Interleukin-2 Receptor Expression Research Articles

Defective interleukin-2-dependent STAT5 signalling in CD8 T lymphocytes from HIV-positive patients: restoration by antiretroviral therapy.

CD8 T lymphocytes are critical in the control of HIV replication and disease progression. Our previous studies demonstrated that CD8 T cells from chronically infected patients with high virus load proliferated poorly in response to interleukin-2 (IL-2), a cytokine critical in CD8 T cell growth and differentiation, even though relatively high levels of IL-2 receptor (IL-2R) were expressed. This suggested that signal transduction defects in response to IL-2 might be involved. The STAT5 transcription factor is important in IL-2-dependent biological responses and it is known that there are defects in unstimulated CD3 and CD4 cells in HIV-infected patients. To investigate whether the induction of STAT5 by IL-2 is altered in the CD8 T cells from HIV-positive individuals and the impact of highly active antiretroviral therapy (HAART) on its status. CD8 T lymphocytes were purified from the peripheral blood mononuclear cells of HIV-positive patients before and after HAART. Ex vivo IL-2-induced STAT5 activation was evaluated by immunoblotting and electrophoretic mobility shift assays. CD8 T cells from a subset of untreated HIV-positive patients were unable to activate STAT5a and STAT5b proteins functionally in response to IL-2. This defect was not a result of alterations in IL-2R expression but correlated with an impaired activation of the upstream kinase Jak-3, known to mediate STAT5 activation. Overall, HAART restored Jak/STAT signalling in such patients. These results further uncover a potential mechanism by which CD8 T cell function is impaired in HIV-infected patients.

Effects of selenium on peripheral blood mononuclear cell membrane fluidity, interleukin-2 production and interleukin-2 receptor expression in patients with chronic hepatitis

To study the effect of selenium on peripheral blood mononuclear cell (PBMC) membrane fluidity and immune function in patients with chronic hepatitis. PBMCs were pretreated with selenium (1.156x10(-7) mol/L) for 6 h in vitro or extracted directly from patients after administration of selenium-yeast continuously for 8-12 wk (200 microg/d), and then exposed to Con-A for 48 h. The membrane fluidity, interleukin-2 (IL-2) production and interleukin-2 receptor (IL-2R) expression in PBMCs and malondialdehyde (MDA) concentration in medium and lipid peroxide (LPO) in plasma were determined. The PBMC membrane fluidity, IL-2 production and IL-2R expression in patients with chronic hepatitis were significantly lower than those in healthy blood donators (particle adhesive degree R, 0.17+/-0.01 vs 0.14+/-0.01, P<0.01; IL-2, 40.26+/-9.55 vs 72.96+/-11.36, P<0.01; IL-2R, 31.05+/-5.09 vs 60.58+/-10.56, P<0.01), and the MDA concentration in medium in patients with chronic hepatitis was significantly higher than that in healthy blood donators (1.44+/-0.08 vs 0.93+/-0.08, P<0.01). Both in vitro and in vivo administration of selenium could reverse the above parameters. Supplement of selenium can suppress lipid peroxidation, and improve PBMC membrane fluidity and immune function in patients with chronic hepatitis.

Clinical graft evolution of lymphocytes, polymorphonuclear cells, and antigen expression in tubular renal cells in the urine sediment of 20 renal allograft recipients

Urinary samples from 20 kidney transplant recipients were studied to determine the cellular composition of the sediments using an immunocytological (IC) technique. The expression of HLA class I (A, B, C) and class II (DR, DQ, DP), CD2, CD3, CD4, CD8, and interleukin (IL)-2 receptor (IL-2R) on lymphocytes was assessed using a panel of monoclonal antibodies. The results were correlated with graft function and with the number of episodes of acute renal graft rejection (AR) during a period of 6 months posttransplantation. The cellular infiltration of lymphocytes (LC) and polymorphonuclear cells (PMNC) also was studied using a standard cytology (SC) technique. During this period, 17 of 30 episodes of graft dysfunction due to AR occurred in 12 patients: 8 to acute tubular necrosis (ATN) (n = 8); 4 to cyclosporine (CsA) toxicity (n = 4) and 1 to amphotericin toxicity (n = 1). The diagnosis of AR was made clinically by 3 independent observers, using biopsy in some cases. The immunocytology showed a significantly increased expression of HLA-DR, DO, and DP namely, greater than 20% positivity in 10% of samples on the tubular epithelial cells (TEC) of patients presenting with versus without AR ( P ≤ .001). In addition, a high correlation was observed between the expression of IL-2R and the presence of AR (p ≤ .002). The standard cytology results showed a significantly increased percentage of LC and decreased percentage of PMNCs in samples obtained 2 days prior to the clinical manifestations of patients who developed AR ( P = .001). A greater level of expression of antigen determinants was observed prior to AR. These results suggest that immunocytology of urinary sediments, which is a noninvasive technique, has enormous clinical potential for the differential diagnosis of AR, ATN, and CsA toxicity. In our study, the use of HLA class IL–specific monoclonal antibodies (Abs) gave a 100% specificity, 95% sensitivity, and 95% predictability. Although our results also indicate a potential value in the increased IL-2R expression, these findings must be confirmed by further studies. Furthermore, the combination of both immunologic and SC techniques in urinary sediments allows early detection of AR and is cost effective and simple features that could be used routinely for follow-up of renal transplant recipients.

The immunophenotype and activation status of the lymphocytic infiltrate in human breast cancers, the role of the major histocompatibility complex in cell-mediated immune mechanisms, and their association with prognostic indicators

Background The aims of this study were to characterize, phenotypically, the immune infiltrate in human breast cancers, to assess the activation status of tumor-infiltrating lymphocytes (TIL), and to define the association of these findings with established prognostic indicators. Methods Immunohistochemistry was performed on frozen sections of 60 primary breast cancers by use of monoclonal antibodies to T lymphocytes (CD3), T-helper cells (CD4), cytotoxic T-cells (CD8), natural killer cells (CD56), interleukin-2 receptors (IL-2R), and major histocompatibility (MHC) class I antigen (HLA-ABC) and MHC class II antigen (HLA-DR). Results All tumors stained positive for CD3, CD4 and CD8, but with marked variation in the intensity of the infiltrate. In tumors with a moderate infiltrate of TIL, there was a trend toward a greater representation of T–helper cells. However, as the intensity of TIL increased, there was a decline in the proportion of T–helper cells and a concomitant rise in the relative proportion of cytotoxic T cells. There was a relative paucity of natural killer cells. A significant association was found between the intensity of TIL and the number of positive nodes ( P = .02) and the intensity of the infiltrate of both T–helper cells and cytotoxic T cells with ER expression ( P = .03 and .05, respectively). Most tumors stained positive for IL-2R. The expression of IL-2R was associated with the intensity of TIL ( P<.0001), T–helper cells ( P<.002), cytotoxic T cells ( P = .01) and natural killer cells ( P = 0.04), and also with the degree of lymph node positivity ( P = .02) and histologic tumor grade ( P = .05). MHC class II expression was variable, and a large proportion of the tumors showed limited expression in individual cancer cells. There was an association between the expression of HLA-DR in tumor cells and the activation status of TIL ( P = .03). Conclusion An immune infiltrate is an invariable finding in breast cancers, and the intensity of the infiltrate is greater in node positive tumors. Additionally, TIL may well be activated, albeit partially, in most tumors, suggesting that cell-mediated immune mechanisms are functionally intact.

Identification and Functional Characterization of Voltage-dependent Calcium Channels in T Lymphocytes

In T lymphocytes, sustained calcium (Ca2+) influx through Ca2+ channels localized in the plasma membrane is critical for T cell activation and proliferation. Previous studies indicated that voltage-dependent Ca2+ channels (VDCCs) play a role in Ca2+ mobilization during T lymphocyte activation. However, the role of VDCCs in otherwise nonexcitable cells is still poorly understood. We used RT-PCR to identify a transcript encoding the pore-forming alpha1F-subunit of an L-type Ca2+ channel in T lymphocytes. Its identity was confirmed by DNA sequencing. To further investigate the contribution of Ca2+ influx through VDCCs, we assessed the effects of the 1,4-dihydropyridine L-type Ca2+ channel agonist, (+/-) Bay K 8644, and antagonist, nifedipine, on the human Jurkat T cell leukemia line, human peripheral blood T lymphocytes and mouse splenocytes. We found that treatment of T lymphocytes with (+/-) Bay K 8644 increased intracellular Ca2+ and induced the activation of phosphoextracellular-regulated kinase 1/2 (Erk1/2), whereas nifedipine blocked Ca2+ influx, the activity of Erk1/2 and nuclear factor of activated T cells (NFAT), interleukin-2 (IL-2) production, and IL-2 receptor expression. Nifedipine also significantly suppressed splenocyte proliferation in an in vitro mixed lymphocyte reaction and the proliferation of male antigen (H-Y)-specific T cell receptor-transgenic CD8+ T cells in transplanted male mice in vivo. Taken together these novel findings indicate that an L-type Ca2+ channel plays a significant role in the Ca2+ influx pathways mediating T lymphocyte activation and proliferation in vitro and in vivo.

The enhancement of T cell proliferation by l-dopa is mediated peripherally and does not involve interleukin-2

Although several recent studies suggest that dopamine may have a significant role in the regulation of immune cell function, the mechanisms involved remain unclear. We undertook this study to clarify the actions of dopamine in vivo on lymphocyte proliferation and to determine whether such effects are mediated by alterations in interleukin-2 (IL-2) production and/or IL-2 receptor (IL-2R) expression. Administration of the dopamine precursor, l-dopa, for 5 days to BALB/c mice significantly increased Concanavalin A (ConA)-induced proliferation of lymphocytes. This effect was blocked by treatment with dopamine receptor antagonists as well as a peripheral dopamine synthesis inhibitor, suggesting a peripheral action of dopamine. l-dopa treatment had no effect on IL-2 production or IL-2R expression, indicating that the effects of dopamine on lymphocyte proliferation are not indirectly mediated by alterations in the IL-2 system.

Plasticity of IL-2 and IL-2 receptor chains in rat lymphoid tissues in situ after stimulation with staphylococcal enterotoxin A.

Although the effects of mitogens on the synthesis of interleukin-2 (IL-2) and IL-2 receptor (IL-2r) have been described, a detailed in situ analysis of the spatio-temporal changes of the expression of the IL-2 gene and the three IL-2r components in lymphoid tissues is still missing. Therefore, we analyzed the IL-2 and IL-2r expression after a staphylococcal enterotoxin A (SEA)-induced T cell activation on a cellular and anatomical basis in the Wistar rat. SEA caused a rapid induction of IL-2 mRNA in T cells of spleen, lymph node, and thymus, followed by the appearance of high systemic IL-2 serum levels (5 ng/ml), and a significant increase of CD25 on CD4+ and CD8+ lymphocytes. The histotopographic analysis of the IL-2r chains revealed a strong upregulation of IL-2r alpha (alpha) and IL-2r beta (beta) mRNAs in similar T cell specific compartments of spleen, lymph node, and thymus as seen for IL-2 mRNA. The abundant constitutive expression of IL-2r gamma (gamma) mRNA was unaffected by SEA. The parallel upregulation of IL-2, IL-2ralpha, and beta chains in conjunction with the continuous presence of the IL-2rgamma chain predominantly in T cell regions of immune organs suggests that the biological effects of IL-2 are essentially limited to T cells, at least after superantigen stimulation.

Expression of IL-2 receptor beta and gamma chains by human gingival fibroblasts and up-regulation of adhesion to neutrophils in response to IL-2.

To investigate the role of human gingival fibroblasts (HGF), the major constituents of gingival tissue in periodontal inflammatory disease, the expression of interleukin-2 receptor (IL-2R) alpha, beta, and gamma chains was examined. Immunohistochemistry showed a pronounced accumulation of CD8(+) T cells in the inflamed lamina propria of gingival tissue from patients with adult periodontitis. HGF express IL-2Rbeta and IL-2Rgamma at mRNA and protein levels, but the expression of IL-2Ralpha could not be detected, as assessed by reverse transcriptase-polymerase chain reaction and flow cytometry. IL-2Rbeta, and -gamma expressed on HGF were functionally active, as addition of neutralizing anti-IL-2Rbeta and -gamma antibodies caused inhibition of the IL-2-induced production of monocyte chemoattractant protein-1 (MCP-1), and addition of IL-2 induced phosphorylation of Janus tyrosine kinase 3, which is critical in signaling through IL-2Rgamma in HGF. The IL-2-induced MCP-1 production was significantly inhibited by pretreatment with neutralizing antibody to IL-15. Addition of IL-2 also induced a marked up-regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of HGF, which in turn, significantly augmented the adhesion of human neutrophils, which were inhibited by an anti-ICAM-1 antibody. These results suggest that HGF express functional IL-2Rbetagamma, respond to IL-2 from infiltrated T cells, and actively participate in the inflammatory process in the periodontal region and that IL-15 produced by HGF sustains IL-2-mediated signaling in HGF.

Interleukin 12-augmented T cell proliferation of peripheral blood mononuclear cells from HIV-seropositive individuals is associated with interleukin 12 receptor beta 2 upregulation.

Interleukin 12 (IL-12) production is believed to be impaired in individuals with HIV infection and this impairment manifests early in disease, when the CD4(+) cell counts are within normal values. The reduced antigen-specific and mitogen-stimulated T cell-proliferative responses that occur in HIV infection can be corrected by the addition of recombinant human interleukin 12 (rhIL-12). As the IL-12 receptor (IL-12R) is central to the IL-12 signaling pathway, we examined whether the augmentation of antigen-specific proliferation of HIV(+) peripheral blood mononuclear cells (PBMCs) related to altered IL-12R expression. rhIL-12 augmented antigen-specific proliferation of HIV(+) PBMCs but not of HIV(-) PBMCs. Examination of resting PBMCs from HIV(+) and HIV(-) donors showed that neither of these populations expressed IL-12R beta 1 or IL-12R beta 2 chains on their cell surface as detected by flow cytometry. However, examination of mRNA showed that both IL-12R beta 1 and IL-12R beta 2 mRNAs were markedly reduced in HIV(+) PBMCs when compared with HIV(-) PBMCs. After mitogen activation there was an increase in IL-12R beta 1 expression on the cell surface of HIV(+) and HIV(-) PBMCs and this level was not altered by coculture with rhIL-12 or interferon gamma (IFN-gamma). However, coculture of phytohemagglutinin (PHA)-activated HIV(+) or HIV(-) PBMCs with rhIL-12 (but not IFN-gamma) increased IL-12R beta 2 expression on the cell surface of both populations. Examination at the message level showed a correction of IL-12R beta 1 to normal levels with activation that was further enhanced by rhIL-12 coculture for both the HIV(+) and HIV(-) PBMCs. However, although the level of IL-12R beta 2 for the HIV(+) PBMCs was normalized by PHA, rhIL-12 caused a further augmentation. This information provides a strong link between IL-12R upregulation, and the significant improvement in antigen-specific HIV-proliferative responses seen with the addition of rhIL-12. It also reveals that the dysfunction in IL-12R expression seen in cells from HIV(+) patients occurs at the transcriptional level. In addition, we provide further evidence that IL-12R beta 1 and IL-12R beta 2 regulation in human PBMCs is independent of IFN-gamma.

CD2-CD48 interactions promote cytotoxic T lymphocyte induction and function: anti-CD2 and anti-CD48 antibodies impair cytokine synthesis, proliferation, target recognition/adhesion, and cytotoxicity.

The role of CD2 signaling in cytotoxic T lymphocyte (CTL) development was examined by stimulating mouse T cells with anti-CD3 monoclonal antibody (mAb) in the absence or presence of anti-CD2 mAb or anti-CD48 mAb or both. Induction of nonspecific CTL and interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) synthesis were impaired in the absence of CD2-CD48 interactions. Anti-CD2 mAb also inhibited activation-induced expression of the high-affinity IL-2 receptor (IL-2R). In contrast, IFN-gamma receptor (IFNGR) expression was increased in the presence of anti-CD2 mAb. Reduced cytotoxicity by CTL induced in the absence of CD2-CD48 interactions was associated with a diminished ability of CTL to conjugate with target cells and reduced expression of granzyme B and perforin. Anti-CD2 mAb did not affect expression of Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by anti-CD3-activated T cells. Cytotoxic effector function and granzyme B and perforin expression were rescued when exogenous IL-2 and IFN-gamma were added in combination with anti-CD2 mAb to anti-CD3-activated T cells at initiation of culture. We conclude that CD2-CD48 interactions during T cell activation are critical for the synthesis of sufficient IL-2 and IFN-gamma to drive CD8(+) T cells to differentiate into functional cytotoxic effector cells.

DAB389IL2 (ONTAK®) fusion protein therapy of chronic lymphocytic leukaemia

Despite multiple new therapeutic options for patients with chronic lymphocytic leukaemia (CLL), the prognosis of patients with relapsed disease is poor. After first-line therapy with fludarabine, alkylating agents, rituximab or combinations of these agents, most patients relapse within a few years. While second-line therapy with alemtuzumab, or other combinations of the above agents, have produced remissions, most of these are partial responses lasting months rather than years. Patients commonly die from progressive disease or infections related to the underlying disease or treatment. The authors sought to develop a novel therapeutic with the capacity to kill chemotherapy-resistant CLL cells and with reduced toxicities to normal tissues. Based on the presence of high affinity interleukin-2 receptor (IL-2R) on CLL cells, therapy of relapsed CLL patients with a diphtheria fusion protein targeting IL-2R – DAB389IL2 (ONTAK®,Seragen, Inc., Hopkinton, MA, USA) – was tested in a pilot Phase II study. Biological activity and a partial remission were observed with modest drug-related side effects. Based on these encouraging findings, alternative schedules and combinations with agents that may enhance CLL cell expression of IL-2R are being tested. Hopefully, the use of these targeted therapeutic approaches will provide additional therapeutic options with fewer side effects for this common and incurable condition.

In vitro responses to purified protein derivate of caprine T lymphocytes following vaccination with live strains of Mycobacterium avium subsp. paratuberculosis

Live attenuated vaccines provide protection against intestinal lesions in goats infected with Mycobacterium avium subsp. paratuberculosis. To examine the role of different T lymphocyte subsets in the development of this protective immunity, CD4 +, CD8 + and γδ T cell receptor (TCR) + cells from peripheral blood of goat kids vaccinated with live attenuated strains of M. a. paratuberculosis were studied. After in vitro stimulation with purified protein derivate, the expression of γ-interferon (IFN-γ) and the activation marker interleukin-2 receptor (IL-2R) was analysed by flow cytometry. A depletion experiment was performed, where the phenotypes and IL-2R expression was studied after stimulation of cultures depleted of a T lymphocyte subpopulation. Close to all of the IFN-γ producing cells were of the CD4 + subset, while only a small number were CD8 + cells. The γδ TCR + cells were highly activated, but did not produce IFN-γ after in vitro stimulation. Depletion of CD4 + cells lead to a decrease in the percentage of total γδ TCR + cells and γδ TCR +IL2-R + cells. Removing the γδ TCR + cells increased the relative numbers of CD4 +, but not the CD4 +IL-2R + cells. Insight into the in vitro recall responses of T cell subsets from animals vaccinated with live paratuberculosis vaccines is essential in the development of more efficient vaccines.

The flame retardants tetrabromobisphenol A and tetrabromobisphenol A–bisallylether suppress the induction of interleukin-2 receptor α chain (CD25) in murine splenocytes

Polybrominated flame retardants (PBF) are frequently used additives in electronical equipment. They are ubiquitous environmental contaminants which bioaccumulate with several health effects for humans and the environment. This study investigated immunotoxic effects of the PBF tetrabromobisphenol A (TBBP A), tetrabromobisphenol A-bisallylether (TBBP A-AE), tetrabromobisphenol A-bis-(2,3-dibromopropyl-ether) (TBBP A-PE), decabromodiphenylether (DBDE), and 2,4,6-tribromophenol (TBP) in vitro . The structurally related polychlorinated aromatic hydrocarbon 3,4,3′,4′-tetrachlorobiphenyl (PCB77) and dioxins mediate their immunotoxicity via the Ah-receptor gene complex. A highly relevant function of the Ah receptor, the induction of CYP 1A1 in hepatocytes of C57BL/6 mice by the established inducers 3-methylcholanthrene (MC) and PCB77 was compared to the effect of PBF by measurement of ethoxyresorufin- o-deethylase (EROD) activity. The PBF did not show any induction of CYP 1A1, while EROD activity of hepatocytes exposed to MC and PCB77 was induced 10.8- and 8.7-fold, respectively. To investigate immunotoxic effects of the flame retardants, splenocytes of C57BL/6 mice were incubated with subtoxic doses of the flame retardants and PCB77 and activated by concanavalin A (Con A). The flame retardants TBBP A and TBBP A-AE significantly inhibited the expression of interleukin-2 receptor α chain (CD25) in contrast to TBBP A-PE, DBDE, TBP, and PCB77 as shown by immunohistochemistry and quantitative analysis by laser scanning cytometry. None of the substances had any effect on the Con A-induced production of cytokines. Hence, TBBP A and TBBP A-AE may act as immunotoxic compounds by specifically inhibiting the expression of CD25.

Zinc enhances the expression of interleukin-2 and interleukin-2 receptors in HUT-78 cells by way of NF-κB activation

Production of interleukin (IL)-2 is decreased in zinc-deficient human beings, and zinc is essential to IL-2-mediated T-cell activation. We used a human Th0 malignant lymphoblastoid cell line, HUT-78, to study the effect of zinc on IL-2 production in PHA/PMA activated T-cells. In zinc-deficient cells, the gene expression of IL-2 was decreased by 50% compared with that in zinc-sufficient cells. The effect of zinc was specific and at the transcriptional level. We also showed a significant effect of zinc on the gene expression of IL-2 receptors α and β. Binding of NF-κB (a zinc-dependent transcription factor) to DNA was decreased in zinc-deficient cells. Using transfection of expression vectors of anti-sense NF-κB p105 (precursor of NF-κB p50) in cells, we showed that a decrease in gene expression of IL-2 and IL-2 Rα may be partly due to decreased activation of NF-κB in zinc-deficient cells. Our studies demonstrate, for the first time, the role of zinc in gene expression of IL-2 and its receptors in HUT-78 cells. We also document that the binding of NF-κB to DNA was adversely affected, thereby decreasing the gene expression of IL-2 and IL-2 Rα in zinc-deficient HUT-78 cells. (J Lab Clin Med 2002;140:272-89)

Cowdria ruminantium antigens of around 15 kDa are potent inducers of IFN-gamma.

Cellular responses induced in two Creole goats by vaccination with killed Cowdria ruminantium (Cowdria) were confirmed by IFN-gamma production and interleukin-2 receptor (IL-2R) expression. Both CD4+ and CD8+ but not WC1+ T cells showed a substantial increase in cell surface expression of IL-2R molecules in response to whole Cowdria lysate. Cowdria (Welgevonden strain) proteins were fractionated using continuous-flow electrophoresis and tested for their ability to induce IFN-gamma production by PBMC collected three weeks after the first inoculation and one week after the booster injection. Pooled fractions of around 15, 22, and 24 kDa were found to induce significant IFN-gamma production in both vaccinated animals on one of the two occasions. Antigens of around 15 kDa induced substantially higher IFN-gamma production than any other fractions in both animals. These pilot experiments pave the way towards the identification of proteins/genes that have potential for the development of a recombinant vaccine against heartwater.

Immunomodulatory effects of RXR rexinoids: modulation of high-affinity IL-2R expression enhances susceptibility to denileukin diftitox

Rexinoids binding to both the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families of rexinoid receptors have demonstrated clinical activity in hematologic malignancies and have been shown to mediate genes associated with both growth and differentiation. RXR rexinoids have demonstrated efficacy in the treatment of cutaneous T-cell lymphomas, but the mechanism of action is unclear. We explored the immunomodulatory effects of RAR and RXR rexinoids in human T- and B-cell leukemia cells and demonstrated that RXR rexinoids are capable of up-regulating high-affinity interleukin-2 receptor (IL-2R) expression. Exposure to 10(-6) to 10(-10) M bexarotene or Panretin for 48 hours was associated with increased expression of both the p55 and p75 subunits of the IL-2R in T-cell leukemias and p75 in B-cell leukemias. Furthermore, rexinoid exposure enhanced susceptibility of the cells to denileukin diftitox fusion toxin-targeting and -intoxicating cells expressing high-affinity IL-2R. These results suggest a rationale for combining rexinoids with IL-2R-targeted therapies in lymphoid malignancies as well as possibly in autoimmune diseases.

Localisation of CD25+ cells and MHCII+ cells in lymph nodes draining Mycobacterium avium subsp. paratuberculosis vaccination granuloma and the presence of a systemic immune response

Vaccination of goat kids against paratuberculosis protects against lesions and clinical disease. The systemic cellular response was studied in goat kids 3–9 weeks after vaccination. Peripheral blood cells showed increased interferon-γ production and expression of interleukin-2 receptor (CD25) after stimulation with Mycobacterium avium subsp. paratuberculosis antigens. The lymph node draining the vaccination granuloma was studied three weeks after vaccination in a parallel group of goat kids. In deep cortex, MHCII+ cells were observed surrounded by CD4+ T-cells, while follicular hypertrophy and hyperplasia were prominent in the subcapsular region and along connective tissue trabecula. Comparison of the local and systemic immune responses revealed an inverse relationship between CD25+ T-cells in the lymph node deep cortex and cells in peripheral blood that up-regulate CD25 upon in vitro stimulation, suggesting that activated and regulatory T-cells in the local lymph node influence the level of circulating antigen-specific T-cells following vaccination against paratuberculosis in goats.

Research Communication: Immune Functions Are Maintained in Healthy Men with Low Zinc Intake

Although immunity is impaired during severe zinc deficiency, there is limited information about the effects of mild zinc depletion on immune response in humans. We evaluated the effects of a zinc-restricted diet (4.6 mg/d) on several indices of immunity in 8 healthy men. The subjects consumed zinc supplements with 9.1 mg/d during the 5-wk baseline (BL) and 5-wk repletion (RP) periods, and placebos during the 10-wk zinc-restriction (ZR) period. Leukocyte numbers and functions were studied at the end of each metabolic period. After ZR, there were reductions in the proliferation of peripheral blood mononuclear cells (PBMNC) stimulated with phytohemagglutinin (PHA, 1.2, 2.5, 5.0, 10.0 [corrected] and 20.0 mg/L; P < 0.01) and in the in vitro secretion of interleukin-2 receptor (IL-2R) (PHA, 2.5 mg/L; P = 0.058). These variables remained reduced (P < 0.05) even after 5 wk of zinc repletion. The amount of zinc consumed did not alter the numbers of circulating neutrophils, monocytes and lymphocytes, the in vitro PBMNC secretion of interferon-gamma and tumor necrosis factor-alpha or neutrophil superoxide production. The results suggest that changes in lymphocyte proliferation and IL-2R expression may be early markers of mild zinc deficiency.

Arginine butyrate increases the cytotoxicity of DAB 389IL-2 in leukemia and lymphoma cells by upregulation of IL-2Rβ gene

DAB 389IL-2 (ONTAK) is a fusion protein consisting of the ADP-ribosyltransferase and membrane translocating domains of native diphtheria toxin and the full-length sequence for interleukin-2 (IL-2) gene. In vitro data demonstrates that DAB 389IL-2 is cytotoxic to cells expressing the high affinity IL-2 receptor (IL-2R). In Phases I and II clinical trials of patients whose tumor cells express a component of the IL-2R, the response rates were 18% for B-cell non-Hodgkin lymphoma (NHL) and 30% for cutaneous T-cell lymphoma (CTCL). In this study, we examined the effects of arginine butyrate on IL-2R expression and susceptibility of leukemia cells to intoxication by DAB 389IL-2. We demonstrate that the p75 subunit of the IL-2R (IL-2Rβ) is upregulated in the presence of low concentrations of arginine butyrate (0.06 mM) which had no direct growth inhibitory effect on the cells. To explore mechanisms of this upregulation, we examined the effect of 0.06 mM arginine butyrate on relevant transcriptional elements and on histone deacetylase and found activation of cAMP response element (CRE) but not NFAT or NFKB, as well as inhibition of histone deacetylase (HDAC). Our results suggest that the effects of physiologically achievable concentrations of butyrate on IL-2R expression could be exploited to enhance the susceptibility of intermediate and low-affinity IL-2R expressing leukemia cells to DAB 389IL-2.

Receptor upregulation enhances cell surface receptor targeted therapies

A number of new therapeutic agents for hematologic malignancies have been approved in the last 5 years that target cell surface receptors on the malignant cell. These include a mouse–human chimeric monoclonal antibody to CD20 (rituximab), a 90Y-labeled mouse monoclonal antibody to CD20 (ibritumomab tiuxetan), a humanized antibody to CD52 (alemtuzumab), an immunoconjugate consisting of a humanized antibody to CD33 coupled to calicheamicin (gemtuzumab ozogamicin), and a diphtheria fusion protein (denileukin diftitox) directed at the high affinity interleukin-2 receptor (IL2R) complex [1–5]. In addition, there are a number of targeted protein molecules in various stages of clinical development reactive with various hematopoietic cell surface receptors including CD20, CD22, CD33, HLA-DR, the granulocyte–macrophage colonystimulating factor receptor, among others (see Table 1). Many of these biologics show impressive clinical activity, but most treated patients relapse. To improve the remission rate and duration, one approach has been to combine the biologic compound with cytotoxic drugs [6]. This has yielded outstanding results with rituximab. Another approach is to selectively enhance the potency of the biologic on the target malignant stem cell. Based on both preclinical and clinical data, tumor cell kill with toxin immunoconjugates is proportional to receptor density [7,8]. Foss and coworkers have studied methods to increase the expression of high affinity IL2R on malignant hematopoietic cells to enhance killing by denileukin diftitox [9–11]. In this issue, her group documents that arginine butyrate increases the expression of the subunit of the IL2R on leukemia cells and that this leads to increased sensitivity to denileukin diftitox [12]. Upregulation of receptors on malignant cells is an attractive strategy. This may involve enhanced gene transcription, improved protein stability, or increased protein trafficking to the cell surface. Compounds have been found which modify each of these pathways for a number of receptors (see Table 2). The molecular mechanisms for receptor upregulation by most of these compounds are poorly characterized.