Impaired Interleukin-2 Production Research Articles

Altered Maturation Status and Possible Immune Exhaustion of CD8 T Lymphocytes in the Peripheral Blood of Patients Presenting With Acute Coronary Syndromes.

Inflammation in response to oxidized lipoproteins is thought to play a key role in acute coronary syndromes (ACS), but the pattern of immune activation has not been fully characterized. We sought to perform detailed phenotypic and functional analysis of CD8 T lymphocytes from patients presenting with ACS to determine activation patterns and potential immunologic correlates of ACS. We used polychromatic flow cytometry to analyze the cytokine production profiles of naïve, effector, and memory CD8 T cells in patients with ACS compared with control subjects with stable coronary artery disease. ACS was associated with an altered distribution of circulating CD8(+) T-cell maturation subsets with reduced proportions of naïve cells and expansion of effector memory cells. ACS was also accompanied by impaired interleukin-2 production by phenotypically naïve CD8 T cells. These results were validated in a second replication cohort. Naïve CD8 cells from patients with ACS also had increased expression of programmed cell death-1, which correlated with interleukin-2 hypoproduction. In vitro, stimulation of CD8 T cells with oxidized low-density lipoprotein was sufficient to cause programmed cell death-1 upregulation and diminished interleukin-2 production by naïve CD8 T cells. In this exploratory analysis, naïve CD8(+) T cells from patients with ACS show phenotypic and functional characteristics of immune exhaustion: impaired interleukin-2 production and programmed cell death-1 upregulation. Exposure to oxidized low-density lipoprotein recapitulates these features in vitro. These data provide evidence that oxidized low-density lipoprotein could play a role in immune exhaustion, and this immunophenotype may be a biomarker for ACS.

OP0104 Low-Dose Interleukin-2 Therapy Caused Selective Expansion of Regulatory T Cells Together with RAPID Reduction of Disease Activity in A Patient with Severe Refractory Sle

BackgroundSLE is characterized by the breakdown of immune tolerance towards nuclear autoantigens. Aberrant activation of autoreactive T and B cells leads to uncontrolled inflammation and production of autoantibodies ultimately resulting...

A variable degree of autoimmunity in the pedigree of a patient with type 1 diabetes homozygous for thePTPN22 1858T variant

We investigated whether the PTPN22 C1858T polymorphism is associated with the autoimmune conditions present in the family of a child affected by type 1 diabetes (T1D) carrying the TT genotype (index patient) and the potential immunological effect of the variant. We found that nine family members carried the CT genotype and five suffered from autoimmunity. Interestingly, anti-ZnT8 antibodies were detected in T1D patients and in three healthy relatives. In the TT patient, we showed diminished T-cell proliferation and reduced interleukin-2 (IL-2) and interferon-gamma (IFN-γ) production. A marked reduction of IL-2 was also observed for all CT relatives with autoimmunity and a lack of IFN-γ production was observed for the younger brother of the index patient, heterozygous for the polymorphism. In this family, the C1858T variant might confer a high risk of autoimmunity. Moreover, our data confirm that impaired IL-2 production upon T-cell receptor stimulation is associated with autoimmunity in the carriers of the polymorphism. This study might prompt to extend the panel of risk markers in relatives of subjects affected by T1D.

Impaired Production of Interleukin-2(IL2) in Patients with Graves' Disease by Newly Developed IL2 Radioimmunoassay

We have developed a sensitive, reproducible and specific radioimmunoassay for human interleukin-2. Using 125I-labeled interleukin-2 and polyclonal rabbit antisera raised against recombinant human interleukin-2, a competitive inhibition assay was described which could detect 1 U/ml of human interleukin-2. Substances such as interleukin-1 alpha, interferon beta, nerve growth factor, tissue necrotizing factor, various hormones, peptides and lectins did not affect the assay. Interleukin-2 was measured in supernatants of culture media of stimulated human blood mononuclear cells. Kinetics of interleukin-2 production in seven normal lymphocytes revealed that in both PHA- and Con A-stimulations, the peak levels of interleukin-2 were seen at the end of 72 hours (113.9 +/- 54.4 U/ml, 111.6 +/- 37.3 U/ml, respectively) and then declined. Interleukin-2 levels in PHA- and Con A-stimulations of untreated Graves' disease were significantly lower (14.5 +/- 15.5 U/ml, 12.3 +/- 12.7 U/ml, respectively) than normal controls. However, the improvement of decreased interleukin-2 production in methimazole-treated patients with Graves' disease was observed (38.2 +/- 28.1 U/ml, 48.0 +/- 35.6 U/ml, respectively). The present study demonstrates the usefulness of quantitating human interleukin-2 produced by human blood mononuclear cells and that there exists an impaired production of interleukin-2 in Graves' disease.

Interleukin-2 production is altered by copper deficiency.

Copper is an essential nutrient for optimal function of the immune system; deficiency results in impairment of both humoral and cell-mediated components. Copper deficiency in rodents results in decreased numbers of CD4+ (helper) and total T cells. This defect has been traced to impaired production of interleukin-2, a cytokine essential for T-cell division and differentiation. Impairment of quiescent cell proliferation is reversed by both in vivo and in vitro copper supplementation.

MTOC translocation modulates IS formation and controls sustained T cell signaling

The translocation of the microtubule-organizing center (MTOC) toward the nascent immune synapse (IS) is an early step in lymphocyte activation initiated by T cell receptor (TCR) signaling. The molecular mechanisms that control the physical movement of the lymphocyte MTOC remain largely unknown. We have studied the role of the dynein–dynactin complex, a microtubule-based molecular motor, in the process of T cell activation during T cell antigen–presenting cell cognate immune interactions. Impairment of dynein–dynactin complex activity, either by overexpressing the p50-dynamitin component of dynactin to disrupt the complex or by knocking down dynein heavy chain expression to prevent its formation, inhibited MTOC translocation after TCR antigen priming. This resulted in a strong reduction in the phosphorylation of molecules such as ζ chain–associated protein kinase 70 (ZAP70), linker of activated T cells (LAT), and Vav1; prevented the supply of molecules to the IS from intracellular pools, resulting in a disorganized and dysfunctional IS architecture; and impaired interleukin-2 production. Together, these data reveal MTOC translocation as an important mechanism underlying IS formation and sustained T cell signaling.

Mechanism of a lymphocyte abnormality associated with HLA-B8/DR3: role of interleukin-1

Lymphocytes from normal individuals with the histocompatibility antigens HLA-B8 and DR3 have impaired proliferative responses when stimulated with suboptimal concentrations of mitogens. We have previously shown that an important factor in the impaired response is a failure to produce normal quantities of interleukin-2 (IL-2). To examine the mechanism of decreased responsiveness further, we measured interleukin-1 (IL-1) production of low responder subjects compared with controls. The peripheral blood mononuclear cells of five low responder individuals with HLA-B8/DR3 stimulated with 0.05 micrograms/ml of phytohaemagglutinin (PHA) accumulated only 0.036 U/ml of IL-1 compared with 0.32 U/ml for normal responders. There was a highly significant correlation between the PHA-stimulated IL-1 concentration at 12 h and the subsequent IL-2 concentration at 48 h(r = 0.89, P less than 0.0001) suggesting a role of decreased IL-1 production in the impaired response. A study of unfractionated or column-fractionated culture supernatants revealed no evidence that the decreased IL-1 activity in the supernatants of low responder subjects was related to increased IL-1 inhibitor concentrations. These results suggest that impaired IL-2 production and lymphocyte proliferation in healthy subjects with HLA-B8/DR3 may be mediated at least in part by decreased IL-1 production, and implicates a defect of a very early event in lymphocyte activation.

A Novel ERK-dependent Signaling Process That Regulates Interleukin-2 Expression in a Late Phase of T Cell Activation

Engagement of the T cell antigen receptor (TCR) rapidly induces multiple signal transduction pathways, including ERK activation. Here, we report a critical role for ERK at a late stage of T cell activation. Inhibition of the ERK pathway 2-6 h after the start of TCR stimulation significantly impaired interleukin-2 (IL-2) production, whereas the same treatment during the first 2 h had no effect. ERK inhibition significantly impaired nuclear translocation of c-Rel with a minimum reduction of NF-AT activity. Requirement for sustained ERK activation was also confirmed using primary T cells. To induce sustained activation of ERK, T cells required continuous engagement of TCR. Stimulation of T cells with soluble anti-TCR antibody resulted in activation of ERK lasting for 60 min, but failed to induce IL-2 production. In contrast, plate-bound anti-TCR antibody activated ERK over 4 h and induced IL-2. Furthermore, T cells treated with soluble anti-TCR antibody produced IL-2 when phorbol 12-myristate 13-acetate, which activates ERK, was present in the culture medium 2-6 h after the start of stimulation. Together, the data demonstrate the presence of a novel activation process following TCR stimulation that requires ERK-dependent regulation of c-Rel, a member of the NF-kappaB family.

P59 fyn is upregulated in anergic CD8 + T cells

The ultimate goal in clinical transplantation is achievement of graft tolerance. Despite long-term immunosuppression, alloantigens on transplants elicit alloresponses that can initiate organ rejection. Acute rejection is mediated by CD8 + cytotoxic T cells, whereas chronic rejection is a result of many factors including non-immunological events. The aim of this study was to examine the molecular requirements of T cell anergy, a cellular state that is an integral component of tolerance in vivo. In vitro, the tolerant state is usually best represented by T cell anergy, which is defined by loss of the ability of T cells to produce and secrete interleukin-2 upon restimulation. In the literature, molecular changes in anergic CD4 + T cells have been studied in great detail, but only little is known about functional and biochemical characteristics of anergic CD8 + T lymphocytes. In this study, we demonstrate, that CD8 + T cells are rendered anergic by TCR stimulation without costimulation. They exhibit impaired interleukin-2 production and tyrosine-phosphorylation, but markedly upregulated p59 fyn expression, which could be shown to be an early event during anergization. Anergic CD8 + T lymphocytes show elevated surface expression of early activation markers as well as costimulatory molecules, especially that of CTLA4. These results, are an important component for the discovery of potential molecular targets, which contribute to the development and maintenance of tolerance.

Influence of antigen dose and costimulation on the primary response of CD8+ T cells in vitro.

The influence of costimulation on the primary response of CD8+ T cells to class I alloantigens was studied with the aid of a T cell receptor transgenic model and defined peptides as antigen. With small doses of antigen, the proliferative response of CD8+ cells was high early in culture but was of brief duration and declined to low levels by day 4; this abbreviated response was associated with limited production of interleukin 2 (IL-2) and was strongly dependent upon costimulation via CD8-major histocompatibility complex class I and CD28-B7 interactions. The response to large doses of antigen was quite different in two respects. First, large doses of antigen inhibited the early (day 3) proliferative response but caused a marked elevation of the response late in culture (day 5); these altered kinetics were associated with increased production of IL-2. Second, the initial proliferative response to large doses of antigen did not require costimulation: indeed, blocking costimulation with CTLA4lg or anti-CD8 monoclonal antibody enhanced the early proliferative response. However, blocking costimulation impaired IL-2 production and prevented the late proliferative response. These findings indicate that the requirement for costimulation of T cells can be partly overcome by increasing the dose of antigen to a high level. However, costimulation plays a key role in prolonging the response, presumably by triggering strong and sustained production of IL-2.

Interleukin 2 induces CD8+ T cell-mediated suppression of human immunodeficiency virus replication in CD4+ T cells and this effect overrides its ability to stimulate virus expression.

The nonlytic suppression of human immunodeficiency virus (HIV) production from infected CD4+ T cells by CD8+ lymphocytes from HIV-infected individuals is one of the most potent host-mediated antiviral activities observed in vitro. We demonstrate that the pleiotropic cytokine interleukin 2 (IL-2), but not IL-12, is a potent inducer of the CD8+ HIV suppressor phenomenon. IL-2 induces HIV expression in peripheral blood or lymph node mononuclear cells from HIV-infected individuals in the absence of CD8+ T cells. However, IL-2 induces CD8+ T cells to suppress HIV expression when added back to these cultures, and this effect dramatically supersedes the ability to IL-2 to induce HIV expression. Five to 25 times fewer CD8+ cells were required to obtain comparable levels of inhibition of viral production if they were activated in the presence of IL-2 as compared with IL-12 or no exogenous cytokine. Furthermore, IL-2 appeared either to induce a qualitative increase in HIV suppressor cell activity or to increase the relative frequency of suppressor cells in the activated (CD25+) CD8+ populations. Analyses of proviral levels in peripheral blood mononuclear cells suggest that CD8+ T cell-mediated lysis of in vivo infected cells is not induced by IL-2. These results have implications for our understanding of the effects of impaired IL-2 production during HIV disease as well as the overall effects of IL-2-based immunotherapy on HIV replication in vivo.

Mechanism of the impaired T-cell proliferation in adult rats exposed to alcohol in utero

Although attempts have been made to assess the effect of ethanol on the immune responses in individuals with fetal alcohol syndrome, there is no consensus as to the effect of ethanol on the immune system. Evidence that fetal alcohol-exposed (FAE) humans and animals have diminished proliferative response of T-cells to mitogenic lectins is well established. However, little is known about the mechanism of a toxic effect of ethanol on T-cell growth. Thus, a rat model was used to delineate the mode of ethanol action on T-cell proliferation. We found that the diminished T-cell proliferation in young adult FAE rats was due to a decreased responsiveness to interleukin 2 (IL2), but not to an impaired production of IL2 and expression of IL2 receptors (IL2R). Furthermore, the decreased proliferative response did not result from the presence of an excessive suppressor T-cell activity. Measurements of [Ca +2] i and T-cell proliferation were concurrently performed in batches of cells from the same animals. It was demonstrated that an increase in [Ca +2] i induced by Concanavalin A (Con A) in T-cells from FAE rats was not impaired, although the T-cell proliferation induced by Con A was significantly diminished. The results of the IL2-binding study showed that the K d values and the number of both high- and low-affinity IL2R binding sites on the T-cells of FAE rats were comparable to those of pair-, or chow-fed rats. Finally, the results of the kinetics and rate of the internalization of IL2 showed that (1) the amount of the internalized IL2 was significantly reduced in T-cells from FAE rats, and (2) the half-time (t 1 2 ) for dissociation of IL2 from the receptors in the T-cells from FAE rats was also greater than that of the control rats. These results taken together indicate that ethanol suppresses T-cell proliferation by interfering with events following the IL2-IL2R interaction.

Transfusion of recombinant human erythropoietin-induced autologous blood and peri-operative changes in production of interleukin-2 in esophageal surgery

Surgical stress-induced and allogeneic blood transfusion-induced immunosuppression may promote the rapid regrowth of residual minimum cancer and may accelerate metastasis to distant sites; it is important to develop countermeasures to protect against such post-operative immunosuppression. The effects of recombinant human erythropoietin (rH-EPO)-induced autologous blood donation and transfusion were examined in 14 patients with esophageal cancer, and the peri-operative changes in production of interleukin-2 (IL-2) were analyzed and compared to those of 11 control patients. Pre-operative blood was donated by all 14 rH-EPO-treared patients; the mean volume of blood donated was 824 ml. Allogeneic blood transfusion was performed in only 2 of the 14 patients, whereas it was necessary in 8 of 11 patients in the control group. The administration of rH-EPO resulted in a pre-operative trend towards increased production of IL-2 and the rapid recovery from impaired post-operative production of this cytokine. Our results indicate that preoperative administration of rH-EPO contributes both to the avoidance of allogeneic blood transfusion and to rapid recovery from post-operative impaired production of IL-2 after esophageal surgery.

The liver of MRL/ lpr mice contains defective accessory cells and a population of immunosuppressive lymphocytes

Immunoregulatory abnormalities in the MRL/ lpr mouse strain include activation of macrophages and hepatic natural killer cells, spontaneous production of tumor necrosis factor, defective oral tolerance, and impaired production of interleukin-2. Because the liver is the major organ responsible for the clearance, degradation, and presentation of foreign antigens from the gastrointestinal tract, we have investigated antigen presentation activity of hepatic nonparenchymal cells (NPC) from MRL/ lpr, MRL/++, and C3H/HeN female mice in the primary immune response as measured by stimulation of allogeneic one-way mixed lymphocyte response (MLR), and allogeneic cell-mediated lympholysis (CML). Whereas adherent NPC from C3H/HeN, MRL/++, and young MRL/ lpr mice were effective stimulators, NPC from MRL/ lpr mice older than 9 weeks were defective stimulators of both MLR and CML responses. This abnormality was not observed in splenic accessory cells from these mice. Moreover, a population of hepatic NPC from older MRL/ lpr mice are immunosuppressive: mixing of MRL/ lpr NPC with splenic stimulators from MRL/++ mice profoundly inhibited primary allogeneic CML responses. The inhibitory hepatic nonparenchymal cell population was nonadherent, radioresistant and was removed by pretreatment with antibodies to either asialoAGM-1 or Lyt-2 plus complement. This inhibition was not observed with the addition of MRL/++ NPC or supernates from cultured MRL/ lpr NPC. These findings suggest a selective organ-specific and age-dependent impairment of antigen presentation and the presence of an immunosuppressive lymphocyte population in the liver of MRL/ lpr mice which may contribute to the autoimmune process.

Impaired interleukin-2 production by T-lymphocytes in polycythemia vera.

T-lymphocyte function was investigated in a group of eight patients with polycythemia vera (PV) and 13 normals. Responsiveness of T-cells to stimulation by mitogen was significantly impaired in the PV patients (p = .02). Analysis of immune cell subpopulation composition by assessment of cell surface phenotype did not reveal imbalances that could account for impaired T-cell proliferative ability in PV patients. Addition of recombinant human interleukin-1 to PV mononuclear cell cultures did not enhance proliferative ability. Addition of recombinant human interleukin-2 to cultures had no effect on maximum mitogen stimulation in normals but significantly improved responses in PV patients (p less than .02), correcting them to normal levels. Measurement of interleukin-2 production by stimulated cultured mononuclear cells confirmed subnormal IL-2 production by PV T-cells (p less than .00005). Impaired IL-2 production could conceivably contribute to the pathogenesis of PV by limiting the ability of IL-2-dependent cells to regulate hematopoiesis.

Defect in lectin-induced interleukin 2 production by peripheral blood lymphocytes of patients with invasive urinary bladder carcinoma

The production of interleukin 2 (IL-2) by phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) from 21 patients with transitional cell carcinoma of the urinary bladder (BTCC) and 16 control blood donors was measured with a solid phase enzyme immunoassay based on the dual antibody immunometric sandwich principle. PBMC from patients with invasive BTCC (grade III–IV) showed a defect in the production of IL-2. The concentration of IL-2 in the supernatants of PBMC cultures from these patients was substantially lower (0.4 ± 0.1 U/ml) than that observed in the supernatants of PBMC cultures from patients with non-invasive BTCC, grade II (1.5 ± 0.7 U/ml), and from tumour-free controls (1.4 ± 0.8 U/ml). These results suggest an immune dysfunction based on quantitatively impaired IL-2 production in patients with invasive BTCC and indicate that exogenous IL-2 could be used as an immunological response modifier for the treatment of these patients.

Alteration of Lymphocyte Functions by 8-Methoxypsoralen and Long-Wave Ultraviolet Radiation. II. The Effect of In Vivo PUVA on IL-2 Production

Our previous studies demonstrated that psoralen plus long-wave ultraviolet radiation (PUVA) treatment inhibited certain T-lymphocyte functions, such as locomotive ability. To further analyze the effects of PUVA on T-lymphocyte function, we investigated the ability of mouse spleen cells to produce interleukin 2 (IL-2) after treatment of the mice in vivo with PUVA. Interleukin 2 production was impaired in cells from PUVA-treated mice compared with those from UVA-irradiated, 8-methoxypsoralen-treated, or normal mice. This impairment was not dose dependent, over the dose range of UVA (2-20 J/cm2) examined. Interleukin 2 production was markedly suppressed on day 3 after PUVA and returned to normal by day 7 after the treatment. Topical treatment of the mice with croton oil did not affect IL-2 production of their spleen cells. This result indicates that cutaneous inflammation per se may not be responsible for the suppressive effect of PUVA on IL-2 production. Addition of exogenous IL-1 did not reconstitute the decreased ability of spleen cells to produce IL-2 in vitro, indicating that PUVA affected primarily IL-2 producing cells. These suggest that impaired IL-2 production may account for some of the immune dysfunction observed in PUVA-treated animals.

Effect of human recombinant interleukin (IL-2) on peripheral blood mononuclear cells of burned patients

Interleukin-2 (IL-2) has been suggested to play a key role in the immune response. To investigate the effect of human recombinant IL-2 on proliferative and cytotoxic responses in burn patients. peripheral blood mononuclear cells from 20 severely burned patients (body surface area (BSA)>20 per cent) were studied in this way. Proliferative response measured by tritiated thymidine uptake was impaired from week 2 following injury. IL-2-activated cytotoxicity to NK-resistant target T24 was significantly impaired from week 2. To clarify if the defect in cytoloxic response is due to a decreased number of cell precursors or due to reduced responsiveness to IL-2, a limiting dilution assay was introduced. However, there was no reduction in precursor cell numbers. These data suggest that, in addition to impaired IL-2 production, there is an endogenous defect in IL-2 responsiveness. This may be of clinical importance since it is not possible to reverse this defect, at least in vitro.

Immunoregulatory lymphokines in rheumatoid joints. I. Search for interleukin 2 in synovial fluid.

Since there have been conflicting findings about the presence of interleukin 2 (IL-2) in synovial fluid (SF) of patients with rheumatoid arthritis (RA), we decided to study this subject ourselves. In a sensitive assay utilizing CTLL-2 as indicator cells, no IL-2-like activity was found in SF from any of the 15 RA patients studied. Treatment of SF with hyaluronidase, dialysation, heating to 56 degrees C for 1 h, preincubation of CTLL-2 with SF prior to washing and incubation all resulted in no detection of any IL-2. SF itself did not inhibit the CTLL-2 assay. Failure to detect IL-2 may to some extent be explained by in vivo absorption of any IL-2 by IL-2 receptor-positive T cells in SF. The failure is discussed along with new evidence from various reports suggesting that impaired IL-2 production by activated T cells may be a feature of several autoimmune diseases.

Differential expression of asialo GM1 on alloreactive cytotoxic T lymphocytes and lymphokine-activated killer cells

The present study was undertaken to examine the differential expression of asialo GM1 (AsGM1) on the responding cells and effectors of alloreactive cytotoxic T lymphocytes (CTL) and lymphokine-induced activated killers (LAK). It was found that AsGM1 was expressed on the 3-day-cultured LAK effectors. Its expression gradually disappeared to the extent that AsGM1 became undetectable after 5 to 6 days of culturing. In contrast, AsGM1 was detected on 3-day CTL generated in mixed-lymphocyte cultures (bulk cultures); however, the levels of AsGM1 expression remained the same for at least 7 days. When examining the expression of AsGM1 on the responding cells, the reciprocal results were obtained. AsGM1 was expressed on the LAK responders, but we were unable to demonstrate AsGM1 on CTL responders. Depletion of AsGM1 + cells from the responding population reduced subsequent CTL responses; however, CTL responses could be restored by adding conditioned media containing both interleukin 2 (IL-2) and other helper-T-cell factors and could not be restored by purified IL-2 alone adding at comparable doses. Reconstituting the AsGM1 -depleted responders with Lyt-2-depleted splenocytes also restored the CTL response. Furthermore, depletion of AsGM1 cells from the responding population did not reduce the precursor frequency of allo-CTL, whereas the precursor frequency of LAK cells was reduced 42-fold. These findings show that the reduction of CTL responses after depletion of AsGM1 + cells was not due to the removal of precursors; instead, the defect appeared to be in the helper population. We further found that the helper defect was not due to impaired IL-2 production, because the endogenous production of IL-2 in AsGM1-depleted responders was not reduced. Therefore, AsGM1 + cells may play a role in the helper pathway other than IL-2 production.