Parameters Of T-cell Activation Research Articles

CTL ELISPOT Assay and T Cell Detection.

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T-cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T-cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T-cell (CTL) studies have taken advantage with this high-throughput technology by providing insights of quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T-cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T-cells is of relevance for immune diagnostic. The most utilized Elispot assay is the Interferon-gamma (IFN-γ) test, a marker for CD8+ CTL activation, but Elispot can be also used to distinguish different subsets of activated T-cells by using other cytokines such as T-helper (Th) 1 type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21 and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10 and IL-13), and Th17 (IL-17) cells.The reliability of Elispot generated data, by the evaluation of T-cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot Assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Milteny cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T-cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot Assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and Perforin Elispot assays will be described.

The role of the reduction of extracellular calcium ion concentrations in activation of human peripheral blood T cells

Calcium plays a fundamental role in many essential functions of a living organism. It is well known that many infections can reduce extracellular calcium concentrations ([Ca2+]o) (hypocalcaemia) in human blood. The importance of Ca2+ for the T lymphocyte activation has been known for a long time, but the optimal range of [Ca2+]o levels has not been found yet. The main goal of this work was the study of functional activities of CD4+ cells in human peripheral blood (HPB) in vitro in the conditions of hypocalcemia. Polyclonal activation was induced in whole blood of healthy donors by immobilized monoclonal antibodies (mAbs) toward CD3 molecules (epsilon chain of the T cell receptor) and dissolved mAbs toward CD28 (the major molecule of T cell costimulation). Dependencies of T-cell activation parameters on varied [Ca2+]o in HPB in vitro were studied. Production of interleukins-2 (IL-2), IL-4, IL-17A, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) as well as CD4+CD69+, CD4+CD25+, and CD4+(CYTOKINE)+ T cells were used as activation parameters of the T cell polyclonal activation in HPB. EGTA was used as a tool for the reduction of [Ca2+]o levels. The results demonstrated that the EGTA-stimulated binding of extracellular calcium in HPB caused a nonlinear dependence of T cell activation on [Ca2+]o. No EGTA influence on resting T cells was found. The dependence of the number of CD4+CD69+ T cells on [Ca2+]o was represented as a nonlinear curve with two maxima and a local minimum in between. One of the maxima was in the area of the HPB lower [Ca2+]o normal level, whereas the other one was even lower, which is characteristic for pathological states. The T cell-induced production of IL-2, IL-4, IL-17A, IFN-γ, and TNF-α as well as the populations of CD4+CD25+ and CD4+(CYTOKINE)+ T cells were analyzed in HPB in vitro under the same activation conditions. All the dependencies obtained were similar in shape to that of the CD4+CD69+ T cell population versus EGTA concentration in HPB in vitro and was observed as curves with two maxima. The dependencies of this type were described first. It is likely that the presence of two maxima and a local minimum in between for the dependence of HPB T cell activation parameters on [Ca2+]o concentrations is of great physiological importance because hypocalcemia has been associated with many pathological states.

Sudden Cardiac Death in a Young HIV-Positive Man on Effective Antiretroviral Therapy

We describe the case of a young HIV-positive man on effective HAART with excellent viro-immunological control who presented a massive cardiac infarction. Despite the presence of clinical risk factors for cardiovascular disease, the patient had normal arterial carotid IMT values, known to be strong predictors of atherosclerosis and stroke. Interestingly, parameters of T-cell activation (CD8+CD38+) were shown to increase just before the onset of myocardial infarction. As T-cell activation is known to mediate atherosclerosis, the authors suggest that surrogate immunologic markers should be identified to better assess cardiovascular risk in the setting of HIV infection.

Ultraviolet B Suppresses Immunity by Inhibiting Effector and Memory T Cells

Contact hypersensitivity is a T-cell-mediated response to a hapten. Exposing C57BL/6 mice to UV B radiation systemically suppresses both primary and secondary contact hypersensitivity responses. The effects of UVB on in vivo T-cell responses during UVB-induced immunosuppression are unknown. We show here that UVB exposure, before contact sensitization, inhibits the expansion of effector CD4+ and CD8+ T cells in skin-draining lymph nodes and reduces the number of CD4+ and IFN-gamma+ CD8+ T cells infiltrating challenged ear skin. In the absence of UVB, at 10 weeks after initial hapten exposure, the ear skin of sensitized mice was infiltrated by dermal effector memory CD8+ T cells at the site of challenge. However, if mice were previously exposed to UVB, this cell population was absent, suggesting an impaired development of peripheral memory T cells. This finding occurred in the absence of UVB-induced regulatory CD4+ T cells and did not involve prostaglandin E2, suggesting that the importance of these two factors in mediating or initiating UVB-induced immunosuppression is dependent on UVB dose. Together these data indicate that in vivo T-cell responses are prone to immunoregulation by UVB, including a novel effect on both the activated T-cell pool size and the development of memory T cells in peripheral compartments.

Multiple CD4 and CD8 T-cell activation parameters predict vaccine efficacy in vivo mediated by individual DC-activating agonists

A systematic comparison of the immunostimulatory capacity of TLR 2, 3, 4, 5, 7 and 9 agonists and an agonistic CD40-specific antibody was performed in a single long peptide vaccination model. All adjuvants activated DC in vitro but not all induced a strong functional T-cell response in vivo. Optimal clonal CD8+ T-cell expansion depended on the capacity of agonists to mature pro-inflammatory DC and the duration of their in vivo stimulatory effect. Strong agonists promoted the induction of both antigen-specific IFNγ-producing CD4+ T-helper cells and high numbers of IFNγ producing CD8+ effector T-cells that killed target cells in vivo. Importantly, the capacity of an agonist to function as an adjuvant depended on the vaccine strategy used. Collectively, the multi-parameter system presented here can be used as a general road map to develop therapeutic vaccines.

Effects of iEW Synthetic Peptide Isomers on Bone Marrow Colony-Forming Capacity In Vivo

Studies of cooperative effects of D- or L-isomers of iEW dipeptide and neuraminidase on the number of splenic CFU, comparison of the peptide effects on CD34+ cells and splenic CFU, and evaluation of the effects of iEW D- or L-isomers on parameters of T-cell activation showed that interactions of L-(iEW) and D-(iEW) with CD34+ surface receptors are realized through the same mechanism, while their different biological effects on hemopoietic precursor cells in vivo can be explained by their different influence on T-component of the microenvironment.

Evidence for systemic immune system alterations in sporadic amyotrophic lateral sclerosis (sALS)

Sporadic amyotrophic lateral sclerosis (sALS) is a progressive neuroinflammatory disease of spinal cord motor neurons of unclear etiology. Blood from 38 patients with sALS, 28 aged-match controls, and 25 Alzheimer's disease (AD) patients were evaluated and activated monocyte/macrophages were observed in all patients with sALS and AD; the degree of activation was directly related to the rate of sALS disease progression. Other parameters of T-cell activation and immune globulin levels showed similar disease associated changes. These data are consistent with a disease model previously suggested for AD, wherein systemic immunologic activation plays an active role in sALS.

Selective blockade of voltage-gated potassium channels reduces inflammatory bone resorption in experimental periodontal disease.

The effects of the potassium channel (Kv1.3) blocker kaliotoxin on T-cell-mediated periodontal bone resorption were examined in rats. Systemic administration of kaliotoxin abrogated the bone resorption in conjunction with decreased RANKL mRNA expression by T-cells in gingival tissue. This study suggests a plausible therapeutic approach for inflammatory bone resorption by targeting Kv1.3. Kv1.3 is a critical potassium channel to counterbalance calcium influx at T-cell receptor activation. It is not known if Kv1.3 also regulates RANKL expression by antigen-activated T-cells, and consequently affects in vivo bone resorption mediated by activated T-cells. Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein-specific Th1-clone cells were used to evaluate the expression of Kv1.3 (using reverse transcriptase-polymerase chain reaction [RT-PCR] and Western blot analyses) and the effects of the potassium channel blocker kaliotoxin (0-100 nM) on T-cell activation parameters ([3H]thymidine incorporation assays and ELISA) and expression of RANKL and osteoprotegerin (OPG; flow cytometry, Western blot, and RT-PCR analyses). A rat periodontal disease model based on the adoptive transfer of activated 29-kDa outer membrane protein-specific Th1 clone cells was used to analyze the effects of kaliotoxin in T-cell-mediated alveolar bone resorption and RANKL and OPG mRNA expression by gingival T-cells. Stimulated 29-kDa outer membrane protein-specific Th1 clone cells were transferred intravenously on day 0 to all animals used in the study (n = 7 animals per group). Ten micrograms of kaliotoxin were injected subcutaneously twice per day on days 0, 1, 2, and 3, after adoptive transfer of the T-cells. The control group of rats was injected with saline as placebo on the same days as injections for the kaliotoxin-treated group. The MOCP-5 osteoclast precursor cell line was used in co-culture studies with fixed 29-kDa outer membrane protein-specific Th1-clone cells to measure T-cell-derived RANKL-mediated effects on osteoclastogenesis and resorption pit formation assays in vitro. Statistical significance was evaluated by Student's t-test. Kaliotoxin decreased T-cell activation parameters of 29-kDa outer membrane protein-specific Th1 clone cells in vitro and in vivo. Most importantly, kaliotoxin administration resulted in an 84% decrease of the bone resorption induced in the saline-treated control group. T-cells recovered from the gingival tissue of kaliotoxin-treated rats displayed lower ratios of RANKL and OPG mRNA expression than those recovered from the control group. The ratio of RANKL and osteoprotegerin protein expression and induction of RANKL-dependent osteoclastogenesis by the activated T-cells were also markedly decreased after kaliotoxin treatments in vitro. The use of kaliotoxin or other means to block Kv1.3 may constitute a potential intervention therapy to prevent alveolar bone loss in periodontal disease.

Allergen immunotherapy with cat allergen peptides.

Desensitising therapy for allergic diseases has changed little over almost a century of practice. Administration of increasing doses of extracts of allergen source material has been shown to be reproducibly effective when patients are carefully selected and appropriate concentrations of allergen employed. However, specific immunotherapy is limited by the interaction of specific IgE with allergen, leading to a relatively high frequency of adverse events including anaphylaxis and death. Several strategies have been developed to tackle this issue. Most of these rely on reducing the allergenicity of the treatment, whilst maintaining the immunogenicity. The use of short, synthetic peptide sequences corresponding to T-cell epitopes from the allergen has been shown to modify surrogate markers of allergy including cutaneous responses to allergen challenge and ex vivo parameters of T-cell activation. This review discusses recent advances in our understanding of the mechanisms and potential efficacy of this form of therapy.

T-cell immune parameters and depression in patients with Crohn's disease.

The causes of Crohn's disease (CD) are still considered to be unknown. It is likely that an immune defect leads to an increase in T helper 1 (Th1) cytokine responses, which may then contribute to the characteristic morphologic changes. Increased values in distinct immune activation parameters connected with a Th1 immune response have also been found in patients who are depressed. Because various clinical observations have asserted that a depressive disposition may have some connection with the development of CD, we examined whether a relationship exists between T-cell activation parameters and depressive personality characteristics in patients with CD. Seventy-one patients (62% women; age, 38 +/- 13 years) with CD (23 with CD Activity Index [CDAI] >150 and 48 with CDAI < or =150) were included in this study. Sixty patients were re-examined after 4.4 +/- 1.8 months. The T cell parameters CD2, CD3, CD26, CD28, and CD95 and the activity of dipeptidyl peptidase IV (DPP IV) in serum were examined in the peripheral blood. The patient's subjective health status was assessed by means of a standardized psychometric instrument (Short-form Inflammatory Bowel Disease Questionnaire [SIBDQ]). The presence of various characteristics indicative of a depressed mood was also assessed (Center of Epidemiological Studies-Depression Scale, Berliner Stimmungsfragebogen), as well as indicators of a personality disposed to depression (Giessentest) and a depressive illness coping strategy (Freiburger Krankheitsverarbeitungs-Fragebogen), all using the above-mentioned standardized instruments. The immune parameters connected with the activation of Th1 (CD25+, CD25+/CD26+) are increased in patients, depending on the stage of illness. In addition, DPP IV activity was significantly lower in patients with an active disease, as was their subjective health status (SIBDQ). A connection with an indicator of mental depression could not be found. The observed changes in immune parameters support the idea that morphologic changes could be connected with an increased Th1 response and that DPP IV activity could play an immunomodulatory role. A relationship between the measured immune parameters and individual characteristics of depression could not be found.

Atopy patch test reactions are associated with T lymphocyte-mediated allergen-specific immune responses in atopic dermatitis.

Positive test reactions to epicutaneous application of aeroallergens served as a model of 'early' eczema in atopic dermatitis (AD) in a number of dermato-immunological studies. However, no quantitative evaluation has been performed so far comparing specific T-cell activation parameters in the peripheral blood of AD patients with positive or negative atopy patch tests (APT). The purpose of this study was to investigate specific immunological parameters in patients with atopic dermatitis showing positive or negative atopy patch tests reactions. APT results (n = 96) were compared with allergen-specific IgE, specific lymphocyte proliferation, and the expression of 'activation' markers on peripheral blood T-cells upon in vitro stimulation with house dust mite, cat or grass pollen allergens. Only a subpopulation (48%) of patients sensitized to aeroallergens (i.e. specific IgE > 0.7 kU/L) developed APT-reactions to the corresponding allergen. APT reactions were, however, significantly associated with allergen specific lymphocyte proliferation (p < 0.0001), and a higher number of CD54+ or CD30+ T-cells (p < 0.05) upon in vitro stimulation. The association of delayed skin reactions with allergen specific T-cell parameters in the blood points to an immunologically mediated mechanism leading to positive reactions in the APT.

Cholera toxin can inhibit FF-mas induced germinal vesicle breakdown in mouse oocytes cultured in vitro

The mechanism by which purinergic agonists modulate murine T-lymphocyte activation and proliferation was investigated. Adenosine and other compounds such as ATP and 2-chloroadenosine (ClAdo) were found to block T-cell mitogenesis induced by concanavalin A (Con A) in a dose-dependent fashion. The nonmetabolizable adenosine analog ClAdo was the most potent agent capable of inhibiting T-cell mitogenesis. Extracellular addition of the permeable cAMP analog dibutyrl cyclic AMP (dbcAMP) also led to a dose-dependent blockade of T-cell mitogenesis, although with less efficiency when compared to ClAdo. Addition of IL-2-enriched fluids failed to reverse blockade of T-cell mitogenesis by ClAdo or dbcAMP. ClAdo blocked T-cell enlargement induced after 20 hr of culture with Con A. We analyzed the effect of micromolar concentrations of ClAdo on interleukin-2 (IL-2) production, expression of IL-2 receptors (7D4 and 3C7 surface antigens), and induction of IL-2 responsiveness after in vitro cultivation with Con A. ClAdo inhibited both IL-2 secretion and induction of IL-2 responsiveness up to control levels in the same dose range it inhibited T-cell mitogenesis. However, cell surface expression of IL-2 receptors was not affected. Short incubations of resting splenic T cells with ClAdo led to a dose-dependent accumulation of cyclic AMP in responding cells. This effect was markedly reduced by the purinergic antagonist 3-isobutyl-1-methylxanthine (IBMX) but was not prevented by the adenosine uptake blocker dipyridamole. ClAdo elicited cAMP accumulation in the same dose range it inhibited T-cell activation events. Extracellular administration of dbcAMP to splenic T cells stimulated by Con A mimicked the effects of ClAdo on T-cell activation parameters, as revealed by a dose-dependent blockade of both IL-2 secretion and IL-2 responsiveness induction, without affecting IL-2 receptor expression. Short incubations of Con A-activated T-cell blasts with ClAdo also led to a dose-dependent accumulation of cAMP. We then analyzed the effect of purines and dbcAMP on IL-2-mediated activated T-cell growth. Purines caused a dose-dependent inhibition of IL-2-mediated T-cell proliferation and ClAdo was the most potent purinergic agonist tested. The effect of ClAdo on Con A-induced T blasts was shifted to the right, if compared to earlier T-cell activation steps. Extracellular addition of dbcAMP mimicked the effect of ClAdo on IL-2-dependent T-cell proliferation, with a marked shift to the right in its inhibitory potency. Concomitant addition of IBMX to activated T-cell cultures competitively antagonized the inhibitory effect of ClAdo on IL-2-dependent proliferation. On the other hand, the IL-2-dependent long-term T-cell line CTL-L was markedly resistant to the inhibitory effect of ClAdo on IL-2-dependent proliferation, although this cell line was still blocked by dbcAMP. Loss of sensitivity to ClAdo in CTL-L cells correlated with loss of the ability of ClAdo to elicit cAMP accumulation. Together, these data indicate that the selective effects of purinergic agonists on T-cell activation and proliferation parameters are carried out through intracellular cAMP as a second messenger.

Estimation of maximal affinities between T-cell receptors and MHC\\peptide complexes

Recognition of peptide\\MHC complexes by T-cell receptors (TCRs) is a critical step for T-cell activation. We studied T-cell activation as a function of this interaction using a mathematical model. Unlike other models analysing TCR-MHC\\peptide interactions, this study takes into account that both TCRs and MHC\\peptide complexes are anchored in membranes and not in solution. The proposed model quantitatively predicts several essential features of antigen-specific T-cell activation, including the experimentally determined rate of TCR-downregulation during peptide-specific T-cell stimulation. In addition, the model offers an explanation as to why the affinity of the TCR for MHC\\peptide complexes is low in general and it correctly predicts the on-rates of the TCR-MHC\\peptide interaction observed in different model systems. Thus, the proposed model predicts key parameters of T-cell activation and offers an explanation for the surprisingly low affinity of the TCR for its antigen.

Immunogenicity of biliary epithelium: Investigation of antigen presentation to CD4+ T cells

The intrahepatic biliary epithelium is susceptible to extensive T-cell- mediated damage during primary biliary cirrhosis, primary sclerosing cholangitis, and hepatic allograft rejection. During these processes, human intrahepatic biliary epithelial cells (HIBEC) become activated and express high levels of the lymphocyte adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and lymphocyte-associated antigen (LFA)-3, and of class II MHC antigens. It follows that activated HIBEC may also play a direct role in the activation of antigen-specific CD4+ T lymphocytes. The capacity of class II MHC antigen-expressing HIBEC to present antigen and induce specific proliferation of CD4+ T cells was examined in this study. Lines of purified HIBEC were activated by culture with the proinflammatory cytokines interferon gamma (IFN-gamma) and tumor necrosis factor a and were mixed in coculture with allogeneic CD4+ T cells. The result of interaction between these cells was assessed by measurement of lymphoproliferation and IL-2 production. Class II MHC antigen-expressing HIBEC failed to induce either lymphoproliferation or IL-2 production. However, both of these parameters of T-cell activation were positive in cocultures when a costimulation signal was delivered to T cells by adding bivalent anti-CD28 antibodies. The antigen-specific activation of these T cells was further enhanced by the addition of a cross-linking secondary antibody that caused CD28 receptor aggregation. The failure of cytokine-stimulated HIBEC to induce T-cell activation is consistent with the observation that HIBEC do not express the costimulatory CD28 ligands B7-1 or B7-2 at either mRNA or protein levels. It may be concluded that HIBEC are unlikely to play a direct role in activation of antigen-specific CD4+ T lymphocytes within the inflamed liver. (Hepatology 1996 Sep;24(3):561-7)

Immunogenicity of biliary epithelium: investigation of antigen presentation to CD4+ T cells.

The intrahepatic biliary epithelium is susceptible to extensive T-cell-mediated damage during primary biliary cirrhosis, primary sclerosing cholangitis, and hepatic allograft rejection. During these processes, human intrahepatic biliary epithelial cells (HIBEC) become activated and express high levels of the lymphocyte adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and lymphocyte-associated antigen (LFA)-3, and of class II MHC antigens. It follows that activated HIBEC may also play a direct role in the activation of antigen-specific CD4+ T lymphocytes. The capacity of class II MHC antigen-expressing HIBEC to present antigen and induce specific proliferation of CD4+ T cells was examined in this study. Lines of purified HIBEC were activated by culture with the proinflammatory cytokines interferon gamma (IFN-gamma) and tumor necrosis factor a and were mixed in coculture with allogeneic CD4+ T cells. The result of interaction between these cells was assessed by measurement of lymphoproliferation and IL-2 production. Class II MHC antigen-expressing HIBEC failed to induce either lymphoproliferation or IL-2 production. However, both of these parameters of T-cell activation were positive in cocultures when a costimulation signal was delivered to T cells by adding bivalent anti-CD28 antibodies. The antigen-specific activation of these T cells was further enhanced by the addition of a cross-linking secondary antibody that caused CD28 receptor aggregation. The failure of cytokine-stimulated HIBEC to induce T-cell activation is consistent with the observation that HIBEC do not express the costimulatory CD28 ligands B7-1 or B7-2 at either mRNA or protein levels. It may be concluded that HIBEC are unlikely to play a direct role in activation of antigen-specific CD4+ T lymphocytes within the inflamed liver.

Cellular and humoral immune responses to well-defined blood stage antigens (major merozoite surface antigen) of Plasmodium falciparum in adults from an Indian zone where malaria is endemic.

Conserved and variant regions of two blood stage vaccine candidate antigens of Plasmodium falciparum, merozoite surface antigen (MSA-1) and ring-infected erythrocyte surface antigen (Pf155/RESA), have been shown to be immunogenic. However, the relative immunogenicity of these immunogens in different populations has not been studied. The conserved N-terminal region of MSA-1 was investigated for its immunogenicity by studying cellular (T cell) and humoral (B cell) immune responses in P. falciparum-primed individuals, living in malaria-hyperendemic areas (Orissa State, India), where malaria presents an alarming situation. MSA-1-derived synthetic peptides contained sequences that activated T cells to proliferate and release gamma interferon in vitro. There was considerable variation in the responses to different peptides. However, the highest responses (51% [18 of 35] by proliferation and 34% [12 of 35] by gamma interferon release) were obtained with a synthetic hybrid peptide containing sequences from conserved N- and C-terminal repeat regions of MSA-1 and Pf155/RESA, respectively. Antibody reactivities in an enzyme immunoassay of plasma samples from these donors to different peptides used for T-cell activation were heterogeneous. In general, there was poor correlation between DNA synthesis and either gamma interferon release or antibody responses in individual donors, underlining the importance of examining several parameters of T-cell activation to assess the total T-cell responsiveness of a study population to a given antigen. However, the results from our studies suggest that synthetic constructs containing sequences from the N- and C-terminal regions of MSA-1 and Pf155/RESA representing different erythrocytic stages of the P. falciparum parasite are more immunogenic in humans living in malaria-hyperendemic areas of India who have been primed by natural infection.

Anti-CD3-induced T-cell activation in vivo—I. Flow cytometric analysis of dose-responsive, time-dependent, and cyclosporin a-sensitive parameters of CD4 + and CD8 + cells from the draining lymph nodes of C57B1/6 mice

An in vivo model to assess the effects of chemicals on T-cell activation has been characterized and validated using the immunosuppressive drug, cyclosporin A (CsA). The dose response and kinetic effects of the hamster anti-mouse monoclonal antibody 145-2C11 (anti-CD3) on various parameters of T-cell activation were examined in cells from the draining popliteal and inguinal lymph nodes of C57B1/6 mice. Parameters of anti-CD3-induced T-cell activation included 3H-TdR incorporation (± recombinant murine IL-2), and flow cytometric analysis of CD3 and IL-2 receptor (IL-2R) expression on CD4 + and CD8 + cells. Increases in the percentage of lymphocyte subsets in the S/G 2M phase of the cell cycle and total cell recovery following anti-CD3 are also reported. Increased 3H-TdR incorporation was maximal over a dose range of 0.25–25 μg anti-CD3, while maximal increases in the percentage of CD4 + and CD8 + cycling occurred after a dose of 2.5 μg anti-CD3. At 24 h after anti-CD3 treatment, CD3 expression on both CD4 + and CD8 + cells was dose dependently down-modulated while IL-2R expression and IL-2 driven 3H-TdR incorporation were dose dependently increased. In addition, total cell recovery increased at 24 h and correlated with an increase in the percentage of B220 + cells present in the lymph nodes. There was a corresponding decrease in the percentage of Thy 1.2 +, CD4 +, and CD8 + cells. No increase in cycling of B220 + cells was observed, suggesting an influx of B220 + cells into the node rather than proliferation. Elevation in 3H-TdR incorporation occurred as early as 4 h after anti-CD3 treatment, while increases in the percentage of CD4 + and CD8 + cells cycling were not apparent until 24 h. At 48 h, the percentage of CD8 + cells cycling doubled while the percentage of CD4 + cells cycling remained constant. Down-modulation of CD3 expression on CD4 + and CD8 + cells was apparent as early as 1 h after treatment with less than 10% of CD4 + and CD8 + cells expressing CD3 by 12 h. Induction of IL-2R expression and IL-2-driven 3H-TdR incorporation was maximal at 12 h after anti-CD3. The immunosuppressive drug, CsA (25, 50, or 100 mg/kg, i.p.) decreased anti-CD3-induced 3H-TdR incorporation. Concurrently, anti-CD3-induced increases in the percentage of CD4 + and CD8 + cells cycling were inhibited by CsA. Likewise, IL-2 responsiveness and IL-2R expression on both T-cell subsets were inhibited by CsA. Two novel effects of CsA were found: (1) CsA increased anti-CD3 down-modulation of CD3 expression on CD4 + and CD8 + cells; and (2) CsA inhibited the anti-CD3-induced increase in the level of CD8 expressed on CD8 + cells. These results suggest that this in vivo model will be useful in determining the mechanism(s) by which chemicals impair T-cell function.

Immunopathology and altered immunity in posterior uveitis in man: a review.

Posterior uveitis is thought to be a T-cell mediated disease since active foci of inflammation, identified in eyes enucleated for the complications of intraocular inflammation, are found to be predominantly composed of CD4+ T-cells. Few B-cells and little immunoglobulin are found suggesting that antibody and immune complex deposition do not play a major role in perpetuating the inflammatory process. As ocular biopsy is not a feasible method for monitoring disease activity and response to treatment, parameters of T-cell activation and retinal damage have been studied in the peripheral blood. These have included antibody and T-cell sensitisation to retinal S-antigen, serum soluble IL-2 receptors and IL-2 receptors on activated T-cells. none of these parameters, however, have been found to be useful in the monitoring of ocular disease activity alone or in the prediction of disease relapse.

Hexadecylphosphocholine-mediated enhancement of T-cell responses to interleukin 2

The effect of low-dose hexadecylphosphocholine (He-PC) on normal peripheral mononuclear cells (PMNC) was studied. Interferon-gamma (IFN-g) production, interleukin 2 (IL-2) receptor, and HLA-DR antigen expression were investigated, representing typical T-cell activation parameters. In PMNC cultures, He-PC dose-dependently enhanced the production of IFN-g, provided IL-2 had been added exogenously. Without IL-2 He-PC was ineffective. In some cultures, at a concentration of 8 μg/ml He-PC stimulated the secretion of IFN-g more than 20-fold compared to untreated controls. Although He-PC by itself lacked mitogenic activity, this compound also stimulated IFN-g production in the presence of suboptimal doses of phytohemagglutinin (PHA). Immunofluorescence studies demonstrated that He-PC also increased IL-2 receptor and HLA-DR antigen expression under these experimental conditions. Taken together, these results indicate that He-PC may possess immunomodulatory activity also in vivo, acting as a costimulator for the IL-2-mediated T-cell activation process.

Production by activated human T cells of interleukin 4 but not interferon-gamma is associated with elevated levels of serum antibodies to activating malaria antigens.

T cells play a crucial role in antibody-mediated and antibody-independent immunity against Plasmodium falciparum malaria. Therefore, a vaccine immunogen should include parasite-derived B- and T-cell epitopes capable of giving rise to protective responses in both systems. The P. falciparum antigen Pf155/ring-infected erythrocyte surface antigen (RESA), a vaccine candidate, contains immunodominant T- and B-cell epitopes located in the central (5') and C-terminal (3') invariant repeat regions of the molecule. To relate Pf155/RESA-peptide-specific responses of T cells to function, T cells from P. falciparum immune donors were activated with peptides corresponding to these immunodominant regions. Activation was measured as induction of interferon-gamma secretion, T-cell proliferation (DNA synthesis), or transcription and translation of interleukin 4 (IL-4) mRNA. Peptides from both regions were shown to induce interferon-gamma, IL-4, proliferation, or any combination. In individual donors, there was no correlation between these different activities. Rather, they were negatively correlated, demonstrating the importance of examining multiple parameters of T-cell activation when estimating the proportion of individuals responding to a given epitope. However, IL-4 mRNA and intracellular IL-4 could be induced in T cells of donors who had elevated concentrations of serum antibodies to the same peptide that was used for T-cell activation. These results suggest that a causal relationship exists between the activation of IL-4-producing T-cell subsets and production of the anti-Pf155/RESA-specific antibodies in individuals in which immunity has been induced by natural infection. This finding has implications that should be considered for the selection of immunogens to be included in a future P. falciparum subunit vaccine and for vaccine development in general.