CAMP Is Involved In Low-Dose Cyclophosphamide-Reversed Immune Evasion In B Cell Lymphoma Mouse Model

Non-Hodgkin’s lymphoma (NHL) is a group of malignant tumors with immune disorders, deriving from B and T lymphocytes. The lymphoma cells mobilize many mechanisms to escape from the immune system. Now there is substantial evidence that the CD4+CD25+regulatory T cells (Tregs) play key roles in the control of tumor immune tolerance and immune evasion. Suppression of the number and/or function of Tregs may be critical for successful immunological therapy in lymphoma. Many researches focus on the removal of Tregs in animal models, resulting in improved function of CD4+effector cells (Teff), ultimately enhance the antitumor responses. Therefore, elucidating the mechanism involved in Treg-induced immune evasion in tumor progression, may have important implications in the design of effective immunotherapeutic approaches against cancer. Bopp T et al show that Tregs can transfer cyclic adenosine monophosphate (cAMP) to Teff cells through cell contact-dependent gap junction, followed by increase of intracellular concentration of cAMP in Teff cells, inhibiting the interleukin2(IL-2) synthesis in Teff cells. So the Treg cells may suppress the immune network through the intercellular transport of cAMP, indicating the correlation between intracellular cAMP concentration and immune-evasion role of Tregs.Many researches focus on the depletion of Treg by anti-CD25monoclonal antibody (PC61) or low dose CY in tumor and some autoimmune conditions. Being an alkylating drug agent, CY is used in lymphoma chemotherapy. But there isn’t investigation of low-dose CY in lymphoma animal models and patients. In our study, we chose A20B cell NHL lymphoma mouse as an aggressive tumor model, to investigate the depletion of Tregs by low-dose CY (20mg/kg). In the progress of lymphocytic malignancy several signal pathways are involved. Balance of the signaling pathways decides the fate of malignant cells, to survive or to undergo apoptosis. Recently, the role of the cyclic adenosine monophosphate (cAMP) signal pathway in the pathogenesis of lymphoma and apoptosis-inducing therapy has been researched. Increased intracellular cAMP levels induce cell-cycle arrest and apoptosis in several types of lymphoma cells. In our study, we established an aggressive transplantable murine lymphoma model with A20cells and investigated the effects of low-dose CY on tumor immunity. Treatment with20mg/kg CY inhibited tumor growth, increased the survival rates of A20tumor-bearing BALB/c mice, and ultimately increased the IL-2secretion of CD4+Teff cells. The IL-2increase was synchronized with the FOXP3decrease. With their potential roles in reversing tumor immune evasion and promoting apoptosis, and due to the potential clinical cooperation between the cAMP pathway and the CY-Tregs pathway, cAMP analogs/PDE4B inhibitors and low-dose CY may be good candidate agents for lymphoma therapy. Part I:Investigation of low-dosecyclophosphamide-reversed immune evasion in B cell non-Hodgkin’s lymphoma mouse modelObjective:In our study, we chose A20B cell NHL lymphoma mouse as an aggressive tumor model, to investigate the depletion of Tregs by low-dose CY (20mg/kg). We observed the tumor diameters and survival rates of tumor-bearing mice. Then MACS isolation was done to obtain purified Tregs and/or Teffs. Foxp3and IL-2expression of immune cells was compared by Western Blot and RT-PCR between CY-treated and tumor-beared mice.Material and methods:1. Cell culture and animal experiments2. Single cell preparation3. Proin extraction and Western Blot4. RNA extraction and RT-PCR5. Treg cells and Teff cells isolation6. Flow cytometric analysis7. Statistical AnalysisResults:1. A20cells culture and lymphoma mouse model The mouse B-cell lymphoma cell line, A20, was a generous gift from Zhongshan University (Guangzhou, China). This tumor cell line is derived from a spontaneous reticulum cell neoplasm in BALB/c mice[1].The A20cells were cultured in RPMI1640medium (Hyclone, Logan, UT) that was supplemented with10%heat-inactivated fetal bovine serum,100U/ml penicillin,100μg/ml streptomycin and2mM L-glutamine at37℃in a5%CO2-humidified incubator. A20lymphoma cells were implanted into BALB/c mice at concentrations of5×106cells per mouse in100μl of RPMI1640by subcutaneous injection into the left flanks. After7days, the tumor-bearing mice received20mg/kg of CY or NS via the intraperitoneal (i.p.) route each week. 2. The tumor growth of BALB/c mice. We measured the tumor diameters and calculated the survival proportion as shown in Fig2. The treatment of20mg/kg CY caused slight delay on the growth of tumors, ultimately prolonged survival proportion of A20-bearing BALB/c mice.3. FACS analysis. Lymphocytes of A20tumor-bearing or control mice with and without CY treatment (20mg/kg) were analyzed by flow cytometry for the presence of Tregs. The subcutaneous A20tumors increased the percentage of Tregs in the draining lymph nodes,4days after CY injection, the percentage of Tregs decreased and remained low on day8, then increased on day14.After the MACS isolation, we performed FACS to validate the purity of isolated Tregs and Teff cells. The purity of Teffs was higher than98%, and the purity of Tregs was higher than90%.4. Foxp3expression was transiently affected by low-dose CY. To confirm the nature of Tregs alteration, we performed Western Blot and Real-time PCR for the analysis of the Treg-specific marker Foxp3. We detected (3-actin content to assess cell equivalency among the samples, then detected Treg-specific marker Foxp3mRNA or protein expression using specific primers or mAb. Treatment with CY was found markedly to decrease the Foxp3expression from day4, reduced the mRNA and protein expression of Foxp3to a minimum level on day8, then recovered or exceeded to pretreatment levels in lymphoid organs by day14.5. IL-2expression was affected by low-dose CY.Along with the decrease of Tregs, we found the increase of IL-2expression of T effector cells. Tumor-challenge decreased the secretion of IL-2of CD4+effecter T cells (p<0.0001), but CY-treatment developed the function of effecter T cells (Fig.4). The elevation peaked on day8(p<0.0001), then decreased gradually, disappeared after14days. Then the transient interval may provide chance for effective anti-immune response which needs Teff cells activation.Conclusions:1. A20B cell NHL lymphoma mouse model was a good NHL animal model.2. A20lymphoma model had increased Tregs which induced the immue-evasion.3. Low-dose CY could transiently decrease the function and number of the Tregs and increase the expression of IL-2, ultimately reversed the immune-evasion mediated by Tregs in B cell lymphoma mouse model.4. Loe-dose CY could prolong the survival of mouse model, proved the immune-enhancing function of low-dose CY in the treatment of lymphoma mouse model. Part Ⅱ:Involvement of cAMP in the low-dosecyclophosphamide-reversed immune evasion and role of cAMP analogs in apoptosis of NHL cellsObjective:Loe-dose CY could prolong the survival of mouse model, proved the immune-enhancing function of low-dose C Y in the treatment of lymphoma mouse model. We further investigated intracellular cAMP concentration of immune cells with or without CY treatment, in order to provide theoretical foundation for the cooperation of cAMP analogs and low-dose CY. Balance of the signaling pathways decides the fate of malignant cells, to survive or to undergo apoptosis. Recently, the role of the cyclic adenosine monophosphate (cAMP) signal pathway in the pathogenesis of lymphoma and apoptosis-inducing therapy has been researched. In our study, we injected the cAMP analogs into A20NHL mice model, then detected the change of AKT, in order to investigate the apoptosis mechanism of lymphoma. cAMP analogs/PDE4B inhibitors and low-dose CY may be good candidate agents for lymphoma therapy.Material and methods:1. Cell culture and animal experiments2. Intracellular cAMP concentration assay3. Proin extraction and Western Blot4. Flow cytometric analysis5. Immunohistochemistry6. Statistical AnalysisResults:1. The change of intracellular cAMP concentration of immune cells. In this study, we measured intracellular cAMP concentration of immune cells with or without CY treatment. Consistent with the change of Foxp3and IL-2after CY treatment, the intracellular cAMP concentration altered regularly after CY-treatment. We found that tumor-challenge increased the cAMP concentration of immune cells. CY-treatment decreased the cAMP concentration from day4, and T effecter cells harbored the lowest cAMP concentration on day8. The down-regulation was temporary, faded on day14. Then20mg/kg CY created a transient window for optimal T effecter’s differentiation and postponing the tumor growth.2. Apoptosis of A20cell line The mouse B-cell lymphoma cell line, A20, showed increased apoptosis rate, under role of cAMP analogs3. The change of Akt phosphorylation protein wasn’t affectecd by low-dose CY.4. The change of Akt phosphorylation protein was investigated by immunohistochemistryConclusions:1. cAMP analogs induce A20B cell line to apoptosis.2. The change of Akt phosphorylation protein wasn’t affectecd by low-dose CY.3. cAMP analogues are good candidate agents for lymphoma therapy.4. cAMP is involved in low-dose CY-reversed immue evasion.5. Cooperation of cAMP analogs and low-dose CY maybe protective candidate for the immune-therapy of B cell lymphoma.