Blocking antibodies induced by allergen-specific immunotherapy ameliorate allergic airway disease in a human/mouse chimeric model.

BACKGROUND: Allergen-specific immunotherapy (AIT) induces specific blocking antibodies (Ab), which are claimed to prevent IgE-mediated reactions to allergens. Additionally, AIT modulates cellular responses to allergens, for example, by desensitizing effector cells, inducing regulatory T and B lymphocytes and immune deviation. It is still enigmatic which of these mechanisms mediate(s) clinical tolerance. We sought to address the role of AIT-induced blocking Ab separately from cellular responses in a chimeric human/mouse model of respiratory allergy. METHODS: Nonobese diabetic severe combined immunodeficient γc-/- (NSG) mice received intraperitoneally allergen-reactive PBMC from birch pollen-allergic patients together with birch pollen extract and human IL-4. Engraftment was assessed by flow cytometry. Airway hyperresponsiveness (AHR) and bronchial inflammation were analyzed after intranasal challenges with allergen or PBS. Sera collected from patients before and during AIT with birch pollen were added to the allergen prior to intranasal challenge. The IgE-blocking activity of post-AIT sera was assessed in vitro. RESULTS: Human cells were detected in cell suspensions of murine lungs and spleens indicating successful humanization. Humanized mice displayed a more pronounced AHR and bronchial inflammation when challenged with allergen compared to negative controls. Post-AIT sera exerted IgE-blocking activity. In contrast to pre-AIT sera, the presence of heterologous and autologous post-AIT sera significantly reduced the allergic airway inflammation and matched their IgE-blocking activity determined in vitro. CONCLUSION: Our data demonstrate that post-AIT sera with IgE-blocking activity ameliorate allergic airway inflammation in a human/mouse chimeric model of respiratory allergy independently of AIT-induced cellular changes.

Distinct epitope structures of defensin-like proteins linked to proline-rich regions give rise to differences in their allergenic activity.

BACKGROUND: Art v 1, Amb a 4, and Par h 1 are allergenic defensin-polyproline-linked proteins present in mugwort, ragweed, and feverfew pollen, respectively. We aimed to investigate the physicochemical and immunological features underlying the different allergenic capacities of those allergens. METHODS: Recombinant defensin-polyproline-linked proteins were expressed in E. coli and physicochemically characterized in detail regarding identity, secondary structure, and aggregation status. Allergenic activity was assessed by mediator releases assay, serum IgE reactivity, and IgE inhibition ELISA using sera of patients from Austria, Canada, and Korea. Endolysosomal protein degradation and T-cell cross-reactivity were studied in vitro. RESULTS: Despite variations in the proline-rich region, similar secondary structure elements were observed in the defensin-like domains. Seventy-four percent and 52% of the Austrian and Canadian patients reacted to all three allergens, while Korean patients were almost exclusively sensitized to Art v 1. This was reflected by IgE inhibition assays demonstrating high cross-reactivity for Austrian, medium for Canadian, and low for Korean sera. In a subgroup of patients, IgE reactivity toward structurally altered Amb a 4 and Par h 1 was not changed suggesting involvement of linear epitopes. Immunologically relevant endolysosomal stability of the defensin-like domain was limited to Art v 1 and no T-cell cross-reactivity with Art v 125-36 was observed. CONCLUSIONS: Despite structural similarity, different IgE-binding profiles and proteolytic processing impacted the allergenic capacity of defensin-polyproline-linked molecules. Based on the fact that Amb a 4 demonstrated distinct IgE-binding epitopes, we suggest inclusion in molecule-based allergy diagnosis.

Rapid decline in insulin antibodies and glutamic acid decarboxylase autoantibodies with ibrutinib therapy of chronic lymphocytic leukaemia.

WHAT IS KNOWN AND OBJECTIVE: Ibrutinib is inhibiting the Bruton's tyrosine kinase (BTK), thereby influencing B-cell development. We describe an unexpected side effect of ibrutinib in two patients with chronic lymphocytic leukaemia concerning the vigorous decrease of two different diabetes-associated antibodies. CASE DESCRIPTION: Two weeks after onset of ibrutinib therapy, patient A frequently noticed symptoms of hypoglycaemia such as dizziness and blurred vision. Blood glucose declined to 35-40 mg/dL. He had to lower his insulin dose step by step. High levels of insulin antibodies which had developed during insulin therapy were detected. Seven weeks after start of ibrutinib, his insulin antibodies level had dropped by 54.6%. Patient B had a 54.1% decrease in his glutamic acid decarboxylase autoantibodies level after 7 weeks. WHAT IS NEW AND CONCLUSION: The inhibitory effect of ibrutinib on the levels of insulin antibodies and glutamic acid decarboxylase autoantibodies is a novel finding and may have implications for diabetes care.

NI-1: a novel canine mastocytoma model for studying drug resistance and IgER-dependent mast cell activation.

BACKGROUND: Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organs, mediator-related symptoms, and a poor prognosis. Kit mutations supposedly contribute to abnormal growth and drug resistance in these patients. METHODS: We established a novel canine mastocytoma cell line, NI-1, from a patient suffering from MC leukemia. RESULTS: NI-1 cells were found to form mastocytoma lesions in NOD/SCID IL-2Rgamma(null) mice and to harbor several homozygous Kit mutations, including missense mutations at nucleotides 107(C→T) and 1187(A→G), a 12-bp duplication (nucleotide 1263), and a 12-bp deletion (nucleotide 1550). NI-1 cells expressed several MC differentiation antigens, including tryptase, Kit, and a functional IgE receptor. Compared to the C2 mastocytoma cell line harboring a Kit exon 11 mutation, NI-1 cells were found to be less responsive against the Kit tyrosine kinase inhibitors (TKI) masitinib and imatinib, but were even more sensitive against proliferation-inhibitory effects of the mammalian target of rapamycin (mTOR) blocker RAD001 and PI3-kinase/mTOR blocker NVP-BEZ235. The Kit-targeting multikinase inhibitors PKC412 and dasatinib were also found to override TKI resistance in NI-1 cells, and produced growth inhibition with reasonable IC(50) values (<0.1 µM). CONCLUSION: NI-1 may serve as a useful tool to investigate IgE-dependent reactions and mechanisms of abnormal growth and drug resistance in neoplastic MC in advanced mastocytosis.

Leukosialin (CD43)-major histocompatibility class I molecule interactions involved in spontaneous T cell conjugate formation.

Resting T cells spontaneously adhere in a selective manner to potent accessory cells, such as dendritic cells (DC) and lymphoblastoid B blasts (LCL). Here we demonstrate that leukosialin (CD43) and major histocompatibility complex class I molecules (MHC-I) might play a critical role in this process. T cell conjugate formation with monocyte-derived DC (md-DC) and LCL could be strongly inhibited by either preincubating T cells with Fab fragments of CD43 monoclonal antibody (mAb) 6F5 or by preincubating md-DC or LCL with MHC-I mAb W6/32. Intact CD43 mAb 6F5, in contrast to monovalent Fab fragments, enhanced T cell adhesiveness by transactivating CD2 binding to CD58 molecules. Interestingly, induction of this proadhesive signal via CD43 with intact 6F5 mAb was found to revert mAb W6/32-mediated inhibition of T cell conjugate formation. These observations indicated that CD43 cross-linkage mimics and monovalent mAb 6F5 inhibits interaction of T cell CD43 with a stimulatory ligand on opposing cells, presumably MHC-I. For the demonstration of direct physical interaction between CD43 on T cells and MHC-I-coated beads it was necessary, however, to ligate CD2 on T cells with a stimulatory pair of CD2 mAbs (VIT13 plus TS2/18). This suggests that CD2 ligation crosswise upregulates CD43 binding avidity for MHC-I and that both adhesion molecule pairs (CD43/MHC-I and CD2/CD58) act in concert to induce and mediate T cell conjugate formation with certain cell types.

Neutrophil granulocyte-committed cells can be driven to acquire dendritic cell characteristics.

Polymorphonuclear granulocytes (PMNs) are thought to fulfill their role in host defense primarily via phagocytosis and release of cytotoxic compounds and to be inefficient in antigen presentation and stimulation of specific T cells. Dendritic cells (DCs), in contrast, are potent antigen-presenting cells with the unique capacity to initiate primary immune responses. We demonstrate here that highly purified lactoferrin-positive immediate precursors of end-stage neutrophilic PMN (PMNp) can be reverted in their functional maturation program and driven to acquire characteristic DC features. Upon culture with the cytokine combination granulocyte/macrophage colony-stimulating factor plus interleukin 4 plus tumor necrosis factor alpha, they develop DC morphology and acquire molecular features characteristic for DCs. These molecular changes include neo-expression of the DC-associated surface molecules cluster of differentiation (CD)1a, CD1b, CD1c, human leukocyte antigen (HLA)-DR, HLA-DQ, CD80, CD86, CD40, CD54, and CD5, and downregulation of CD15 and CD65s. Additional stimulation with CD40 ligand induces also expression of CD83 and upregulates CD80, CD86, and HLA-DR. The neutrophil-derived DCs are potent T cell stimulators in allogeneic, as well as autologous, mixed lymphocyte reactions (MLRs), whereas freshly isolated neutrophils are completely unable to do so. In addition, neutrophil-derived DCs are at least 10,000 times more efficient in presenting soluble antigen to autologous T cells when compared to freshly isolated monocytes. Also, in functional terms, these neutrophil-derived DCs thus closely resemble "classical" DC populations.

Targeting of mTOR is associated with decreased growth and decreased VEGF expression in acute myeloid leukaemia cells.

BACKGROUND: The mammalian target of rapamycin (mTOR) has recently been implicated in leukaemic cell growth, tumour-associated angiogenesis and expression of vascular endothelial growth factor (VEGF). We examined whether mTOR plays a role as regulator of growth and VEGF-expression in acute myeloid leukaemia (AML). Three mTOR-targeting drugs, rapamycin, everolimus (RAD001) and CCI-779, were applied. The effects of these drugs on growth, survival, apoptosis and VEGF expression in primary AML cells and various AML cell lines were examined. MATERIALS AND METHODS: Growth of AML cells and AML-derived cell lines was assessed by (3)H-thymidine incorporation, survival was examined by light- and electron microscopy, by Tunel assay and by AnnexinV-staining, and the expression of VEGF by Northern blotting, RT-PCR and ELISA. RESULTS: Rapamycin was found to counteract growth in the AML cell lines U937 and KG1a as well as in primary AML cells in 14/18 patients examined. The effects of rapamycin and its derivatives were dose-dependent (IC(50): 10 pM-100 nM). It was also found that exposure to mTOR-targeting drugs resulted in apoptosis and in decreased expression of VEGF in leukaemic cells. CONCLUSIONS: mTOR-targeting drugs exert antileukaemic effects on AML cells in vitro through multiple actions, including direct inhibition of proliferation, induction of apoptosis and suppression of VEGF. Based on this study and other studies, mTOR can be regarded as a potential drug target in AML.

Danon disease: case report and detection of new mutation.

Danon disease is an X-linked disorder resulting from mutations in the lysosome-associated membrane protein-2 (LAMP2) gene. We report a male patient with skeletal myopathy, mental retardation, and massive hypertrophic obstructive cardiomyopathy necessitating heart transplantation. Immunohistochemistry of skeletal muscle and leukocytes, western blot analysis of leukocytes and cardiac muscle, flow cytometry, and DNA sequencing were performed. Muscle biopsy revealed autophagic vacuolar myopathy and lack of immunohistochemically detectable LAMP-2. Diagnosis of Danon disease was confirmed by western blot analysis of myocardial tissue and peripheral blood sample of the patient showing deficiency of LAMP-2 in myocardium and leukocytes. Moreover, absence of LAMP-2 in lymphocytes, monocytes and granulocytes was shown by flow cytometric analysis. Genetic analysis of the LAMP2 gene revealed a novel 1-bp deletion at position 179 (c.179delC) at the 3' end of exon 2, resulting in a frameshift with a premature stop codon.

Evaluation of antileukaemic effects of rapamycin in patients with imatinib-resistant chronic myeloid leukaemia.

BACKGROUND: Recent data suggest that the mammalian target of rapamycin (mTOR) is involved in the regulation of growth of neoplastic cells in chronic myeloid leukaemia (CML). PATIENTS AND METHODS: We treated six patients with imatinib-resistant CML in haematological relapse (leukocytes > 20,000 microL(-1)) with rapamycin at 2 mg per os daily for 14 consecutive days, with dose-adjustment allowed to reach a target rapamycin serum concentration of 10-20 pg mL(-1). RESULTS: A major leukocyte response with decrease to less than 10,000 microL(-1) was obtained in two patients, and a minor transient response was seen in two other patients. In responding patients, we also observed a decrease in vascular endothelial growth factor (VEGF) mRNA levels in circulating leukaemic cells. Side effects during rapamycin treatment were mild in most patients. In one patient, pneumonia developed. Rapamycin was also found to counteract growth of CML cells in vitro as determined by (3)H-thymidine incorporation. Moreover, rapamycin inhibited the growth of Ba/F3 cells exhibiting various imatinib-resistant mutants of BCR/ABL, including the T315I variant that exhibits resistance against most currently available BCR/ABL kinase inhibitors. CONCLUSIONS: Rapamycin shows antileukaemic effects in imatinib-resistant CML in vitro and in vivo. Larger trials with rapamycin or rapamycin-derivatives in combination with other targeted drugs are warranted to further determine clinical efficacy in CML.

Efficient retrovirus-mediated gene transfer of dendritic cells generated from CD34+ cord blood cells under serum-free conditions.

A retroviral-vector encoding the low affinity nerve growth factor receptor (LNGFR) was used to transduce dendritic cells (DCs) generated from CD34+ cord blood (CB) progenitor cells under serum-free conditions. Transduction efficiency was monitored by flow cytometry (FACS) using a specific monoclonal antibody. Prior to retroviral infections, CD34+ CB cells were stimulated for 60 h in a serum-free medium containing a DC differentiation inducing cytokine cocktail: stem cell factor (SCF), granulocyte/macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFalpha), and transforming growth factor beta 1 (TGF-beta1). Addition of flt3-ligand (FL) to the aforementioned growth factors significantly enhanced cell expansion (41.7+/-11.5 fold vs. 22.5+/-4.7 fold without FL) and generation of CD1a+ DCs (mean 45.7+/-9.8% vs. 28+/-6.5% without FL, n = 4,p = 0.01). Furthermore, FL significantly increased the proportion of CD1a+LNGFR+ cells (mean 10%+/-4.4% vs. 6%+/-2.4 without FL n = 4, p = 0.03). When serum-free viral supernatants were used to infect DCs progenitors under entirely serum-free conditions and with the most potent cytokine combination, approximately one-third of the CD1a+ DCs generated co-expressed the LNGFR gene. Moreover, the transduced gene was also identified in more mature CD1a+CD80+ and CD1a+CD86+ DCs after 12-14 days of culture. In addition, transduced CD1a+ DCs maintained their functional properties, stimulating allogeneic T cells with similar efficiency as nontransduced CD1a+ DCs. Thus, the serum-free system described allows efficient generation and transduction of CD1a+ DCs derived from CD34+ progenitor cells and may be very useful for future therapeutic applications of DCs.

Association between chronic cutaneous lupus erythematosus and HLA class II alleles.

CCLE, a disease entity at the benign end of the lupus spectrum, is characterized by marked photosensitivity and skin lesions in sun-exposed areas. The histopathology of lesions resembles hypersensitivity type IV reactions. We have asked whether an association between class II alleles and CCLE exists. RFLP analysis of HLA-DQA genes revealed a Taq I HLA-DQA1 allelic restriction fragment overrepresented in a group consisting of 26 patients as compared to healthy control individuals. This result was corroborated by typing with oligonucleotide probes. The presence of the DQA1*0102 allele in the patients' group led to a relative risk of 4.57, with a statistical significance of p < 0.05 after correction for 36 comparisons. Although not statistically significant, it is interesting that all patients possess in at least one of their HLA-DQA1 alleles a nucleotide sequence coding for the amino acid glutamine at position 34 of the DQ alpha molecule. The expected frequency of these alleles in the control population amounts to 82%. The HLA-DRB1*16 allele, which is found in linkage disequilibrium with the HLA-DQA1*0102 allele, is also observed at an increased frequency in the patient's group, though the association was not significant after correction for the number of comparisons. However, no associations of CCLE with alleles at the HLA-DPB1 locus was found. The association of CCLE with certain HLA class II alleles points to an involvement of HLA-DQ and/or -DR molecules in the pathogenesis of the disease. Alternatively, genetic loci in linkage disequilibrium may code for elements which contribute to the development of CCLE.(ABSTRACT TRUNCATED AT 250 WORDS)

HLA-DR1-positive patients suffering from rheumatoid arthritis are at high risk for developing mucocutaneous side effects upon gold therapy.

Population studies suggest an association between RA and, depending on the ethnic background, HLA-DR1 and/or -DR4. One standard regimen for the treatment of RA is the use of gold compounds like SATM to arrest progression of the disease. In the present study, the immunogenetic background of RA patients developing side effects upon SATM treatment was determined. A total of 53 patients under SATM therapy were tested for their HLA-DRB and -DQ alleles by DNA typing; a significantly higher frequency of HLA-DR1 (p < 0.004, uncorrected) was observed in patients presenting with mucocutaneous side effects (MCT) when compared with patients without MCT. The RR was 6.85. Thus, HLA-DR1 seems to be a marker for the susceptibility of gold adverse reactions.

Association between IgE response against Bet v I, the major allergen of birch pollen, and HLA-DRB alleles.

The association of the human IgE response against Bet v I, the major allergen of birch pollen, and the HLA-DR and DQ phenotype was studied. Birch pollen allergic patients showed a typical case history, positive skin-prick test, and positive RAST with birch pollen extracts. They were divided into two groups. Group I (n = 37) consisted of individuals generating IgE antibodies that selectively reacted with Bet v I. Their serum IgE did not react with minor allergens from birch pollen as tested by immunoblot analysis, nor did they show a response against allergens from a panel of grass and other tree pollen or perennial allergens from animals and fungi as determined by skin-prick test. Patients belonging to group II (n = 34) possessed IgE reacting with Bet v I plus one or more additional allergens. The control group consisted of 637 healthy blood donors. Comparison of the frequencies of RFLP-defined HLA-DR and DQ alleles in patients and the control group revealed that the distribution of DRB3 alleles in group I patients differed significantly from that in the control group: A higher frequency of the DRw52a/c alleles in comparison to the control group (pcorr less than 0.02) was observed. In addition, alleles defined by nucleotide sequences coding for the amino acid sequence tyrosine-phenylalanine-histidine at positions 30-32 of the beta chain of DR molecules were found with a higher frequency in patient group I (pcorr less than 0.02), too. These alleles comprise DRw52a/c and some DRB1 alleles.(ABSTRACT TRUNCATED AT 250 WORDS)

The CDw65 monoclonal antibodies VIM-8 and VIM-11 bind to the neutral glycolipid V3FucnLc8Cer.

At the IVth and Vth Workshop on Human Leukocyte Differentiation Antigens a group of monoclonal antibodies recognizing myeloid cells was found to bind to the ganglioside X3-NeuAcVII3FucnLc10Cer (VIM-2 dodecasaccharide). These antibodies were given the provisional cluster of differentiation designation CDw65. Three antibodies of this cluster (VIM-2, VIM-8, and VIM-11) have now been studied in detail at the molecular and the cellular level. Binding of VIM-2 is abolished after treatment of cells with Vibrio cholerae neuraminidase, whereas VIM-8 and VIM-11 show enhanced binding to neuraminidase-treated cells. We investigated binding of the three mAbs to glycolipid antigens with shorter carbohydrate chains. Distinct differences were observed in the binding of CDw65 antibodies to VIII3-NeuAcV3FucnLc8Cer (VIM-2 decasaccharide). VIM-2 strongly bound to this antigen, whereas no binding was observed with the other two mAbs. Conversely, the asialoganglioside of the VIM-2 decasaccharide, V3FucnLc8Cer, was not recognized by VIM-2, but this antigen bound strongly VIM-8 and VIM-11. Thus, VIM-2 and the other CDw65 antibodies represented two different antigen specificities.

Dendritic Cell Generation from Highly Purified CD14+Monocytes.

Dendritic cells (DC) play a pivotal role in the function of the immune system, for they are the primary antigen-presenting cells (APC) in the activation of naive T-lymphocyte responses (1). Recent studies have uncovered complexity in the DC lineage with several subsets, functions, and maturational stages. Although it is generally accepted that human DC derive from hematopoietic progenitor cells (2-9), it is not clear at present whether DC cells and their precursors represent a separate hematopoietic lineage or whether DC should be seen as specialized macrophages with particular morphological, molecular, and functional features. Several lines of evidence point to DC and monocytes/ macrophages being offspring of the same CD34(+) hematopoietic progenitor cell (3-5,12-14, and reviewed in ([10,11].) DC committed precursor cells have also been identified in peripheral blood (15-18).

[The use of highly polymorphic DNA systems in the demonstration of mixed chimerism following bone marrow transplantation]. / Der Einsatz hochpolymorpher Systeme der DNA zum Nachweis des gemischten Chimärismus nach Knochenmarkstransplantation.

The demonstration of restriction fragment length polymorphism (RFLP) of the highly polymorphic systems MS1, MS31, g3, and MS43 to detect mixed chimerism after bone marrow transplantation is discussed. Degree of heterozygosity, somatic stability and sensitivity are the parameters investigated to demonstrate the practicability of this method. Examples of mixed chimerism after bone marrow transplantation are shown.

[Identification of an old frozen alcohol blood sample using a genetic probe technique]. / Identifikation einer alten eingefrorenen "Alkohol"-Blutprobe mittels Gensondentechnik.

Determination of ethanol concentration in a blood sample drawn from a person who caused a serious car crash showed a level which was markedly above the upper limit tolerated legally i.e. 0.08%. At the court hearing the accused car driver challenged the drunken driving charge and claimed that there might have been a mix up of the blood samples, whereby his was replaced by another blood sample, since the tube containing his blood was not marked with his name. The blood sample had been stored without anticoagulants for about 6 months at -20 degrees C. Due to haemolysis it was impossible to determine conventional haemogenetic marker systems. We therefore tried to extract DNA from the blood sample and to determine the restriction fragment length polymorphism (RFLP) by means of five DNA probes recognizing highly polymorphic single-locus systems as described by Jeffreys et al. We analyzed the RFLP's of both the old blood sample and of fresh blood drawn from the accused car driver and we were able to identify the blood sample as certainly having been taken from the accused.

[Regulation of cytokine production by human T-lymphocytes in allergic immune response]. / Regulation der Zytokinproduktion humaner T-Lymphozyten in der allergischen Immunantwort.

A network of cytokines regulates the growth and function of the cells of the immune system. T cells possess a dominant role in this network since they are the main source of many cytokines. The production of different cytokines is specifically regulated by means of cell interactions and cytokines and depends largely on the state of differentiation of the T cell. The production of certain cytokines is, therefore, restricted to defined subpopulations of T cells. IL2, IL4, IL5, IL10, IL12 and Interferon gamma (IFN gamma) play a central role in allergic immune response. The production of these cytokines can be restricted to specialized T cells. Two different types of differentiated T cells can be characterised according to the pattern of cytokine production of T cells: IL2 and IFN gamma are typically produced by T helper 1 (Th 1) cells, whereas predominantly T helper 2 (Th 2) cells produce IL4, IL5 and IL10. The selective activation of either Th 1 or Th 2 type cells depends on the antigen and is influenced by cytokines produced partly by antigen-presenting cells and partly by T helper cells. Typically, allergens induce an immune response which is dominated by Th 2 cells. These cells then stimulate the production of IgE by B cells.

[Usefulness and cogency of gene probes in paternity questions (Personal experiences: 1989/90]. / Brauchbarkeit und Beweisert von Gen-Sonden in Abstammungsfragen (Eigene Erfahrungen: 1989/90).

In cases of disputed parentage usually more than 20 polymorphic systems (red and white cell antigens, serum markers and enzyme markers) have to be analyzed using a battery of different techniques. A more recent method involving analysis of restriction fragment length polymorphism in highly polymorphic genes allows assignment of offspring to their parents. To test the latter method, 28 paternity cases were studied in parallel using conventional systems and detection of RFLPs of probes MS 1, MS 31, MS 43, and g 3, developed by Jeffreys et al. Furthermore, the cogency of exclusion of parentage, the average power of exclusion and the probability of parentage is calculated using published mutation rates and gene frequencies of the four probes. In conclusion, use of the four gene probes has both theoretically and practically turned out to be a powerful method for parentage testing.

Azithromycin suppresses CD4(+) T-cell activation by direct modulation of mTOR activity.

Advanced macrolides, such as azithromycin (AZM) or clarithromycin (CLM), are antibiotics with immunomodulatory properties. Here we have sought to evaluate their in vitro influence on the activation of CD4(+) T-cells. Isolated CD4(+) T-cells were stimulated with agonistic anti-CD3/anti-CD28 monoclonal antibodies in the presence of 0.6 mg/L, 2.5 mg/L, 10 mg/L or 40 mg/L AZM or CLM. Cell proliferation, cytokine level in supernatants and cell viability was assessed. Intracellular signaling pathways were evaluated using reporter cell lines, FACS analysis, immunoblotting and in vitro kinase assays. AZM inhibited cell proliferation rate and cytokine secretion of CD4(+) T-cells in a dose-dependent manner. Similarly, high concentrations of CLM (40 mg/L) also suppressed these T-cell functions. Analysis of molecular signaling pathways revealed that exposure to AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target of mTOR. This effect was also observed at 40 mg/L CLM. In vitro kinase studies using recombinant mTOR showed that AZM inhibited mTOR activity. In contrast to rapamycin, this inhibition was independent of FKBP12. We show for the first time that AZM and to a lesser extent CLM act as immunosuppressive agents on CD4(+) T-cells by inhibiting mTOR activity. Our results might have implications for the clinical use of macrolides.