T cells express a phagocyte-type NADPH oxidase that is activated after T cell receptor stimulation

T cell receptor (TCR) stimulation induces rapid generation of reactive oxygen species, although the mechanisms for this are unclear. Here we found that T cells expressed a functional phagocyte-type nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. TCR crosslinking induced oxidase activation through the recruitment of preformed Fas ligand and Fas. TCR stimulation induced three separable events generating reactive oxygen species: rapid hydrogen peroxide production independent of Fas or NADPH oxidase; sustained hydrogen peroxide production dependent on both Fas and NADPH oxidase; and delayed superoxide production that was dependent on Fas ligand and Fas yet independent of NADPH oxidase. NADPH oxidase-deficient T cells showed enhanced activation of the kinase Erk and a relative increase in T helper type 1 cytokine secretion. Thus, mature T cells express a phagocyte-type NADPH oxidase that regulates elements of TCR signaling.     

Decline in mitogen induced proliferation of lymphocytes with increasing age.

We previously reported that mitogen-induced proliferative responses of lymphocytes in the elderly were significantly lower than in young individuals. To determine when this decline occurs, we evaluated the responses of 26-30 subjects of each decile from the third to the tenth decile to the T cell mitogens, phytohaemagglutinin (PHA) and concanavalin A (ConA), and the T dependent B cell mitogen, pokeweed mitogen (PWM). There was a significant decrease in the responses of the 70-, 80- and 90-year-olds to PHA and ConA (less than 40% of the 20-year-olds; P less than 0.01). The 80- and 90-year-olds also showed a decreased response to PWM (approximately 50%; P less than 0.01). The 60-year-olds showed a decreased response to all three mitogens but only the PHA and ConA responses were significantly decreased (P less than 0.01). The 50-year-olds showed a decreased response to ConA, while the 40-year-olds showed decreased responses to both PHA and ConA; both significant at P less than 0.01. The decreased response of the 40-year-olds, however, was only seen in the females. This may be due to the hormonal changes associated with menopause. The general trend of the data suggests that there is a gradual decrease in mitogen-induced proliferative responses with increasing age, large differences become apparent at the age of 60, with a further decrease in the 70s, and most importantly, they remain fairly constant thereafter. Of interest is that only three of the 111 subjects less than 60 years old failed to mount a proliferative response and in each case this was to only one mitogen, while 42 of 118 subjects greater than 60 years old did not respond to at least one mitogen. Ten of these older subjects (2/28 of the 60-year-olds) did not respond to any of the three mitogens (P less than 0.01). This lack of response may be important since we have found a significant association between the lack of response to all three mitogens and increased mortality.     

Age-related changes of expression of IL-2 receptor subunits and kinetics of IL-2 internalization in T cells after mitogenic stimulation

The age-associated changes of the expression of IL-2 binding molecules p55/Tac(alpha chain) and p70/75(beta chain) were examined after phytohemagglutinin (PHA) stimulation. The expressions of both p55/Tac molecules and p70/75 molecules were significantly reduced in the aged compared with those in the young persons. The amounts of p55/Tac and p70/75 molecules on T cells from the aged were 55% and 59% of those on young ones, respectively. The ratio of the amount of p70/75 to that of p55/Tac in aged T cells was 0.28 and that in young ones was 0.26. The ratio was somewhat higher in the aged but not significantly. We also examined the kinetics of IL-2 internalization mediated by its receptor. The calculated t1/2 of receptor-mediated IL-2 internalization was 17 min in the aged and 16 min in the young, respectively. There was no kinetic difference between the 2 groups. The percentage of the internalized IL-2 to the sum total was 58.2% in the aged and 73.4% in the young (P less than 0.02). the amount of internalized IL-2 in T cells from the aged was 48.6% of that from the young (P less than 0.01).     

Phorbol myristate acetate and calcium ionophore A23187-stimulated human T cells do not express high-affinity IL-2 receptors

Phorbol myristate acetage (PMA) and Ca2+ ionophore A23187 mimic early signal transduction pathways and activate purified human T cells to secrete large quantities of interleukin-2 (IL-2) and to proliferate. Despite producing 50-100-fold more IL-2 than phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL), PMA/A23187-stimulated human T cells proliferate less than cells activated by PHA. Washing the cells to remove PMA/A23187 was found to increase cellular proliferation two to five-fold. High-affinity IL-2R (HA-IL-2R) were found to be expressed by human T cells that had been washed 24 hr after PMA/A23187 stimulation and recultured without stimulus for an additional 48 hr, but not by T cells constantly exposed to PMA/A23187 for 72 hr. Radioligand binding studies with [125I]IL-2 demonstrated that while the alpha (p55) and beta (p70-75) subunits of HA-IL-2R were both present on the constant PMA/A23187-stimulated T cells, they did not appear to associate to form functional HA-IL-2R. This defect in the expression of bio-active HA-IL-2R on constant PMA/A23187-stimulated human T cells seems to account for their low proliferative response.     

Low dose of deoxyguanosine increases IL-2 receptors of IL-2-dependent cultured T cells

Deoxyguanosine ( dGuo ) is a purine nucleoside phosphorylase (PNP) substrate which has been shown to inhibit T lymphoblast growth, PHA-induced cell proliferation and suppressor T cell activity. Low dGuo concentrations (0.5-5 microM) increase interleukin-2 (IL-2) sensitivity of IL-2-dependent cultured T cells (CTC). dGuo alone has no direct mitogenic effect on CTC. The increased IL-2 sensitivity of CTC was more marked in the presence of low IL-2 concentrations than high ones. CTC incubated with dGuo at 37 degrees C for 24 hrs absorbed more exogenous IL-2 than control non-treated CTC. CTC incubated at 4 degrees in the presence of dGuo did not absorb any more IL-2 than control CTC. FACS analysis further showed increased Tac expression on CTC due to dGuo . These findings indicate that a 37 degrees incubation of CTC with dGuo increases the number of CTC IL-2 receptors. dGuo was not found to increase IL-2 production by PHA-stimulated PBM. Therefore, low dGuo concentrations selectively augment the sensitivity of cells responding to IL-2 by increasing IL-2 receptors.     

Detection of lymphokines and lymphokine receptors in pulmonary sarcoidosis. Immunohistologic evidence that inflammatory macrophages express IL-2 receptors.

In an investigation of the immunopathogenesis of sarcoidosis, the authors undertook the tissue localization of those lymphokines and lymphokine receptors which are known to play a central role in T-cell and macrophage activation. Using monoclonal antibodies and an ABC immunoperoxidase technique. They determined the distribution of gamma interferon (IFN-g), interleukin-2 (IL-2), and corresponding IL-2 receptors (IL-2R), plus T cells and T cell subsets, B cells, and macrophages within thoracic lymph nodes and lung specimens of 9 patients with active pulmonary sarcoidosis. Epithelioid and multinucleate giant cells within sarcoid granulomas of all specimens showed membrane labeling for IL-2R and IFN-g, in addition to IL-2, suggesting that these cells indeed express functional IL-2 receptors. Infiltrating T cells, largely T4+, were also IL-2R+, and many showed IL-2 and IFN-g labeling. By comparison, macrophages within sections of normal lung or lymph node failed to stain for IL-2, IL-2R, or IFN-g. These immunohistologic studies extend recent in vitro observations by these authors and others that normal human blood monocytes and alveolar macrophages are induced by IFN-g or IL-2 to express functional membrane-bound IL-2 receptors. The in vivo expression of IL-2R by mononuclear phagocytes in pulmonary sarcoidosis is demonstrated, and a new role is suggested for T-cell-derived lymphokines in macrophage activation.     

Normal and certain leukaemic B cells express IL-2 receptors without in vitro activation.

IL-2 receptor expression by B cells has previously been considered to be confined to activated normal B cells and, among the B cell leukaemias, to the hairy cells (HC) of hairy-cell leukaemia. In the present paper, using alpha-Tac monoclonal antibodies in a highly sensitive indirect rosette method, we show that both normal and certain leukaemic B cells other than HC express IL-2 receptors. The density of these receptors is low since they were not detectable by indirect immunofluorescence. Various controls excluded non-specific-reagent or exogenous receptor binding and blocking studies with recombinant IL-2 confirmed the presence of the IL-2 receptors. The significance of the findings is discussed and it is suggested that B cell IL-2 receptor expression without in-vitro activation may be a function of B cell maturity.     

Interleukin production by human colostral cells after in vitro mitogen stimulation

Human colostral cells were pulsed with PHA, Con A, or LPS and cultivated in serum-free medium. The culture supernatants were tested for IL-1 activity in C3H/HeJ thymocyte assay and for IL-2 activity on human lymphoblasts. The IL-1 activity was the highest at the 24th h of cultivation and IL-2 activity at the 48th h of cultivation.     

Suppression by Trypanosoma brucei rhodesiense of the capacities of human T lymphocytes to express interleukin-2 receptors and proliferate after mitogenic stimulation.

We studied the suppressive effects induced in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) by purified blood forms of Trypanosoma brucei rhodesiense. The parasite was found to markedly impair lymphocyte proliferation (measured in terms of [3H]thymidine incorporation). The extent of this effect increased with parasite concentration and was not due to mitogen absorption, depletion of medium nutrients, or PBMC killing by the parasite. Significant reductions in interleukin-2 receptor (IL-2R) expression, determined by flow cytometric analysis, were also observed in PHA-stimulated PBMC cultured in the presence of T. b. rhodesiense as evidenced by marked decreases in the surface density of the receptor. Concomitant decreases in the percentage of IL-2R+ cells were recorded in approximately half of the experiments. A discrete, dimly stained subpopulation of IL-2R+ cells were consistently demonstrable whether or not a reduction in the percentage of IL-2R+ cells occurred. Living, but not glutaraldehyde-fixed, parasites suppressed IL-2R expression. In kinetic studies, a low but reproducible level of suppression of IL-2R was demonstrable as early as 6 h after PHA stimulation; the extent of this effect became considerably more pronounced as additional culture time elapsed. Levels of IL-2 biological activity in cocultures of T. b. rhodesiense with PHA-stimulated PBMC were comparable with or higher than those present in control cultures lacking the parasite. Therefore, insufficient levels of this cytokine would be an unlikely explanation for the noted suppression of IL-2R expression and lymphoproliferation. These effects of T. b. rhodesiense could represent an important component of the mechanism by which immunosuppression develops in African sleeping sickness.     

Human mast cells express receptors for IL-3, IL-5 and GM-CSF; a partial map of receptors on human mast cells cultured in vitro

BACKGROUND: Mast cells have long been recognized as the principal cell type that initiates the inflammatory response characteristic of acute allergic type 1 reactions. Our goal has been to further characterize maturation of progenitors to mast cells. METHODS: Mast cells were cultured from human cord blood derived CD133(+) progenitors. Mast cell function was tested using histamine release. During differentiation mast cells surface marker expression was monitored by flow cytometry. RESULTS: CD133(+) progenitors expressed the early haematopoietic and myeloid lineage markers CD34, CD117, CD13 and CD33. Mature mast cells expressed CD117, CD13 and CD33, and expression of the high affinity immunoglobulin E receptor FcepsilonRI increased during culture. Cytokine receptors interleukin (IL)-5R, IL-3R, granulocyte-macrophage-colony stimulating factor (GM-CSF)R and IL-18R were expressed at high levels during maturation. Chemokine receptors CXCR4 and CXCR2 were highly expressed on both newly purified CD133(+) cells and mature cells. CONCLUSION: Human mast cells can be cultured from a CD34(+)/CD117(+)/CD13(+)/CD33(+) progenitor cell population in cord blood that is tryptase and chymase negative. Developing and mature mast cells express a wide range of chemokine and cytokine receptors. We found high levels of expression of CD123, IL-5R and GM-CSF receptors, also found on eosinophils and basophils, and high levels of expression of the receptor for the inflammatory cytokine IL-18.     

Functional double-negative T cells in the periphery express T cell receptor Vβ gene products that cause deletion of single-positive T cells

A proportion of peripheral T cells lack surface expression of the CD4 or CD8 coreceptor molecules and hence are designated as “double negative” (DN). Most DN T lymphocytes express the Γ/β T cell receptor (TcR), but a minor fraction of them, in both humans and mice, express the α/β TcR. Whereas α/β⁺ DN T lymphocytes are infrequent (< 1%) in conventional lymphoid organs (spleen, blood, lymph node), they account for two-thirds of the T cells residing in adult bone marrow. Analysis of the TcR Vβ repertoire expressed by peripheral DN T cells revealed a high frequency of cells bearing autoreactive TcR that cause deletion of “single-positive” (SP) (CD4⁺CD8^? or CD4^?CD8⁺) T cells. Peripheral DN cells thus represent a cell type that is relatively resistant to clonal deletion. Furthermore, such cells have not been inactivated (anergized) in vivo since they proliferate and secrete interleukins in response to cross-linking by monoclonal antibodies specific for these Vβ gene products that are deleted in SP T cells. These results might help to understand the association of peripheral expansion of DN cells and development of autoimmune diseases.     

Helper interleukins are produced by both CD4 and CD8 splenic T cells after mitogen stimulation.

We have earlier described (Cardell, S. and Sander, B., Eur. J. Immunol. 1990. 20:389) mitogen-induced production of interleukin (IL)2, IL4 and IL5 mRNA by murine spleen cells, analyzed by in situ hybridization. In the present study we have investigated the potential of CD8 T cells to produce these interleukins, normally associated with the helper function of CD4 T cells. When concanavalin A (Con A)-activated spleen cells were restimulated with Con A and phorbol 12-myristate 13-acetate (PMA), higher levels of IL2, IL4 and IL5 mRNA were induced, as detected both by increased frequencies of positive cells, and by more mRNA per cell. Four-to-six-day Con A blasts were enriched for CD4+ or CD8+ T cells, and restimulated with Con A and PMA. Both CD4 and CD8 cells were found to produce all three kinds of mRNA when restimulated. The frequencies of IL2 mRNA-containing CD8 cells were half of those found for CD4 cells (3.5% as compared to 7%). On the average 1% of the CD8 cells were induced to produce IL4 and IL5 mRNA, while 9% and 3% of the activated CD4 cells contained IL4 and IL5 mRNA, respectively. CD4 and CD8 cells displayed different sensitivities to the reagents when tested alone. Con A induced the synthesis of IL4 and IL5 in CD4 cells, but not CD8 cells, independently of PMA. PMA alone induced extensive thymidine incorporation in CD8 cells, but not in CD4 cells, in the absence of detectable lymphokine mRNA. The results suggest that some CD8 cells have the capacity to give help in immune responses, by secretion of IL2, IL4 and IL5.     

Chimeric antigen receptor-modified human regulatory T cells that constitutively express IL-10 maintain their phenotype and are potently suppressive

Clinical trials of Treg therapy in transplantation are currently entering phases IIa and IIb, with the majority of these employing polyclonal Treg populations that harbor a broad specificity. Enhancing Treg specificity is possible with the use of chimeric antigen receptors (CARs), which can be customized to respond to a specific human leukocyte antigen (HLA). In this study, we build on our previous work in the development of HLA-A2 CAR-Tregs by further equipping cells with the constitutive expression of interleukin 10 (IL-10) and an imaging reporter as additional payloads. Cells were engineered to express combinations of these domains and assessed for phenotype and function. Cells expressing the full construct maintained a stable phenotype after transduction, were specifically activated by HLA-A2, and suppressed alloresponses potently. The addition of IL-10 provided an additional advantage to suppressive capacity. This study therefore provides an important proof-of-principle for this cell engineering approach for next-generation Treg therapy in transplantation.     

Characterization of Thy 1.2 positive cells in mouse bone marrow by sedimentation and mitogen stimulation.

By the use of unit gravity velocity sedimentation it was found that the majority of Thy 1.2 positive cells in the bone marrow of BALB/c mice sedimented in the same fractions as small lymphocytes of the marrow. This was shown both in normal, neonatally thymectomized and congenitally athymic mice. In all three groups of mice, two populations of Thy 1.2 positive cells were found. This indicates that these cells are cycling in the bone marrow. Long-lived T cells of normal bone marrow were included in the slowly sedimenting Thy 1.2 positive population (peak at 3 mm/h). Results after stimulation of bone-marrow cells with phytohaemagglutinin or concanavalin A indicated that the majority of Thy 1.2 positive cells in the bone marrow of thymus-deprived mice are effete end-products.     

HL-1 cells express an inwardly rectifying K+ current activated via muscarinic receptors comparable to that in mouse atrial myocytes

An inwardly rectifying K⁺ current is present in atrial cardiac myocytes that is activated by acetylcholine (I_KACh). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gβγ G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrial-derived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbachol-activated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPγS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch.     

小鼠CD4+T细胞活化后IL-10受体I表达的研究

目的:检测抗小鼠CD4+T淋巴细胞活化后表面白细胞介素10受体I(IL-10R1)表达变化,为进一步研究白细胞介素10(IL-10)相关免疫疾病的发病机制及进展提供有力支持.方法:取C57BL/6小鼠脾细胞,溶血后采用尼龙毛去除B淋巴细胞后,流式细胞仪无菌分选CD4+T淋巴细胞,利用anti-CD3和anti-CD28多克隆抗体刺激分选后细胞,分别在第0、12、24、48和72小时流式细胞术检测IL-10R1表达情况.结果:0小时时小鼠CD4+T淋巴细胞不表达IL-10R1,随着刺激时间的延长IL-10R1表达增加,24小时时达到最高,随后表达水平开始下降,72小时时表达情况与0小时基本相同.结论:CD4+T淋巴细胞活化后IL-10R1表达短暂,以24小时时表达最高.     

The CD4+ T cell immunodominant Anaplasma marginale major surface protein 2 stimulates gammadelta T cell clones that express unique T cell receptors

Major surface protein 2 (MSP2) of the bovine rickettsial pathogen Anaplasma marginale is an abundant, serologically immunodominant outer membrane protein. Immunodominance partially results from numerous CD4+ T cell epitopes in highly conserved amino and carboxy regions and the central hypervariable region of MSP2. However, in long-term cultures of lymphocytes stimulated with A. marginale, workshop cluster 1 (WC1)+ gammadelta T cells and CD4+ alphabeta T cells proliferated, leading to a predominance of gammadelta T cells. As gammadelta T cells proliferate in A. marginale-stimulated lymphocyte cultures, this study hypothesized that gammadelta T cells respond to the abundant, immunodominant MSP2. To test this hypothesis, gammadelta T cell clones were isolated from MSP2 vaccinates and assessed for antigen-specific proliferation and interferon-gamma secretion. Seven WC1+ gammadelta T cell clones responded to A. marginale and MSP2, and three of these proliferated to overlapping peptides from the conserved carboxy region. The gammadelta T cell response was not major histocompatibility complex-restricted, although it required antigen-presenting cells and was blocked by addition of antibody specific for the T cell receptor (TCR). Sequence analysis of TCR-gamma and -delta chains of peripheral blood lymphocytes identified two novel TCR-gamma chain constant (Cgamma) regions. It is important that all seven MSP2-specific gammadelta T cell clones used the same one of these novel Cgamma regions. The TCR complementarity-determining region 3 was less conserved than those of MSP2-specific CD4+ alphabeta T cell clones. Together, these data indicate that WC1+ gammadelta T cells recognize A. marginale MSP2 through the TCR and contribute to the immunodominant response to this protein.     

Defective expression of high affinity IL-2 receptors on activated T cells from aged humans

The proliferative response of T cells from aged humans to a number of mitogens is significantly reduced. We report here that there is a decrease in high affinity IL-2 receptor (IL-2R) expression on activated T cells from aged humans. Scatchard analysis of the binding of [125I]IL-2 demonstrates fewer high affinity IL-2 binding sites. Autoradiographic techniques demonstrate that this results from there being fewer activated T cells from old as compared to young donors that express high affinity IL-2R. However, T cells from old donors that do not express high affinity IL-2 binding sites express both the IL-2 binding 55 and 75 kd chains. Thus, although the two IL-2 binding peptides are expressed on activated T cells from old donors, expression of the high affinity IL-2R is reduced. This may explain the decreased ability of T cells from old donors to respond to IL-2. The impaired ability of activated T cells from old donors to express high affinity IL-2R while expressing the 55 and 75 kd chains may provide insights into the mechanisms of IL-2 interactions with its receptor.     

Pathogenic autoantibody-inducing gamma/delta T helper cells from patients with lupus nephritis express unusual T cell receptors.

In previous work, we found that only 59 (15%) of 396 "autoreactive" T cell clones derived from five patients with lupus nephritis had the ability to selectively augment the production of pathogenic anti-DNA autoantibodies and the majority (49/59) of those autoimmune T helper (Th) clones were CD4+. Surprisingly, 7 of those Th clones were CD4-/CD8- and gamma/delta TCR+, capable of augmenting the production of pathogenic anti-DNA autoantibodies up to 125-fold. The gamma/delta Th clones responded in a MHC-nonrestricted manner to some endogenous autoantigen associated with heat shock proteins (HSP60) on the lupus B cells. The gamma/delta TCR genes expressed by 4 of these Th clones were amplified and sequenced here. Three of the 4 Th clones, each from a different lupus patient, expressed a gene from the V gamma 1 subgroup. Moreover, 2 of the Th clones expressed V delta 5, and the others V delta 1 or V delta 3. These TCRs are rarely expressed by peripheral blood gamma/delta T cells of normal adult humans. The predominant gamma/delta T cells in human peripheral blood express V gamma 2 (V gamma 9) and V delta 2 TCR genes, including HSP-responsive T cells. None of the lupus Th clones expressed this combination of TCR genes. In addition, some of these pathogenic autoantibody-inducing Th clones from the lupus patients had limited diversity and few N-nucleotide additions in their gamma/delta TCR junctional regions (CDR3), thus resembling fetal gamma/delta thymocytes early in ontogeny.