Th1 Cytokine IFN Research Articles

A novel bispecific antibody targeting CD3 and prolactin receptor (PRLR) against PRLR-expression breast cancer

BackgroundProlactin receptor (PRLR) is highly expressed in a subset of human breast cancer and prostate cancer, which makes it a potential target for cancer treatment. In clinical trials, the blockade of PRLR was shown to be safe but with poor efficacy. It is therefore urgent to develop new therapies against PRLR target. Bispecific antibodies (BsAbs) could guide immune cells toward tumor cells, and produced remarkable effects in some cancers.MethodsIn this study, a bispecific antibody targeting both tumor antigen PRLR and T cell surface CD3 antigen (PRLR-DbsAb) was constructed by split intein mediated protein transsplicing (BAPTS) system for the first time. Its binding activity was determined by Biacore and Flow cytometry, and target-dependent T cell mediated cytotoxicity was detected using LDH release assay. ELISA was utilized to study the secretion of cytokines by immune cells. Subcutaneous tumor mouse models were used to analyze the in vivo anti-tumor effects of PRLR-DbsAb.ResultsPRLR-DbsAb in vitro could recruit and activate T cells to promote the release of Th1 cytokines IFN- γ and TNF- α, which could kill PRLR expressed breast cancer cells. In xenograft models with breast cancer cell line T47D, NOD/SCID mice intraperitoneally injected with PRLR-DbsAb exhibited significant inhibition of tumor growth and a longer survival compared to mice treated with PRLR monoclonal antibody (PRLR mAb).ConclusionsBoth in vitro and in vivo experiments showed PRLR-DbsAb had a potential therapy of cancer treatment potential therapy for cancer. Immunotherapy may be a promising treatment against the tumor target of PRLR.

Neutrophils express pro- and anti-inflammatory cytokines in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques

Neutrophils are implicated in the pathogenesis of tuberculosis (TB), a disease caused by Mycobacterium tuberculosis infection, but the mechanisms by which they promote disease are not fully understood. Neutrophils can express cytokines that influence TB progression, and so we compared neutrophil and T-cell expression of the Th1 cytokines IFNγ and TNF, the Th2 cytokine IL-4, and regulatory cytokine IL-10 in M. tuberculosis-infected macaques to determine if neutrophil cytokine expression contributes to dysregulated immunity in TB. We found that peripheral blood neutrophils produced cytokines after stimulation by mycobacterial antigens and inactive and viable M. tuberculosis. M. tuberculosis antigen-stimulated neutrophils inhibited antigen-specific T-cell IFNγ production. In lung granulomas, neutrophil cytokine expression resembled T-cell cytokine expression, and although there was histologic evidence for neutrophil interaction with T cells, neutrophil cytokine expression was not correlated with T-cell cytokine expression or bacteria load. There was substantial overlap in the spatial arrangement of cytokine-expressing neutrophils and T cells, but IL-10-expressing neutrophils were also abundant in bacteria-rich areas between caseum and epithelioid macrophages. These results suggest that neutrophils contribute to the cytokine milieu in granulomas and may be important immunoregulatory cells in TB granulomas.

Study on the correlation of the effect of entecavir on Th1/Th2 cytokines level in the treatment of chronic hepatitis

Objective: To explore the expression level of peripheral blood Th1/Th2 type cytokines of chronic hepatitis b (CHB) patients in the entecavir (ETV) antiviral treatment, analyze the relationship between various cytokines, and the correlation among of cytokines and HBV DNA loads. Methods: Luminex Liquid Chip Technology was applied to detect the peripheral blood Th1/Th2 type cytokines expression level of CHB patients; At the same time, liver function was detected by Fully Automatic Biochemical Analyzer; HBV DNA loads were detected by PCR Method; Hepatitis b virology markers were detected by Chemiluminescence Method. F-test and Pearson correlation analysis were used for statistical analysis. Results: Before ETV antiviral treatment, peripheral blood Th1 cytokines IFN gamma expression level in patients with CHB increased significantly (P = 0.010) compared with the healthy control group while TNF alpha expression level having no statistically significant difference (P = 0.095); Th2 type cytokines IL-4, IL-6, IL-10 levels decreased obviously (P = 0.039, P = 0.014, P = 0.026) compared with those in the control group. After 48 weeks of treatment, Th1 cytokines IFN gamma and TNF alpha expression levels were reduced significantly (19.2±5.03 pg/ml vs 24.69±6.51 pg/ml, and 6.09±4.99 pg/ml vs 9.50±7.34 pg/ml, P < 0.001, and P = 0.016) compared with that before treatment; Th2 type cytokines IL-4, IL-6 expression level increased significantly (33.86±22.47 pg/ml vs 21.32±12.84 pg/ml, and 11.65±6.91 pg/ml vs 8.51±4.94 pg/ml, P = 0.004, and P = 0.029) compared with that before treatment while IL-10 expression level having no statistical significance (P = 0.081). There was disorder and irregularity in the correlation between the levels of Th1/Th2 type cytokines. And there was no correlation between the various cytokines and HBV DNA loads in patients with CHB. Conclusion: ETV can not only inhibit HBV DNA replication, reducing HBV DNA loads, but also contribute to regulate Th1/Th2 type cytokines expression level in patients with CHB, but there was no correlation between the levels of various cytokines, various cytokines and HBV DNA loads.

STAT4 and T-bet Are Required for the Plasticity of IFN-γ Expression across Th2 Ontogeny and Influence Changes in Ifng Promoter DNA Methylation

CD4(+) T cells developing toward a Th2 fate express IL-4, IL-5, and IL-13 while inhibiting production of cytokines associated with other Th types, such as the Th1 cytokine IFN- γ. IL-4-producing Th2 effector cells give rise to a long-lived memory population committed to reactivation of the Th2 cytokine gene expression program. However, reactivation of these effector-derived cells under Th1-skewing conditions leads to production of IFN-γ along with IL-4 in the same cell. We now show that this flexibility ("plasticity") of cytokine expression is preceded by a loss of the repressive DNA methylation of the Ifng promoter acquired during Th2 polarization yet requires STAT4 along with T-box expressed in T cells. Surprisingly, loss of either STAT4 or T-box expressed in T cells increased Ifng promoter CpG methylation in both effector and memory Th2 cells. Taken together, our data suggest a model in which the expression of IFN-γ by Th2-derived memory cells involves attenuation of epigenetic repression in memory Th2 cells, combined with Th1-polarizing signals after their recall activation.

P6: Th1/Th2 Cytokines promote Nitrite/H2O2-mediated tyrosine nitration in airway epithelial cells: Potential role in severe asthma

Protein tyrosine nitration is increased in the airways of asthmatics, particularly in severe disease, where the evidence of a complex adaptive immune process with elements of an active Th1 pathway, in combination with a Th2 signature, is increasingly observed. Although airway 3-nitrotyrosine (3NT) formation is generally attributed to peroxynitrite, formed from NO and superoxide, 3NT can also be generated through NO 2 radical, formed from nitrite and H 2 O 2 . Nitrite is an active metabolite of iNOS-derived NO, while in airway epithelial cells, H2O2 is believed to be primarily produced by Dual oxidase (DUOX)-2. In human airway epithelial cells, Th1 and Th2 cytokines induce iNOS, while DUOX2 and H 2 O 2 are upregulated by Th1 cytokine IFN- γ , and further enhanced by Th2 cytokine IL-13 (Voraphani et al., ATS abstract 2012). Whether the concomitant presence of Th1 and Th2 stimuli could augment nitrative stress is not known. We hypothesized the Th1 cytokine (IFN- γ ), in the face of a Th2 (IL-13) background, would enhance nitrite production through a nitrite/H2O2 pathway, which would contribute to increases in 3NT. Epithelial cells (from bronchial brushings) [1 normal control (NC), 12 mild/moderate (MMA), 6 severe asthmatics (SA)] were cultured in air–liquid interface with IL-13 (1,10 ng/ml) or media for 8 days, with/without IFN- γ (10,100 ng/ml) for the last 72 h. This was followed by 1-h incubation with PBS with/without the following: nitrite (10,25 μM), catalase (H 2 O 2 scavenger, 150 U/ml), or the combination. Nitrite and nitrate levels were measured by the Griess reaction. 3NT levels were quantified in lysates by ELISA. iNOS mRNA and protein were evaluated by RT-PCR and Western blot, respectively. Fresh bronchial epithelial cells from 7 NC, 9 MMA, and 10 SA were also analyzed for 3NT expression. Nitrite levels were dose-dependently increased by IFN- γ and IL-13. Although nitrate was the predominant metabolite generated by both cytokines, IFN- γ treatment resulted in a greater ratio of nitrite/nitrate. The presence of IFN- γ , together with low-dose IL-13, synergistically induced iNOS and nitrite expression compared to either cytokine alone. In contrast, the combination did not increase nitrate. Both low-and high-dose IFN- γ increased 3NT levels, while only low-dose IL-13 had an effect. Similar to iNOS and nitrite, 3NT levels were synergistically enhanced in the presence of low-dose IL-13 and IFN- γ . The percent increase in nitrite correlated with the percent increase in 3NT from baseline ( r = 0.7, p < 0.05) with combined low-dose IL-13 and low-dose IFN- γ . Furthermore, 3NT expression increased following nitrite addition, an effect marginally dependent on the presence of IL-13 and IFN- γ , and was attenuated ∼50% with catalase. 3NT levels were elevated in SA epithelial cells ex vivo compared to MMA ( p < 0.05) and NC ( p < 0.001). 3NT expression is increased in SA epithelial cells. In ALI culture, increases in 3NT paralleled increases in iNOS and nitrite, in response to the combination of Th1 and Th2 cytokines. This is similar to our findings on DUOX2 and H 2 O 2 . These results suggest Th1 stimuli, in the presence of a Th2 background, as seen in severe asthma, may play a key role in enhanced nitrative stress through its impact on nitrite/H 2 O 2 -mediated tyrosine nitration. Funding: HL69174, AI 67780, Dellenback Fund.

Abstract 5459: Unexpected Acceleration of Atherosclerosis in Ldlr-null Mice Reconstituted With Group X Secretory Phospholipase A2-Deficient Bone Marrow

Group X (GX) secretory phospholipase A2 (sPLA2) has the most potent hydrolyzing activity toward phosphatidylcholine and elicits a marked release of arachidonic acid among several types of sPLA2. GX is expressed by vascular cells and inflammatory cells like macrophages and CD4+ T lymphocytes and is therefore believed to play a pathogenic role in atherosclerosis. We evaluated the effects of GX deficiency on systemic immune response and the development of atherosclerosis, a vascular disease sustained by lipid modifications and inflammation. GX KO mice showed increased spleen size confirmed by higher splenocyte number compared to C57BL6 controls (P&lt;0.01). LPS/IFN- γ -stimulated GX-deficient splenocytes produced more IL-12 than control splenocytes (3380 vs 1599 pg/ml, P&lt;0.05), with no difference in IL-10. Purified splenic CD4+ T cells from GX KO mice showed enhanced proliferation and production of the Th1 cytokine IFN- γ (5575 vs 1443 pg/ml, respectively, p&lt;0.05) compared with controls. GX-deficient blood mononuclear cells showed enhanced adhesion on activated HUVECs (8.8 vs 3.2 cell/field, P&lt;0.01) and on LDLr KO carotid arteries (4.2 vs 6.4 cell/mm, P&lt;0.01) ex vivo, compared to control cells. When challenged in vitro to test cell death susceptibility to a variety of apoptotic stimuli, bone marrow-derived GX-deficient macrophages displayed a significant increase of apoptosis compared to control cells. Finally, LDLr KO mice were lethally irradiated and reconstituted with either GX KO or wild type bone marrow. After 8 weeks of fat diet, mice reconstituted with GX KO bone marrow showed a ~100% increase of plaque size in the aortic sinus compared to mice with WT bone marrow (294 254 μ m2 vs 142 304 μ m2, respectively, P=0.001), despite similar plasma cholesterol level. This was associated with a more advanced plaque phenotype and marked accumulation of TUNEL-positive apoptotic cells (2.4 % vs 0.08 P&lt;0.001). Immunologic analysis of splenocytes and splenic CD4+ T cells from GX/LDLr chimeric mice showed increased immune cell activation and Th1-related cytokine production. In conclusion, GX sPLA2 deficiency increases innate and adaptive immune responses, enhances macrophage susceptibility to apoptosis and accelerates atherosclerosis in mice.

Differential effects of Chlamydia pneumoniae infection on cytokine levels in human T lymphocyte- and monocyte-derived cell cultures

Continuous cultures of human lymphocyte- and monocyte-derived cell lines were examined for levels of immunoregulatory cytokines important in resistance to the intracellular opportunistic bacterium Chlamydia pneumoniae (Cp), a ubiquitous pathogen widely disseminated in the population and hypothesized to be involved in chronic inflammatory diseases such as atherosclerosis and neurological diseases like multiple sclerosis and Alzheimer's disease. The results of this study showed that the continuous human T lymphocyte cell line MOLT-4 and the continuous monocytic cell line THP-1 were readily infected by Cp in vitro as shown by immunofluorescence microscopy for Cp lipopolysaccharide (LPS). The 16S rRNA expression determined by real-time RT-PCR increased rapidly after infection of either cell line with these bacteria. The THP-1 cells infected with Cp showed increased levels of the immunoregulatory cytokine IL-12 and also of TNF α and IL-10 compared to cultures stimulated with heat-killed Cp (KCp) or Escherichia coli LPS as a control. Stimulation of MOLT-4 cells with KCp or E. coli LPS also induced the Th1 cytokines IFN γ and IL-12 and the Th2 cytokine IL-10, but infection with viable Cp induced higher Th1 cytokine levels. These results suggest that Cp infection induces a predominant Th1 cytokine profile by T cells, in addition to induction of TNF α by monocytes/macrophages. Such effects are likely involved in antibacterial immunity against Cp infection.

612. Determination of Specific Th1 and Th2 CD4 T Cell Responses in AAV2- and AAV8-Mediated Human Factor IX Gene Therapy in Mice

Recombinant AAV is promising for coagulant factor IX (F.IX) gene transfer due to the long-term expression of hF.IX in the liver and weak immune response using a liver specific promoter. However, in humans and some animals the F.IX expression declines over long time periods and immune responses to vector and transgene can occur. It was shown that C3H (H2k) mice express the highest levels of anti-hF.IX antibodies, which requires the IAka and IAk b and the IL-10 region of Chromosome 1 (Blood 105:1029). We utilized MHC class II peptides in the context of I-Ak and I-Ek to specifically detect Th cells in AAV-hF.IX immunized C3H mice. First, we use a prediction program, SYPFEITHI, to predict MHC II binding peptides from hF.IX, AAV2 and AAV8 capsid proteins (Table 1). C3H mice were iv injected with 2|[times]|1011vg of AAV2- or AAV8-hF.IX, in which hF.IX expression is regulated by a liver-specific ApoE/hAAT promoter. The mice were boosted 30 days later. Blood samples were collected for analysis of ALT and plasma hF.IX. The plasma ALT level was significantly elevated (p<0.05) at day 9 after boost with AAV2- or AAV8-hF.IX. There was no significant decrease in plasma hF.IX levels up to 9 days after boost. To determine the frequency of hF.IX, AAV2 and AAV8 specific CD4 Th cells in liver, mononuclear cells (MNC) were prepared and analyzed by peptide specific ELISPOT assays for production of Th1 cytokine IFN|[gamma]| and Th2 cytokine IL-4, respectively. Liver MNCs were co-cultured with irradiated antigen presenting cells pulsed with the peptides specific for hF.IX, AAV2 or AAV8. The number of IFN|[gamma]| spot forming cells (SFC) was determined 48 hr later (Table 1). For both AAV2-hF.IX and AAV8-hF.IX infected mice, an I-Ak restricted hF.IX epitope AA108-122 induced both IL-4 and IFN|[gamma]| production. In AAV2-hF.IX infected mice, an I-Ek restricted AAV2 epitope AA475-489 induced more IL-4 SFCs than IFN|[gamma]| SFCs (p<0.05). For AAV8-hF.IX infected mice, an I-Ek restricted AAV8 epitope AA126-140 was found to induce more IFN|[gamma]| SFCs than IL-4 SFCs (p<0.05). Similar results were seen in spleen of immunized mice. Other predicted peptides failed to induce IFN|[gamma]| or IL-4 SFCs in immunized mice (data not shown). These results indicated that AAV2- or AAV8-hF.IX gene transfer induced both vector and transgene specific Th cells with the highest response in IFN|[gamma]| production to hF.IX epitope peptide AA108-122. Moreover, these epitope peptides could function differently on Th1 and Th2 cells to drive a Th response towards CD8 T cell response or B cell activation. These findings will facilitate a better understanding in CD8 T cell response or antibody response to AAV-hF.IX gene therapy.

Activity and safety of DNA plasmids encoding IL-4 and IFN gamma.

Cytokine-encoding DNA plasmids can act as 'genetic adjuvants', improving the immune response stimulated by co-administered DNA vaccines. We examined whether plasmids encoding the Th1 cytokine IFN gamma (pIFN gamma) or the Th2 cytokine IL-4 (pIL-4) have long-term effects on immune homeostasis when administered to adult mice, or alter immune maturation in neonates. Both plasmids boosted immunity against a co-administered vaccine, with pIFN gamma promoting the development of a Th1 response (characterized by the production of IgG2a antibodies), and pIL-4 preferentially stimulating a Th2 response (characterized by increased IgG1 antibody production). Both pIFN gamma and pIL-4 influenced the ratio of cells actively secreting Th1 versus Th2 cytokines, consistent with an effect on Th cell maturation. Interestingly, this effect persisted for only a few weeks and was not magnified by repeated plasmid administration. Cytokine-encoding plasmids had no long-term effect on the immune response of newborn or adult mice to subsequent antigenic stimulation, nor did they selectively induce the production of pathogenic anti-DNA autoantibodies. These results suggest cytokine-encoding plasmids may be safe as immune adjuvants.

Linomide suppresses both Th1 and Th2 cytokines in experimental autoimmune myasthenia gravis

Suppressive effects of the synthetic immunomodulatory drug Linomide have been shown in several autoimmune models, including antibody-mediated experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia gravis (MG). To define the mechanisms underlying EAMG suppression, we injected Linomide subcutaneously at different doses into Lewis rats immunized with Torpedo acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA), and investigated AChR-specific T and B cell responses, and the levels of lymph node cells expressing mRNA of different cytokines after AChR stimulation in vitro. Both 160 and 16, but not 1.6, mg/kg/day of Linomide effectively suppressed clinical muscle weakness, accompanied by decreased AChR-induced T and B cell responses. Linomide also suppressed the mRNA expression of the Th1 cytokines IFN- γ, IL-12 and TNF- α as well as the Th2 cytokines IL-4 and IL-10, which are important in the immunopathogenesis of EAMG by promoting antibody production. There were no differences for IL-1 β, IL-6, lymphotoxin or TGF- β expression in Linomide-treated vs nontreated control EAMG rats. We conclude that Linomide suppresses clinical EAMG as well as B and T cell responses to AChR by counteracting the production of AChR-induced Th1 and Th2 cytokines.