cells and individual lymphocyte subset. The exact percentage of individual activated lymphocyte subset bearing IL2R was enumerated by photographic counting. There was paucity of IL2R in freshly isolated, unstimulated peripheral blood, PBMC cultured without mitogen, and cultured B lymphocytes. Following pokeweed mitogen stimulation in vitro, 19% of CD4 (T-helper/inducer) lymphocytes and 14% of CD8 (T-suppressor/cytotoxic) lymphocytes expressed IL2R. Similarly, 25% of CD4 lymphocytes and 19% of CD8 lymphocytes expressed IL2R following phytohemagglutinin stimulation in vitro. Contrary to the reported data of Tac-positive cells in human lymphoid tissues, our study revealed that, upon lectin mitogen stimulation, approximately 55% of IL2R positive PBMC were CD4 lymphocytes, and 45% of them were CD8 lymphocytes. These observations imply the plausible notion that interleukin-2 mediated immune activation of T lymphocytes in PBMC is different from that in local lymphoid organs. It was also demonstrated that the release of soluble IL2R (sIL2R) and IL2 production in supernatant from cultured PBMC varied with different lectin stimulation. A significant correlation was demonstrated between the cellular and soluble IL2R but the production of IL2 from activated mononuclear cells bore no good correlation with either the cellular IL2R expression or the release of sIL2R.     

CD4 T lymphocyte activation in acute severe asthma.

The expression of activation molecules on peripheral-blood CD4 and CD8 T lymphocytes and the serum concentrations of two products of activated T lymphocytes [interferon-gamma (IFN-gamma) and soluble interleukin-2 receptor (sIL-2R)] were measured in patients with acute severe asthma (ASA) and controls. Significantly higher percentages of CD4+ cells from patients with ASA expressed IL-2R, HLA-DR and VLA-1 as compared to controls (p less than 0.01). In contrast, CD8+ cells from both asthmatics and controls did not express IL-2R and VLA-1, and their expression of HLA-DR in asthmatics was not increased. Serum concentrations of IFN-gamma and sIL-2R were significantly elevated in patients with ASA as compared to control groups (p less than 0.01). Concentrations decreased as the patients improved clinically following therapy. Significant correlations were observed between the improvements in airways obstruction and (1) the decreases in the percentages of peripheral-blood IL-2R+ T lymphocytes and (2) the decreases in serum concentrations of sIL-2R. These observations suggest that CD4 T lymphocyte activation is important in the pathogenesis of ASA.     

Surface markers on human activated T lymphocytes. IV. Comparison of high-affinity E-rosette receptor expression with the expression of other activation markers (receptor for interleukin 2, MHC class II (antigens).

In order to re-examine the value of high-affinity E rosette receptor (Eh-R) as an activation marker of human T lymphocytes, its existence on resting and activated T cells was compared with the expression of such known activation markers as receptor for interleukin 2 (IL-2R; Tac antigen) and MHC class II antigens (DR/DP and DQ). To this aim expression of the above surface markers on lymphocytes of TEe subset, derived from early E rosettes (Eh-R+) and on lymphocytes of TEl subset, derived from late E rosettes (Eh-R-), was examined immediately after purification of the cells from peripheral blood as well as during cell activation with PHA. The phenotypic studies were done by using monoclonal antibodies and indirect immunofluorescence technique. We confirmed previous observation that in the course of PHA stimulation Eh-R like IL-2R marked currently activated T cells. However, it was also found that in the long-term cultures of lymphocytes activated with PHA, the expression of Eh-R was sustained on the cells which lost their IL-2R and DR/DP antigens. The above findings and the fact that TEe cell subset consisting of Eh-R+ lymphocytes was almost completely depleted from cells bearing IL-2R and MHC class II antigens allowed us to conclude that this subset of peripheral blood T lymphocytes represented not currently activated cells but the cells which had been previously activated in vivo.     

Soluble and cellular markers of immune activation in patients with systemic sclerosis

The peripheral blood lymphocyte pattern, the lymphocyte responses in vitro, as well as the soluble markers of immune activation were studied in 24 patients with systemic sclerosis (SSc patients). The proportions of total T cells (CD3), their CD4 subset, as well as B lymphocytes were within the normal range. The relative proportion of CD8 lymphocytes, however, was significantly reduced. Patients with SSc had a slightly lower percentage of CD4/4B4+ cells, whereas their proportion of CD4/2H4+ cells was elevated as compared to healthy controls. The proportion of lymphocytes expressing the interleukin-2 receptor (IL-2R) was significantly higher in SSc patients. The proliferative responses of peripheral blood mononuclear cells to PHA stimulation were reduced in the patient group, while expression of IL-2R on lymphocytes after such in vitro stimulation was comparable to that of controls. Expression of IL-2R on patient but not control lymphocytes was increased after in vitro exposure to laminin. Such exposure failed to induce IL-2 production or cell proliferative responses. Soluble plasma IL-2R level (sIL-2R) and soluble CD8 (sCD8) molecule levels in SSc patients were significantly elevated. These results indicate the presence of an ongoing lymphocyte activation in this disease process.     

Chemoattractant activity of IL-2 for human lymphocytes: a requirement for the IL-2 receptor beta-chain.

Recombinant human interleukin-2 (IL-2) stimulated locomotion and chemotaxis of human blood lymphocytes as measured by shape change to a polar morphology, by orientation in a chemotactic gradient, and by a collagen gel invasion assays. IL-2 stimulated locomotion of a larger number of lymphocytes than IL-8 or macrophage inflammatory protein (MIP)-1 alpha, but the maximally effective concentration of all three was similar (around 100 ng/ml). Activation of the lymphocytes by culture for 24-48 hr in fetal calf serum (FCS), anti-CD3, or purified protein derivative (PPD) increased the proportion of responsive cells, though even direct from blood, > 20% of lymphocytes showed locomotor responses to IL-2, a figure which was similar to the number of IL-2 receptor (IL-2R) beta+ lymphocytes but higher than the number of IL-2R alpha+ cells. The effect of antibodies to IL-2R alpha and IL-2R beta as inhibitors of these responses was therefore tested. Anti-IL-2R beta (alpha IL-2R beta) completely inhibited the response of both resting and activated cells: alpha IL-2R alpha had no inhibitory effect on the locomotion of lymphocytes direct from blood, and only partially inhibited locomotion after culture for 48 hr in alpha CD3 or PPD. The locomotor response to IL-2 was inhibited by pretreatment of the cells with herbimycin, a protein tyrosine kinase (PTK) inhibitor, an observation consistent with PTK control of cytoskeletal activity following binding of IL-2 to IL-2R beta. These results suggest that the beta-chain of the IL-2R is required for activation of lymphocyte locomotion by IL-2 and that binding of IL-2 to this chain alone is sufficient for a response.     

Human dendritic cells activate T lymphocytes via a CD40: CD40 ligand-dependent pathway

The CD40: CD40 ligand (CD40L) interaction provides T lymphocyte-mediated help for B lymphocyte and monocyte function but has also been shown to serve as a co-stimulus for T lymphocyte activation. In this report, we studied the regulation of CD40 expression and its functional relevance for the human dendritic cell (DC) stimulation of T lymphocytes. Only a small subpopulation of directly isolated blood DC expressed CD40. However, CD40 was rapidly up-regulated by culture, and its expression was further enhanced by interleukin (IL)-1α, IL-1β, IL-3, tumor necrosis factor-α and granulocyte/macrophage-colony-stimulating factor. Expression of CD40L on DC was not detected. The proliferation of T lymphocytes in an allogeneic mixed leukocyte reaction, stimulated by blood DC or epidermal Langerhans cells, was significantly reduced in the presence of the CD40 immunoglobulin (CD40Ig) fusion protein or CD40L monoclonal antibodies. Cross-linking of CD40 on directly isolated DC with mouse CD40L trimer (mCD40LT) markedly augmented CD80 and CD86 up-regulation. Nevertheless, the same cross-linking mCD40LT inhibited DC stimulated T lymphocyte proliferation. When CD40Ig was added simultaneously with CTLA-4Ig, only minimal and variable additional inhibition of DC-stimulated allogeneic T lymphocyte proliferation and IL-2 secretion was observed, compared to each fusion protein alone. These results suggest that both CD80/CD86-dependent and -independent components of DC-T lymphocyte CD40: CD40L co-stimulation exist and further emphasize that the majority of blood DC have to differentiate or be activated to express co-stimulatory molecules.     

CD2-mediated stimulation of the naive CD4+ T-cell subset promotes the development of skin-associated cutaneous lymphocyte antigen-positive memory cells.

Directed migration of lymphocytes from blood into lymph nodes and organ-associated lymphatic tissue, also referred to as homing, is initiated by T-cell adhesion to specialized high endothelial cells of postcapillary vessels. Here, we demonstrate that selective signal transduction pathways specifically modulate the expression of the cutaneous lymphocyte antigen (CLA), the putative skin-homing receptor, during naive to memory transition of CD4+ T cells in vitro. The results show that the expression of CLA is strongly induced by activation via CD2 [T11.1 + T11.2 monoclonal antibodies (mAb)]. Addition of transforming growth factor-beta 1 (TGF-beta 1), interleukin-6 (IL-6), and, to a lesser extent, IL-2 further enhanced the generation of CLA+ T cells, whereas the induction of this antigen was markedly inhibited by IL-4. Periodic restimulation via CD2 and long-term culture of activated cells in the presence of IL-2 and TGF-beta 1 resulted in stable expression of CLA during a culture period of more than 100 days. In contrast, activation of naive CD4+ T cells via CD3, CD28 or by mitogens induced a rapid naive to memory phenotype transition but a much lower percentage of CLA+ T cells showing only weak expression of the antigen. Furthermore, activation of purified CD4+ memory T cells by CD2 strongly induced expression of activation-related antigens CD25 and HLA-DR, but failed to up-regulate CLA expression. Our results show that primary stimulation conditions highly modulate the development of skin-associated T cells and indicate a new functional role for costimulatory adhesion pathways in regulating the expression of molecules associated with T-cell homing.     

Recombinant human interleukin-2 reverses in vitro-deficient cell-mediated immune responses to tuberculin purified protein derivative by lymphocytes of tuberculous patients.

In vitro lymphocyte proliferative response to purified protein derivative of tuberculin (PPD) was investigated in patients with tuberculosis. Peripheral blood lymphocytes (PBL) from patients with advanced, refractory tuberculosis showed a significantly depressed response compared with the response of PBL from patients with newly diagnosed tuberculosis (P less than 0.01). A further characterization of this low responsiveness to PPD revealed that PBL from these advanced tuberculous patients failed to generate interleukin-2 (IL-2) in response to PPD stimulation. IL-2 receptor (Tac antigen) expression on the surface of T cells after PPD stimulation was also impaired, although to a lesser extent, in the patients with advanced, refractory tuberculosis. We attempted to overcome the depressed in vitro response observed in PBL from patients with advanced, refractory tuberculosis and found that the addition of exogenous, recombinant IL-2 returned the depressed PPD-induced PBL proliferation in these patients to the level of response observed in PBL from patients with newly diagnosed tuberculosis. The addition of recombinant IL-2 also had a restorative effect (up regulation) in vitro on the partly impaired PPD-induced IL-2 receptor expression by PBL from the patients with advanced, refractory tuberculosis. Our results suggest that recombinant IL-2 may offer a novel approach to the therapy of advanced, drug-resistant tuberculosis.     

In Vivo Activated Lymphoid Cells (IVALC) Affect the Cloning Efficiency of Human T Lymphocytes Reactive to a Soluble Antigen, Purified Protein Derivative

Peripheral blood mononuclear cells (PBMC) of normal individuals were found to contain a proportion (4-9%) of in vivo activated lymphoid cells (IVALC). These IVALC were characterized by their expression of interleukin 2 (IL-2) receptors, and by the ability to proliferate in the presence of exogenous IL-2. There was a good correlation between the proportion of IVALC in different cell populations and the level of cell proliferation to IL-2. It was found that IVALC isolated from autologous PBMC of Bacillus Calmette-Guerin (BCG)-immunized individuals contained no significant proportion of purified protein derivative (PPD)-reactive lymphocytes. The addition of IVALC markedly enhanced proliferative responses of the autologous T4+T8-IL-2 receptor-negative cell cultures to antigen stimulation. An increased proportion of activated (IL-2 receptor-positive) lymphocytes was generated in PBMC as compared to autologous T4+T8-IL-2 receptor negative cell cultures after stimulation with PPD. Limiting dilution analysis showed that IL-2 responsive IVALC through expansion markedly affected the cloning efficiency of antigen-proliferating T cells of autologous PPD-stimulated PBMC cultures. Only 1 out of every 11-25 blast cells generated in the PBMC cultures could establish itself as a growing colony based on determinations in six BCG-positive individuals. By using a T4+T8- population depleted of IVALC to generate PPD-reactive lymphocytes, a three- to four-fold increase in the cloning efficiency of antigen-specific cells was obtained.     

Selective suppressive effects of Trypanosoma cruzi on activated human lymphocytes.

The acute phase of Chagas' disease is accompanied by immunosuppression. To explore the underlying mechanism(s), we used an in vitro culture system in which the capacities of activated human peripheral blood mononuclear cells to express interleukin-2 receptors (IL-2R) and proliferate are markedly inhibited in the presence of Trypanosoma cruzi, the etiologic agent. The present work was designed to define the earliest time at which T. cruzi-induced suppression is manifested in terms of IL-2R expression on the cell surface and establish whether expression of other lymphocyte activation markers is also suppressed by the parasite. We found that expression of IL-2R by human peripheral blood mononuclear cells cocultured with T. cruzi and stimulated with either phytohemagglutinin or anti-CD3 (a monoclonal antibody specific for an epitope of the T cell receptor complex T3-Ti) was significantly suppressed as early as 12 h after culture initiation. Both the percentage of IL-2R+ cells and the surface density of IL-2R, measured by flow cytometry, were affected. However, expression of EA1, a human lymphocyte activation antigen known to be expressed 4 to 6 h after stimulation, was not altered by T. cruzi whether phytohemagglutinin or anti-CD3 was used. On the other hand, expression of transferrin receptors (TfR), which first occurs between 20 and 24 h after lymphocyte activation, was markedly suppressed by T. cruzi. This effect was denoted by significant reductions in both the percentage of TfR+ cells and the cell surface density of TfR whether phytohemagglutinin or anti-CD3 was used as the mitogen and was observed at all test times, i.e., at 24, 48, 72, and 96 h. Because expression of IL-2R and TfR is required for lymphoproliferation but that of the EA1 lymphocyte activation marker is apparently not, these results are consistent with the possibility that T. cruzi, at a relatively early stage during lymphocyte activation, selectively affects certain key events on which clonal expansion is dependent. Inhibition of IL-2R and TfR expression by the parasite might play a role in causing the suppressive effects associated with acute Chagas' disease.     

Expression of Fc epsilon receptors on activated human T lymphocytes

Our results clearly demonstrate that the low-affinity receptor for IgE (Fc epsilon R) is an activation antigen transiently expressed on a subpopulation of human T lymphocytes. It can be selectively induced by stimulation with certain antigens or lectins, but it is not found on resting T cells. The increased numbers of activated Fc epsilon R+ T cells observed after stimulation of peripheral blood mononuclear cells (PBMC) from bee venom allergic patients with the specific allergen phospholipase A2 (PLA2) suggest that Fc epsilon R+ T cells might very well be involved in the regulation of the human IgE response against the respective antigen. These results were obtained by the use of two monoclonal antibodies, M-L25 and M-L47, which were raised against the human low-affinity Fc epsilon R in our laboratory. After stimulation of PBMC with phytohemagglutinin a peak of 7.6 +/- 6% Fc epsilon R+ T cells was observed on day 3, with pokeweed mitogen of 0.8 +/- 0.8% on days 2 and 3, and with concanavalin A of 0.6 +/- 0.7% Fc epsilon R+ T cells on day 2. Stimulation of PBMC with tetanus toxoid (TT) induced Fc epsilon R on maximally 0.6 +/- 0.8% of the total T cells (day 4), stimulation with purified protein derivative from tuberculin (PPD) on 0.2 +/- 0.6% of the T cells (day 2). In contrast to these antigens, stimulation of PBMC from bee venom allergic patients with PLA2 induced as a peak 2.5 +/- 2.5% of the total T cells to express Fc epsilon R (day 5), although the stimulated T cell population was much smaller than with TT or PPD, as was shown by their stimulation indices. The allergen-stimulated Fc epsilon R+ T cells were exclusively T4+. The Fc epsilon R-expression index was determined, which for a specific antigen or lectin correlates the percentage of Fc epsilon R+ T cells to the stimulated T cell population, respectively.     

Possible activation of murine T lymphocyte through CD98 is independent of interleukin 2/interleukin 2 receptor system

CD98 is a widely expressed cell surface heterodimeric protein of 125 kDa. Its expression is upregulated during lymphocyte activation induced by mitogen, superantigen, conventional antigen, and a combination of phorbol myristate acetate (PMA) and ionomycin. However, the role of CD98 in the immune system is not so well understood. The role of CD98 in murine T lymphocyte proliferation was investigated, especially in correlation with the interleukin 2 (IL-2)/interleukin 2 receptor (IL-2R) system. Monoclonal antibody (mAb) directed against murine CD98 heavy chain (mCD98HC) suppressed the proliferation of lymphocytes stimulated with concanavalin A (Con A). Anti-mCD98HC mAb did not suppress the expression of IL-2Ralpha. Anti-IL-2Ralpha mAb, which suppressed DNA synthesis, did not inhibit the expression of CD98HC. Murine IL-2 (recombinant), which induced considerable DNA synthesis by lymphocytes stimulated with a sub-optimal dose of Con A, did not induce CD98HC expression in lymphocytes. In addition, anti-mCD98HC mAb did not inhibit the production of IL-2 by lymphocyte stimulated with Con A. Taken together with these findings, it was speculated that the CD98 system is independent of the IL-2/IL-2R system in murine T lymphocyte activation.     

Tuberculin-induced lymphocyte proliferation in whole blood: an antigen specific method for assessing immunosuppressive agents

Antigen-stimulated whole blood cultures have not been used to study the effects of immunosuppressive drugs. The aim of this study was to assess the potential usefulness of tuberculin purified protein derivative (PPD)-stimulated lymphocyte proliferation in whole blood for studying the effects of T cell inhibitory agents. We have investigated whether PPD causes antigen specific T cell proliferation, and the role of the major histocompatibility complex class II (MHC class II), co-stimulation and IL-2 in the development of this response. We have also studied the effects of prednisolone and cyclosporin on lymphocyte proliferation. Heparinised blood from healthy volunteers was diluted in culture medium and incubated with PPD. Cell proliferation, measured by liquid scintillation counting, was maximal using 1000 units/ml PPD incubated in 10% whole blood for 6–7 days. A population of large CD4+ lymphocytes appeared in cultures incubated with PPD, suggesting that the major responding population was composed of T lymphocytes. There was no significant response to the negative control antigen KLH, indicating that proliferation was antigen specific. Monoclonal antibodies (mAbs) against MHC, CD2, CD26, CD28 and IL-2 inhibited proliferation. Prednisolone was more potent than cyclosporin in this assay (IC50 values; prednisolone 20 nmol, cyclosporin 278 nmol). For the first time, this report shows that the PPD causes antigen specific lymphocyte proliferation in whole blood, which is dependent on antigen presentation via MHC class II, co-stimulation and IL-2 production. Because the proliferative response is dependent on the major interactions that lead to T cell activation, this simple assay can be used to assess the effects of novel immunomodulators.     

Limiting dilution analysis of the human T cell response to mycobacterial antigens from BCG vaccinated individuals and leprosy patients.

The number of peripheral blood T lymphocytes responding to soluble mycobacterial antigens from Mycobacterium tuberculosis purified protein derivative (PPD) and M. leprae (MLS) was estimated by limiting dilution analysis. Antigen-induced lymphocyte activation was measured by means of [3H]TdR incorporation on day 10 of culture in the presence of suboptimal concentrations of interleukin 2 (IL-2). In the peripheral blood of BCG-vaccinated individuals from the UK, the frequency of T lymphocytes responding to PPD was 1.5 to 4 times greater than to MLS. Frequencies between 1/1970 and 1/13, 982 were observed in response to PPD and between 1/4097 and 1/24, 717 in response to MLS. A proportion of cells in the peripheral blood were also observed to respond to IL-2 only. The frequency of cells observed in limiting dilution analysis for PPD and MLS reflected the relative amounts of proliferation to these two antigens in bulk culture lymphocyte transformation tests. Use of PPD-specific T cell lines suggested that the responsiveness observed to M. leprae antigens in BCG-vaccinated individuals was due to cross-reactivity with antigens shared with M. bovis BCG. In tuberculoid leprosy, the frequency of peripheral blood T lymphocytes responding to M. leprae antigens was either greater than or similar to the frequency of T cells responding to PPD. In contrast, limiting dilution analysis of T lymphocytes from the peripheral blood of lepromatous leprosy patients revealed the complex regulatory heterogeneity of this group. In some patients M. leprae responsive T cells were detected in the presence of exogenous IL-2.     

Lack of Correlation Between Membrane CD30 Expression and Cytokine Secretion Pattern in Allergen-Primed Naive Cord Blood T-Cell Lines and Clones

Various surface molecules are expressed by activated T cells. Among them, the CD30 antigen has been proposed as a reproducible marker that identifies a subset of differentiated and/or activated T lymphocytes that produce T helper (Th)-2-type cytokines, i.e. interleukin (IL)-4 and IL-5. However, because CD30 has mainly been detected on established T-cell clones, it is still unclear whether a priming allergen and/or cytokine can induce its membrane expression on naive T cells, perhaps in parallel with the up-regulation of other relevant activation markers, such as CD25, HLA-DR and L-selectin. It is also unknown whether proper allergen stimulation affects the cytokine secretion pattern by CD30⁺ T-cell clones derived from antigen-unprimed (naive) T lymphocytes. More information on these questions was sought by adopting a model that used cord blood as a source of virgin T cells and exposing them to native cypress allergen or cytokine (IL-2 or IL-4) stimulation, as well as to conventional polyclonal activators such as PHA or anti-CD3. Peripheral blood MC from four adult cypress-sensitive patients was also assayed and used as controls for all culture experiments. Freshly isolated cord and adult T cells did not express the CD30 antigen on their membrane. Many of the stimulating agents tested were able to up-regulate the expression of CD30. However, despite high expression of this molecule, cloned allergen-specific cord CD4⁺ T lymphocytes were unable to produce IFN-γ and/or IL-4. In contrast, they retained the capability to produce IL-2. Thus, expression of the CD30 antigen on virgin T cells does not correlate with a polarized model of T helper (Th)-1 or Th-2 cytokine-producing cells, suggesting that these types of lymphokine-secreting lymphocytes are not a paradigmatic example of T-cell subpopulations that display stable phenotypical features.     

Dietary protein deficiency and Mycobacterium bovis BCG affect interleukin-2 activity in experimental pulmonary tuberculosis.

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated. They were maintained for 6 weeks on defined, isocaloric diets containing either 30% (control animals) or 10% (animals receiving low protein) ovalbumin as the sole protein source. Animals were challenged by the respiratory route with a low dose of virulent M. tuberculosis H37Rv and killed 4 weeks later. Protein-malnourished animals were not protected by previous vaccination with BCG. Lymphocytes isolated from various tissues were tested in vitro for proliferative responses to mitogen (concanavalin A) and antigen (purified protein derivative [PPD]), production of interleukin-2 (IL-2), and response to exogenous recombinant IL-2 (rIL-2). Protein-malnourished guinea pigs responded only weakly to PPD skin tests, and their blood and lymph node lymphocytes exhibited impaired proliferation when cultured with PPD in vitro. IL-2 levels were consistently low in cultures of stimulated blood and spleen lymphocytes from protein-deprived animals. BCG vaccination of nutritionally normal guinea pigs, on the other hand, induced significantly more IL-2 production by PPD- and concanavalin A-stimulated lymphocytes. The addition of exogenous mouse rIL-2 (40 and 80 U/ml) in vitro to PPD-stimulated blood and lymph node cells from nonvaccinated, protein-deprived guinea pigs resulted in no improvement of the proliferative response. Previous vaccination of malnourished guinea pigs did not consistently enhance the response of PPD-stimulated lymphocytes to added rIL-2. Dietary protein deficiency and BCG vaccination appear to modulate antigen-driven cellular immunity in animals with tuberculosis by altering the production of, and the response to, IL-2 by PPD-stimulated lymphocytes.     

Increased plasma levels of soluble IL-2R are associated with severe Plasmodium falciparum malaria.

Plasma samples from children with mild and severe Plasmodium falciparum malaria and from children with unrelated diseases were collected to investigate whether the clinical outcome of infection was associated with plasma factors which reflected the activity of different cells of the immune system. Children with severe P. falciparum malaria had significantly higher plasma levels of soluble IL-2R than children with mild malaria. Plasma levels of IL-2R and levels of parasitaemia were significantly correlated. Neither parasitaemia nor plasma levels of tumour necrosis factor-alpha (TNF-alpha), IL-6, lymphotoxin (LT), interferon-gamma (IFN-gamma), IL-4, soluble IL-4R or soluble CD8 differed significantly between the two groups of children with malaria. High plasma levels of soluble CD8 were associated with failure of lymphocytes to produce IFN-gamma in vitro following stimulation with P. falciparum antigen. We conclude that soluble IL-2R is a useful marker of disease severity independently of the association with levels of parasitaemia, and that functional regulation of different lymphocyte subsets occurs during acute malaria episodes.     

Ability of Pure Resting CD8+ Human T Cells to Respond to Alloantigen

The ability of highly purified resting human CD8 cells to respond to alloantigens in vitro was examined. Necessary conditions for induction of interleukin 2 receptors (IL-2R), IL-2 production, proliferative responses, and various effector functions were determined. Allogeneic non-T cells induced IL-2R expression in a high proportion of resting CD4 and CD8 cells, but only CD4 cells produced detectable amounts of IL-2. CD8 cells also became IL-2 responsive upon stimulation with purified resting allogeneic CD4 or CD8 cells, indicating that HLA class I+, II- cells alone may initiate activation of resting CD8 cells. The activated CD8 cells needed the presence of simultaneously activated CD4 cells or exogenous IL-2 to be able to synthesize DNA. Effector functions like cytotoxicity, mixed lymphocyte culture (MLC) suppression, or gamma interferon (IFN-gamma) production were also only detectable when the CD8 cells were activated in the presence of IL-2.