Anti-IL-12Rβ1 MAb (2.4E6) Regulates Th1 Bias In Bone Marrow Mononuclear Cells From Aplasticanemia Patients In Vitro

The pathophysiology of acquired aplastic anemia(AA)can be characterized by a marked reduction in both the quality and quantity of hematopoietic stem cells resulting in pancytopenia.Pathophysiologically,acquired AA seems to be immune-mediated,causing an organ-specific destruction of bone marrow hematopoietic stem cells and progenitor cells through abnormal T cell activation in most patients.The most convicing evidence for the dominant role of the immune system in pathophysiology of AA was lately supported by the efficacy of immunesuppressive treatment in the majority of patients.Nevertheless,understanding of the initiating and perpetuating mechanisms in AA is still incomplete.The imbalance of Th1/Th2 plays an important role in the onset and developmentof AA.Previous studies have also demonstrated endogenous stimulation in AA T cells and supported the Th1/Tc1 autoimmune basis of this complex disease.Aberrant type 1 immune responses have been linked to the pathogenesis of aplastic anemia,and cytokine drives a highly pathogenic T cell population involved in its initiation. Numerous studies have established IL-12 as an important factor for the differentiation ofnaive T cells into IFN-γ-producing Th1 cells,IL-23,a cytokine that shares the p40 subunit with IL-12,has since prompted reevaluation of the role of IL-12/Th1 response in autoimmunity.These cytokines play a pivotal role in the establishment and maintenance of autoimmune diseases and would be new therapeutic targets. PARTⅠEXPRESSION OF TIM-3 AND ITS LIGAND IN APLASTIC ANEMIA PATIENTSObjective:To investigate the Th1 shift in the cytokines and Th1 specific surface molecule in AA patients and to explore the immune mechanism of TIM-3 in aplastic anemia development.Material and methods:1.Bone marrow and peripheral blood samples were collected from 29 AA patients and 31 patients with non-hematopoietic diseases.2.Bone marrow mononuclear cells and peripheral blood mononuclear cells were prepared by ficoll gradient centrifugation technique.3.Concentration of IFN-γ,IL-18,IL-23 and IL-17 in sera from AA patients and controls were determined by ELISA.4.The peripheral blood was incubated with different combination of cell surface antibodies.The analysis of the TIM-3 expression in CD3~+CD4~+(CD4~+ T cells), CD3~+CD8~+(CD8~+ T cells),CD14~+(monocytes),CD19~+(B cells), CD3~-CD16~+CD56~+(NK cells),and CD3~+CD16~+CD56~+(T cells with NK markers) cells were accomplished with flow cytometry.Intracellular staining was performed for analysis of CD4~+CD25~+Foxp3~+ regulatory T cells.The datas were analyzed using the CellQuest software.5.Real-time quantitative RT-PCR was used for relative quantitation of mRNA in bone marrow mononuclear cells and peripheral blood mononuclear cells. Expression of TIM-3,IFN-γ,galectin-9 and Foxp3 transcripts were normalized respectively toβ-actin mRNA.6.The results were expressed in mean±standard deviation(mean±s).Student's t-test and Spearman's rank test were performed.P value＜0.05 is considerated as significant.Result:1.The serum concentration of IFN-γ,IL-18,IL-23 and IL-17 all increased in AA patients compared with controls(IFN-γ:108.96±35.80 vs.42.41±39.36,pg/ml, p=0.002;IL-18:485.40±227.08 vs.71.59±73.91,pg/ml,p＜0.001;IL-23: 606.88±110.16 vs.213.52±143.10,pg/ml,p＜0.001;IL-17:238.05±173.28 vs. 32.38±29.19,pg/ml,p＜0.001,respectively).They are both higher in SAA than in NSAA(p=0.001,p=0.028,p=0.002,p=0.032,respectively).2.The major cellular source of TIM-3 in peripheral blood in the controls was CD14~+ monocytes and NKT cells.There was remarkable elevation in TIM-3 protein as well as the frequency of TIM-3~+ cells in peripheral blood mononuclear cells from AA patients compared with controls(68.68±14.76 vs 41.66±15.19,p＜0.001; 51.45±10.95 vs 28.11±12.31,%,p＜0.001,respectively).In AA patients,the majority expressing TIM-3 protein was CD4~+ T cells,CD14~+ monocytes and CD8~+ T cells.3.The frequency of CD4~+CD25~+Foxp3~+T cells in AA patients was much decreased compared with that from the controls(0.69±0.52 vs 2.24±0.16,%,p=0.002).The intracellular Foxp3 protein was also significantly reduced in AA patients (51.71±32.12 vs 100.14±60.24,p=0.006).4.Compared with controls,the expression of TIM-3 and IFN-γmRNA was increased in peripheral blood mononuclear cells from AA patients by 3.17-fold and 4.71-fold respectively(both p＜0.001),and by 4.78-fold and 7.13-fold respectively in bone marrow mononuclear cells of AA patients(p=0.001,p＜0.001, respectively).Significant correlation was determined between the mRNA expression of TIM-3 and IFN-γin both peripheral blood mononuclear cells (r=0.806;p＜0.001)and bone marrow mononuclear cells(r=0.811;p=0.001)from AA patients.5.The expression of galectin-9 transcript increased by 4.0-fold in peripheral blood mononuclear cells from AA patients than controls(p=0.03).6.The expression of Foxp3 transcript significantly decreased in peripheral blood mononuclear cells from AA patients than controls(p=0.001). Conclusion:1.Overproduction of IFN-γ,IL-18,IL-23 and IL-17 was observed in sera from AA patients.2.Realtime RT-PCR results revealed significantly higher mRNA expression of TIM-3 in peripheral blood mononuclear cells and bone marrow mononuclear cells from AA patients compared with controls,which well correlated with IFN-γmRNA.3.Decreased number of regulatory T cell and low expression of Foxp3 were observed in AA patients.4.High expression of galectin-9,the ligand of TIM-3,may be implicated in autoimmune marrow failure.PARTⅡANTI-IL-12Rβ1 mAb REGULATES T-bet AND pSTAT4 EXPRESSION IN BMMCs FROM APLASTIC ANEMIA PATIENTS IN VITROObjective:To investigate the effect of anti-IL-12Rβ1 mAb on Th1 shift in bone marrow mononuclear cells from AA patients in vitro.Material and methods:1.Bone morrow samples were collected from 13 AA patients and 10 patients with non-hematopoietic diseases.2.Bone marrow mononuclear cells were prepared by Ficoll gradient centrifugation technique.(1)Freshly isolated bone marrow mononuclear cells from 10 AA patients and 5 controls were incubated for 72 hours in culture media with anti-CD3 mAb (5μg/ml)or without any stimulation.Supernatant was collected.(2)Freshly isolated bone marrow mononuclear cells from 13 AA patients were cultured by stimulation with anti-CD3 mAb(5μg/ml)in the presence or absence of anti-IL-12Rβ1 mAb for 72 hours.Freshly isolated bone marrow mononuclear cells from 10 controls were cultured by stimulation with anti-CD3 mAb(5μg/ml) for 72 hours.Cell pellets and supernatant were collected. 3.The level of IFN-γin culture supernatant was determined by ELISA.4.Real-time quantitative RT-PCR was used for relative quantitation of mRNA in bone marrow mononuclear cells.Expression of GATA-3,IFN-γand T-bet transcripts were normalized respectively toβ-actin mRNA.5.Western blotting was used for determination of protein in bone marrow mononuclear cells.Expression of GATA-3,pSTAT4,STAT4 and T-bet was normalized respectively toβ-actin protein.6.The results were expressed in mean±standard deviation(mean±s).One-way analysis of variance(ANOVA)or Kruskal-Wallis test were performed.P value＜0.05 is considerated as significant.Result:1.Bone marrow mononuclear cells from controls without any stimulation produced lower level of IFN-γin supernatants(18.64±11.95,pg/ml).IFN-γproduction was elevated by 9.29-fold by stimulation with anti-CD3 mAb(p＜0.001).Bone marrow mononuclear cells from AA patients without any stimulation produced increased levels of IFN-γcompared with controls(127.42±60.16,pg/ml,p＜0.001).IFN-γproduction was elevated by 2.36-fold by stimulation with anti-CD3 mAb (p=0.001).2.Anti-IL-12Rβ1 mAb significantly decreased production of IFN-γin culture supernatant in a dose-dependent manner(all p＜0.001).3.Anti-IL-12Rβ1 mAb significantly decreased expression of IFN-γand T-bet at transcript level in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in a dose-dependent manner(p=0.046,p=0.002,p＜0.001 for IFN-γ; p=0.002,p＜0.001,p＜0.001 for T-bet,respectively).4.Anti-IL-12Rβ1 mAb significantly decreased expression of T-bet and pSTAT4 at protein level in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in a dose-dependent manner(all p＜0.001 for T-bet;p=0.048,p＜0.001, p＜0.001 for pSTAT4,respectively). Conclusion:1.There is aberrant activation of Th1 cell in AA resulting in overproduction of IFN-γwithout any prior stimulation.2.Anti-IL-12Rβ1 mAb may significantly decrease expression of IFN-γat both transcript and protein levels in a dose-dependent manner in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in vitro.3.Anti-IL-12Rβ1 mAb may significantly decrease expression of T-bet at both transcript and protein levels in a dose-dependent manner in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in vitro.4.Anti-IL-12Rβ1 mAb may significantly decrease pSTAT4 at protein level in a dose-dependent manner in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in vitro.