Effect Of Indoleamine 2,3-dioxygenase In Immune Tolerace Of Non-Hodgkin's Lymphoma

BackgroundNon-Hodgkin's lymphoma is a malignance of the immune system. Immune tolerance participates in tumor transformation and metastasis, and possesses close relationship with tumor therapy and prognosis. In recent years, increasing evidence has been accumulated that tryptophan catabolism takes part in tumor induced immune tolerance. Indoleamine 2,3-dioxygenase(IDO) is the key enzyme in the tryptophan metabolism, which catalyzes the initial rate-limiting step of tryptophan degradation along the kynurenine pathway and can be expressed in many tissues and cells, but mainly upon cytokine stimulation during infection and with less substrate specificity. IDO activation leads to a tryptophan starvation and an accumulation of downstream breakdown products. Tryptophan starvation by IDO consumption inhibits T-cell activation, while products of tryptophan catabolism, such as kynurenine derivatives and 02-free radicals, regulate T-cell proliferation and survival.In humans, IDO expression has been observed by immunohistochemistry in placenta, tumor-draining lymph nodes and primary tumors, which in turn, is infiltrated by CD4+CD25+ Treg cells. The close relationship between IDO activity and Treg cells have recently been the focus of intense studies due to a positive correlation between the increased numbers of Treg cells and IDO expression in experimental as well as clinical settings. It has been shown that both IDO-expressing dendritic cells(DCs) and IDO-expressing tumor cell can bias naive CD4+ T cells to differentiate into FoxP3+ Tregs in vitro. This important finding thus linked IDO to the potent Treg system, which is known to be a key mechanism of immunosuppression in tumorbearing hosts. A series of studies provided the first indirect evidence that IDO may play an important role in proliferation and conversion of Tregs in tumors. In the present study, we aim to measure the expressions of IDO and FoxP3 in NHL patients and perform in vitro and animal experiments to investigate whether T-cell tolerance in NHL may be induced by directly converting CD4+CD25- T cells into CD4+CD25+Treg cells through an IDO-dependent mechanism.Part I Expression and Significance of IDO and FoxP3 in NHLObjectiveIn this study, we collected samples of NHL and detected the expression of IDO and FoxP3 by IHC staining, western blot and q-PCR. Expression of IDO and FoxP3 were then made analyses with clinical features.MethodsUnstained paraffin sections of NHL(n=57) and normal control(n=18) were obtained from the Pathology Department of Shandong Provincial Hospital. Fresh tumor tissues(n=20) and normal lymph nodes(n=9) were collected during lymph node biopsy or laparotomy. The diagnosis of lymphoma was based on clinical features and pathology. Immunohistochemical (IHC) analyses were performed by the avidin-biotin peroxidase complex method using standard manual methods. Tissue sections were stained with antibodies directed against human IDO and FoxP3.Western blot analyses and Real Time RT-PCR were performed to investigate the protein and mRNA expression of IDO and FoxP3.ResultsIn the IDO-staining sections, positive-staining was observed in tumor cells. In addition, it was clearly demonstrated that IDO was highly expressed in cytoplasm. In contrast, cells of positive-staining in control group were markedly less than that in NHL. In all eighteen normal control sections, the positive cell rate was less than 30%, while in NHL group, eighteen in fifty-seven sections(31.6%) possessed a positive cell rate of no less than 30%. Patients with IDO strong-stained subtype were more likely to be diagnosed at a late phase(P<0.05), present with large tumor (P<0.05) and have a higher level of LDH in serum(P<0.05), all of which may indicate a worse prognosis. We also measured the IDO protein level by western blotting, which was coincidentally higher in NHL(n=20) than controls(n=9). The ratio of IDO toβ-actin were 0.77±0.25 in NHL group, while 0.18±0.18 in couterparts(p<0.05). Corresponding IDO mRNA was evaluated in NHL tumors(n=20) as well as normal control(n=9) with real time RT-PCR. Higher IDO mRNA expression was also observed in NHL group, whereas there was very little IDO mRNA detected in normal control(2-dct:0.00582～0.546 v.0-0.0103, p<0.05).The percentage of FoxP3+ cells was significantly increased in IDO strong-stained NHL samples compared with IDO weak-stained NHL or normal control (P<0.05). Statistics indicated that a positive corelationship existed between IDO and FoxP3 expression(WB:r=0.482; real time PCR:r=0.447).Conclusions1. Both expressions of IDO and FoxP3 are upregulated in NHL,2. Over-expression of IDO is corelated with a later clinical phases and higher LDH values, which indicate a worse prognosis.3. IDO level possess a close relationship with that of FoxP3.PartⅡPurification of Murine CD4+CD25-T-cell Subsets and T-cell Coculture with IDO-expressing A20 Cells in vitroObjectiveIn this part, we aimed to observe whether IDO+ tumor cells could convert CD4+CD25- T cells into Tregs.MethodsMurine CD4+CD25- T-cell was isolated from splenocyte of normal BALB/c mice by MACS. Purified T cells were used for phenotypic assays using Tregs flow cytometry kit. Purifed murine CD4+CD25- with IDO-expressing A20 cell line in the presence and absence of 1-MT, which specially inhibits IDO activity. After 24 hours, cells were collected and used for flow cytometry assays and MACS.The converted CD4+CD25+ T cells were isolated by MACS. To test its suppressive function, the MTT cell proliferation assays were performed. DCs isolated from splenocytes by Dynabeads Mouse DC Enrichment Kit. Splenocytes from naive BALB/c were cultured alone(negtive control group) or cocultured with DCs. The mixed cells were cultured in the presence(experimental group) or absence(positive control group) of converted CD4+CD25+ T cells. Converted CD4+CD25+ T cells was cultured alone as blank control group. The absorbence at 570nm(A value) was recorded by microtiter plate reader. Suppressive rate(SR) was calculated. SR=[1-(Aexperimental group-Ablank control group-Anegtive control group)/(Apositive control group-Anegtive control group)]×100%ResultsConversion of CD4+CD25" T cells to CD4+CD25+ T cells was observed after 24 hours. The mean percentage of CD4+ CD25+T cells was 7.87±1.65% in CD4+CD25-T cells cocultured with A20 cell line, whereas CD4+CD25+ T cells were nearly not detected in CD4+CD25- cells cultured without A20. The addition of 1-MT attenuated conversion of CD4+CD25- T cells into CD4+CD25+ T cells, as we detected the mean percentage of CD4+CD25+ T cells was 3.32±1.19%.In the 3-day MTT proliferation assays, compared with the negtive control group, Apositive control goup was significantly increased(0.8657±0.0353 vs 0.3434±0.0319, p<0.05), which indicated that lymphocyte were stimulated to proliferate. Meanwhile, Aexperimental goup was lower than Apositive control goup.The addition of converted CD4+CD25+ T cells lead to the decreasing of A value(0.4331±0.0280 vs 0.8657±0.0353, P<0.05). The suppressive rate(82.54±7.31%) indicated the suppressive function of converted CD4+CD25+ T cells.Conclusions1. A20 cell line spontaneously high-express IDO.2. MACS is a simple but effective technique to isolate cells into different groups.3. Cocultured with A20 cell line, CD4+CD25- T cells will be converted to CD4+CD25+ T cells which could suppress immune response. The addition of 1-MT could abrogate this conversion. PartⅢStudy on Tumor Model for NHL in MiceObjectiveIn this part, we set up a tumor model for NHL. The contribution of IDO in tumor induced tolerance and the anti-tumor effect of inhibitor 1-MT would be determined by detecting the Tregs in the tumors, lymph nodes and spleens.MethodsTo establish tumors,5×106 live A20 cells were s.c. injected into the right back flank of naive syngeneic BALB/c mice. Tumor growth was monitored three times per week using calipers. For our studies, tumorbearing animals were injected with 1-MT intratumorally every day as soon as the tumors were palpable. PBS was used as a control. Animals bearing tumors were euthanized when tumors reached a size of 20 mm in diameter or earlier if tumors ulcerated or animal showed sign of discomfort. Flow cytometry was performed to measure the percentage of Tregs in tumor, spleen and tumor-draining lymphoma.Results1. The percentage of CD4+CD25+ T cells in lymphocytes and the ratio of CD4+CD25+T cells and CD4+T cells were significantly higher in tumorbearing animals compared with normal controls. The addition of 1-MT could deduce the increasing ratio.2. Data of western blot showed that, IDO expression in tumors, lymph nodes and spleens was upregulated in tumorbearing animals. FoxP3 was over-expressed in tumorbearing animals, but the administration of 1-MT could abrogate this upregulation.Conclusions1. A20 B lymphoma is a useful tumor model in research on IDO and immune tolerance.2. Over-expression of IDO in tumor, lymph nodes and spleens of tumorbearing animals leads to immune tolerance by incresing the percentage of Tregs.3. Administration of 1-MT can decreace the percentage of Tregs and may serve as a potential antitumor agent in the future.