Efficacy And Mechanisms Of Anti-tmuor Immunity Induced By Tumor Vaccination Under Lymphopenia Setting

Classification: R3Tumor immunotherapy has been used in treatment of non-solid tumor, metastasis or late stage tumors and shown the promising application potential in combination with conventional tumor therapy. However, most patients suffered from tumor are considered to be not the candidates for immunotherapy due to the severe lymphopenia induced by operation, chemotherapy, radiotherapy or the combinations. It is controversial whether tumor vaccination under lymphopenia will induce irnmunotolerance or anti-tumor immune response. Accumulated data has proven that lymphocytes could undergo homeostasis proliferation under non-genetic factors induced lymphopenia, which also termed lymphopenia-driven proliferation (LDP). Under lymphopenia setting, lymphocytes undergo a series of burst-like divisions, exhibit a vigorously proliferation and maintain the cell numbers into normal level. During the proliferation, cells yet acquire characteristics of memory and effector phenotype. Thus, lymphopenia setting may facilitate the induction of immune response rather than immune tolerance. We hypothesize that enhanced anti-tumor immune response will be elicited by tumor vaccination under lymphopenia setting, i.e. cancer patients treated with radiotherapy or chemotherapy.Therefore, to determine whether the circumstance of lymphopenia and ensued LDP in cancer patients and mouse model existed, we dynamically detected the numbers of lymphocytes and the proportion of various subsets of lymphocytes after chemotherapy. Tumor vaccine was adopted under lymphopenia setting to prove whether it could induce more powerful anti-tumor response and protect mice from tumor challenge. Furthermore, the mechanisms have also been explored. Thus, our research would provide theoretical and experimental basis for combination of chemotherapy and immunotherapy. 1. Lymphopenia setting in lung cancer patients and the mouse model after chemotherapy.The wildly accepted criteria of lymphopenia has been considered the abnormal reduction of lymphocytes in peripheral blood (adults <1000/μl, children (< 2 years old) <3000/μl). A great deal of factors is involved in the induction of lymphopenia, such as HIV infection, radiotherapy or chemotherapy in tumor. To maintain immunostasis, naive T cells, so do other lymphocytes, undergo homeostasis proliferation under the circumstance of lymphopenia and ultimately results in rebuilding of dynamic balance. We termed the dynamic procedure including cell decrease and increase as lymphopenia setting. To observe whether chemotherapy applied in lung cancer patients are accompanied with the induction of lymphopenia setting, we dynamically detected the numbers of lymphocytes and the proportion of different cell types after chemotherapy. The numbers of white blood cells, neutrophils and lymphocytes in peripheral blood decreased gradually at first 4 to 6 days and recovered for the following 5 days or more. FACS analysis showed that consistant with the decreased number of lymphocytes, proportion of CD4+, CD4+CD25+T cells also dropped for the first 4 to 6 days and recovered later while CD8+, CD4+CD25-T cells remained unchanged.To mimic clinical case, we established a lymphopenia model in B6 mice. According to clinical usage, mice were treated with i.p. injection of 150mg/kg CTX. Changes of lymphocytes in peripheral blood, spleen and LNs were observed. Data showed the similar phenomenon in reduction and recovery of cell numbers after chemotherapy with CTX. Similarly, the proportion of CD4+, CD4+CD25+T subsets were dramatically dropped for the first 4 days and fell into the lowest level at day 4. To confirm chemotherapy induced lymphopenia setting was a general property; we used Taxotere instead of CTX to observe whether the lympnopenia could be induced. Injection of Taxotere i.p at the dosage of 40mg/kg in B6 mice resulted in the same manifest of lymphopenia setting. Therefore, lymphopenia setting induced by chemotherapy was a universal phenomenon rather than the effects of some indicated drugs. Thus, we have successfully established a lymphopenia mouse model which was the basis of our tumor vaccination research.2. The anti-tumor immune response induced by tumor vaccine under lymphopenia setting It has been proved that naive T cells experienced lymphopenia-driven proliferation under lymphopenia setting. However, it remains unclear whether these proliferated T cells could be elicted and enhanced immune response stimulated by tumor vaccination.To address it, mice were treated with injection i.p.of 150mg/kg CTX at day 0. Four days later, mice were immunized with DC-Mut-1 tumor vaccine followed by 3LL tumor cell inoculation at day 11. The lymph-proliferation assay revealed that immunization with DC-Mut-1 under lymphopenia setting induced more significant Mut-1 specific T cell proliferation when re-stimulated with Mut-1 peptide or 3LL in vitro (compared with the group immunized under non-lymphopenia setting or the lymphopenia group without vaccination, p<0.05). Consistent with the results of lymph-proliferation assay, cytokine production and CTL cytotoxicity assay showed that more IFN-γsecretion and higher CTL cytotoxicity were induced in the vaccination under lymphopenia setting. Furthermore, DC-Mut-1 vaccination under lymphopenia setting could elicit enhanced protective anti-tumor immunity in vivo compared with DC-Mut-1 vaccination under non-lymphopenia setting. Mice receiving DC-Mut-1 under lymphopenia setting showed 100% protection from tumor challenge and long-term survival. To exclude the indirect modification of DC-Mut-1 by CTX, we cocultured DC-Mut-1 with sera derived from CTX treated mice and in vivo anti-tumor protection was observed after vaccination. Data showed no enhancement of anti-tumor protection induced by sera cultured with DC-Mut-1. The results as described above suggested that DC-Mut-1 vaccination under lymphopenia setting resulted in a notable enhancement of anti-tumor immune response.However, whether tumor vaccination under lymphopenia setting in tumor bearing mice could elicit enhanced anti-tumor immune response and ultimately result in the regression of established tumor should be more explored. Tumor bearing mice were treated with Taxotere, a chemical drug which has been wildly used for chemotherapy in the clinic, followed by 3LL-GM vaccination and the anti-tumor immune response were observed. Mice receiving combined therapy showed enhanced inhibition of tumor growth and longer tumor free survival compared with chemotherapy or immunotherapy group (P<0.05). Furthermore, the intracellular staining assay conducted in those long survival tumor free mice showed that higher frequency of IFN-γ-producing CD8+T cells were elicited when splenocytes were stimulated with Mut-1 peptide. Rechallenge assay performed 60 days after first tumor inoculation showed complete protection, indicating that specific anti-tumor memory CD8+CTL could be induced when mice were treated with combination therapy.3. The mechanisms involved in enhancement of anti-tumor immune response elicited by tumor vaccination under lymphopenia settingIn the previous study, we have demonstrated that vaccination under lymphopenia setting resulted in the enhancement of anti-tumor immune response. We hypothesized that the reduction of CD4+CD25+Foxp3+Treg cells in lymphopenia setting was the key point and responsible for the successful immune response. We first detected the proportion of Treg cells infiltrated in the tumor, we found that CD4+CD25+Foxp3+Treg cells accumulated in PBMC and TILs of lung cancer patients (n=32). However, the proportion of CD4+CD25+Foxp3+Treg cells decreased gradually while CD4+CD25- and CD8+T cells remained unchanged after patients treated with chemotherapy(n=23), indicating that chemotherapy could selectively decrease the proportion of CD4+CD25+Foxp3+Treg cells. Similar result in decreasing of the proportion of CD4+CD25+Foxp3+Treg was also observed in mouse model after chemotherapy. To address whether the reduced proportion of CD4+CD25+Foxp3+Treg cells was closely related to the enhancement of anti-tumor immune response, CTX treated mice were immunized with DC-Mut-1 at day 1, 4 and 7 followed by 3LL tumor cell challenge. Interestingly, only those day 4-immunized mice showed 100% protection, indicating that the reduction of CD4+CD25+Foxp3+Treg cells under lymphopenia setting was involved in the enhancement of immune response. In vivo anti-CD4 antibody administration to deplete CD4+T cells resulted in an enhanced anti-tumor immunity, implying that the inhibition of tumor growth may due to the further depletion of CD4+CD25+Foxp3+Treg cells.In conclusion, an enhanced anti-tumor immunotherapy was induced by tumor vaccine under lymphopenia setting; indicating that tumor vaccination under lymphopenia setting could not only induce antigen-elicited immune response, but also enhanced the anti-tumor immunity. Contrasted to other report's consider that enhanced immune response under lymphopenia setting is due to the abundant "space", this is the first time we illustrated that the reduced proportion of CD4+CD25+Foxp3+Treg cells after chemotherapy played the key point in the enhancement of anti-tumor immunity. Thus, our research will help to provide a new strategy on tumor immunotherapy combined with other traditional tumor therapy especially chemotherapy and show promising perspective in clinical application.