Interleukin- 2 production during murine infection by Leishmania mexicana amazonensis

Highly susceptible BALB/c mice, resistant C57B1/6 and their F1 progeny (BDF1) were infected subcutaneously in the foot pad with Leishmania mexicana amazonenesis. At various times after infection, spleen or draining popliteal lymph node cells were assayed for their capacity to generate Interleukin-2 (I1-2) by Concanavalin A (ConA) stimulation. In both BALB/c and C57B1/6 strains there was a transient increase in their capacity to produce I1-2, from the 3rd to the 10th week post-infection. Return to pre-infection levels ocurred between 13th to 16th week post-infection in all three strains. BALB/c mice always produced higher titers of 11-2 than C57B1/6, but such differences were statistically significant only at 3 and 10 weeks post-infection. BDF1 mice had titers similar to those observed in BALB/c mice. I1-2 production by ConA-stimulated lymph node cells was lower as compared to the spleen, but with a similar pattern among the three mice strains. Our data show that susceptibility to infection by l. mexicana amazonenesis is not associated with deficient ConA-stimulated I1-2 production.

High antigen concentration inhibits T cell proliferation but not interleukin 2 production: examination of limiting dilution microcultures and T cell clones.

The effect of high antigen dose on the activation of cytochrome c peptide-primed lymph node cells was determined in several strains of mice by a limiting dilution analysis. It was found that proliferation of cytochrome c peptide-specific T cells was completely inhibited at high antigen concentration in C57BL/6 but only partially in DBA mice and had no effect in SJL mice. Clones derived from DBA mice showed a differential capacity to be inhibited by high antigen dose. On the other hand, interleukin 2 production by these clones was not impaired regardless of the antigen concentrations used.

T-cell hybridoma specific for a cytochrome c peptide: specific antigen binding and interleukin 2 production.

T-cell hybridomas were obtained after fusion of BW 5147 thymoma and long-term cultured T cells specific for cytochrome c peptide 66-80 derivatized with a 2,4-dinitroaminophenyl (DNAP) group. The resulting hybridomas were selected for their capacity to specifically bind to soluble radiolabeled peptide antigen. One T-cell hybrid was positive for antigen binding. This hybrid T cell exhibits surface phenotypic markers of the parent antigen-specific T cells. The binding could be inhibited either by an excess of unlabeled homologous antigen or by cytochrome c peptide 11-25 derivatized with a 2-nitrophenylsulfenyl group. Several other peptide antigens tested failed to inhibit binding of the radioactive peptide. This suggests that a specific amino acid sequence, modified by a DNAP group, is the antigenic structure recognized by the putative T-cell receptor. In addition, direct interaction of DNAP-66-80 peptide with the hybridoma cell line induced production of the T-cell growth factor interleukin 2. Furthermore, supernatants derived from syngeneic macrophages pulsed with the relevant peptide also induced the antigen-specific hybridoma to produce interleukin 2.

Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production

T cell receptor ζ (TcRζ)/CD3 ligation initiates a signaling cascade that involves src kinases p56lck and ζ-associated protein 70, leading to the phosphorylation of substrates such as TcRζ, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59fyn, the TcRζ/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59fyn, FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRζ/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists.

Full activation of T cells requires signaling through the T-cell antigen receptor (TCR) and additional surface molecules interacting with ligands on the antigen-presenting cell. TCR recognition of agonist ligands in the absence of accessory signals frequently results in the induction of a state of unresponsiveness termed anergy. However, even in the presence of costimulation, anergy can be induced by TCR partial agonists. The unique pattern of early receptor-induced tyrosine phosphorylation events induced by partial agonists has led to the hypothesis that altered TCR signaling is directly responsible for the development of anergy. Here we show that anergy induction is neither correlated with nor irreversibly determined by the pattern of early TCR-induced phosphorylation. Rather, it appears to result from the absence of downstream events related to interleukin 2 receptor occupancy and/or cell division. This implies that the anergic state can be manipulated independently of the precise pattern of early biochemical changes following TCR occupancy, a finding with implications for understanding the induction of self-tolerance and the use of partial agonist ligands in the treatment of autoimmune diseases.

Cancer vaccines: the interleukin 2 dosage effect.

Cancer vaccines genetically engineered to produce interleukin 2 have been investigated intensively in a series of animal models and are at the point of entering into clinical trials. In this study we demonstrate a strong correlation between the rate of interleukin 2 production and the protection efficiency of murine S91 melanoma cell (clone M-3) vaccines. Best immunization is achieved with vaccines producing medium interleukin 2 levels of 1000-3000 units per 10(5) cells per day. Reduced interleukin 2 production evokes a corresponding decline in the number of successfully treated animals. Unexpectedly, when interleukin 2 expression is raised to high levels of 5000-7500 units per 10(5) cells per day, protection is completely absent because of impaired generation of tumor-specific cytotoxic T lymphocytes. In comparison, granulocyte-macrophage colony-stimulating factor as immunomodulator induces substantial immunization even at a moderate level of secretion and protects all animals at the maximal obtainable level of secretion. Our findings demonstrate the importance of the interleukin 2 level produced by genetically modified tumor cells and may have substantial impact for the clinical application of cancer vaccines.

Therapeutic administration of KM+ lectin protects mice against Paracoccidioides brasiliensis infection via interleukin-12 production in a Toll-like receptor 2-dependent mechanism

KM+ is a mannose-binding lectin from Artocarpus integrifolia that induces interleukin (IL)-12 production by macrophages and protective T helper I immune response against Leishmania major infection. in this study, we performed experiments to evaluate the therapeutic activity of jackfruit KM+ (jfKM(+)) and its recombinant counterpart (rKM(+)) in experimental paracoccidioidomycosis. To this end, jfKM(+) or rKM(+) was administered to BALB/c mice 10 days after infection with Paracoccidiodes brasiliensis. Thirty days postinfection, lungs from the KM+-treated mice contained significantly fewer colony-forming units and little to no organized granulomas compared to the controls. In addition, lung homogenates from the KM+-treated mice presented higher levels of nitric oxide, IL-12, interferon-gamma, and tumor necrosis factor-a, whereas higher levels of IL-4 and IL-10 were detected in the control group. With mice deficient in IL-12, Toll-like receptor (TLR) 2, TLR4, or TLR adaptor molecule MyD88, we demonstrated that KM+ led to protection against P. brasiliensis infection through IL-12 production, which was dependent on TLR2. These results demonstrated a beneficial effect of KM+ on the severity of P. brasiliensis infection and may expand its potential use as a novel immunotherapeutic molecule.

Defective production of interleukin 2 in patients with Chagas' disease: purified IL-2 augments in vitro response in patients with chagasic cardiomyopathy

The production of interleukin 2 (IL-2) by peripheral blood mononuclear cells, from patients with different clinical forms of Chagas disease and healthy controls, was evaluated after stimulation with Trypanosoma cruzi antigen, PPD and PHA. PHA induced higher production of IL-2 in infected patients than healthy controls. No diferences were found between infected groups. With PPD the trend was similar, the only difference was that asymptomatic infected patients (INF) showed higher levels of IL-2 production than patients with cardiomyopathy (CDM). With T. cruzi antigen, most patients showed little or no IL-2 production at 24 hr, a peak at 48 hr and an abrupt fall at 72 hr. A similar pattern of IL- 2 production was observed in INF and CDM. To evaluate the physiologic relevance of the deficit in IL-2 production, we studied the effect of non-mitogenic concentratios of IL-2 in the proliferative response to specific antigens. The addition of IL-2 only enhanced the proliferative response of CDM patients. These observations suggest that patients suffering Chagas' disease, particularly CDM, have a significant reduction in the capacity to produce IL-2. These findings could be of importance in the pathogenesis of Chagas' disease.

Effect of metabolic control on interferon-gamma and interleukin-10 production by peripheral blood mononuclear cells from type 1 and type 2 diabetic patients

The objective of the present study was to evaluate the production of cytokines, interferon-g (INF-g) and interleukin-10 (IL-10), in cultures of peripheral blood mononuclear cells (PBMC) from type 1 and type 2 diabetic patients and to correlate it with inadequate and adequate metabolic control. We studied 11 type 1 and 13 type 2 diabetic patients and 21 healthy individuals divided into two groups (N = 11 and 10) paired by sex and age with type 1 and type 2 diabetic patients. The PBMC cultures were stimulated with concanavalin-A to measure INF-g and IL-10 supernatant concentration by ELISA. For patients with inadequate metabolic control, the cultures were performed on the first day of hospitalization and again after intensive treatment to achieve adequate control. INF-g levels in the supernatants of type 1 diabetic patient cultures were higher compared to type 2 diabetic patients with adequate metabolic control (P < 0.001). Additionally, INF-g and IL-10 tended to increase the liberation of PBMC from type 1 and 2 diabetic patients with adequate metabolic control (P = 0.009 and 0.09, respectively). The increased levels of INF-g and IL-10 released from PBMC of type 1 and 2 diabetic patients with adequate metabolic control suggest that diabetic control improves the capacity of activation and maintenance of the immune response, reducing the susceptibility to infections.

Interleukin-10 production by tumor infiltrating macrophages plays a role in Human Papillomavirus 16 tumor growth

Background: Human Papillomavirus, HPV, is the main etiological factor for cervical cancer. Different studies show that in women infected with HPV there is a positive correlation between lesion grade and number of infiltrating macrophages, as well as with IL-10 higher expression. Using a HPV16 associated tumor model in mice, TC-1, our laboratory has demonstrated that tumor infiltrating macrophages are M2-like, induce T cell regulatory phenotype and play an important role in tumor growth. M2 macrophages secrete several cytokines, among them IL-10, which has been shown to play a role in T cell suppression by tumor macrophages in other tumor models. In this work, we sought to establish if IL-10 is part of the mechanism by which HPV tumor associated macrophages induce T cell regulatory phenotype, inhibiting anti-tumor activity and facilitating tumor growth. Results: TC-1 tumor cells do not express or respond to IL-10, but recruit leukocytes which, within the tumor environment, produce this cytokine. Using IL-10 deficient mice or blocking IL-10 signaling with neutralizing antibodies, we observed a significant reduction in tumor growth, an increase in tumor infiltration by HPV16 E7 specific CD8 lymphocytes, including a population positive for Granzyme B and Perforin expression, and a decrease in the percentage of HPV specific regulatory T cells in the lymph nodes. Conclusions: Our data shows that in the HPV16 TC-1 tumor mouse model, IL-10 produced by tumor macrophages induce regulatory phenotype on T cells, an immune escape mechanism that facilitates tumor growth. Our results point to a possible mechanism behind the epidemiologic data that correlates higher IL-10 expression with risk of cervical cancer development in HPV infected women.

Lactic acid stimulates interleukin-23 production by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide

Lactic acid is the predominant acid present in the vagina. We evaluated the consequences of lactic acid, at physiological levels present in the vagina, on cytokine responses of peripheral blood mononuclear cells (PBMCs) obtained from 10 individuals in the presence or absence of bacterial lipopolysaccharide. Preincubation of PBMCs in 15 mM lactic acid before the addition of lipopolysaccharide resulted in a 246% mean increase in interleukin-23 (IL-23) secretion over that released in the presence of lipopolysaccharide alone (P=0.0068). The lipopolysaccharide-induced production of tumor necrosis factor-alpha, IL-6, IL-10 and IL-12 was unaffected by lactic acid. IL-23 stimulation was not observed if the lactic acid was neutralized before its addition to the culture medium or if hydrochloric acid was substituted for lactic acid. In the absence of lipopolysaccharide, lactic acid did not stimulate the production of IL-23 or any of the other cytokines. The increase in IL-23 production was proportional to the lactic acid concentration over a 15-60 mM range. We conclude that at body sites characterized by lactic acid accumulation, such as in the human vagina, exposure to gram-negative bacteria results in selective IL-23 production, leading to a subsequent preferential stimulation of the Th17 T lymphocyte pathway.

Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients

This study was designed to assess the effect of GB virus (GBV)-C on the immune response to human immunodeficiency virus (HIV) in chronically HIV-infected and HIV- hepatitis C virus (HCV)-co-infected patients undergoing antiretroviral therapy. A cohort of 159 HIV-seropositive patients, of whom 52 were HCV-co-infected, was included. Epidemiological data were collected and virological and immunological markers, including the production of interferon gamma (IFN-γ) and interleukin (IL)-2 by CD4, CD8 and Tγδ cells and the expression of the activation marker, CD38, were assessed. A total of 65 patients (40.8%) presented markers of GBV-C infection. The presence of GBV-C did not influence HIV and HCV replication or TCD4 and TCD8 cell counts. Immune responses, defined by IFN-γ and IL-2 production and CD38 expression did not differ among the groups. Our results suggest that neither GBV-C viremia nor the presence of E2 antibodies influence HIV and HCV viral replication or CD4 T cell counts in chronically infected patients. Furthermore, GBV-C did not influence cytokine production or CD38-driven immune activation among these patients. Although our results do not exclude a protective effect of GBV-C in early HIV disease, they demonstrate that this effect may not be present in chronically infected patients, who represent the majority of patients in outpatient clinics.

Natural killer-like T cells develop in SJL mice despite genetically distinct defects in NK1.1 expression and in inducible interleukin-4 production.

An unusual subset of mature T cells expresses natural killer (NK) cell-related surface markers such as interleukin-2 receptor beta (IL-2R beta; CD122) and the polymorphic antigen NK1.1. These "NK-like" T cells are distinguished by their highly skewed V alpha and V beta repertoire and by their ability to rapidly produce large amounts of IL-4 upon T cell receptor (TCR) engagement. The inbred mouse strain SJL (which expresses NK1.1 on its NK cells) has recently been reported to lack NK1.1+ T cells and consequently to be deficient in IL-4 production upon TCR stimulation. We show here, however, that SJL mice have normal numbers of IL-2R beta+ T cells with a skewed V beta repertoire characteristic of "NK-like" T cells. Furthermore lack of NK1.1 expression on IL-2R beta+ T cells in SJL mice was found by backcross analysis to be controlled by a single recessive gene closely linked to the NKR-P1 complex on chromosome 6 (which encodes the NK1.1 antigen). Analysis of a panel of inbred mouse strains further demonstrated that lack of NK1.1 expression on IL-2R beta+ T cells segregated with NKR-P1 genotype (as assessed by restriction fragment length polymorphism) and thus was not restricted to the SJL strain. In contrast, defective TCR induced IL-4 production (which appeared to be a unique property of SJL mice) seems to be controlled by two recessive genes unlinked to NKR-P1. Collectively, our data indicate that "NK-like" T cells develop normally in SJL mice despite genetically distinct defects in NK1.1 expression and inducible IL-4 production.