The Model And Molecular Mechanism Of Inducing Of CD4~+ Memory T Cells In Vitro

Immunological memory is the most effective mechanism for the body to resistance to external pathogens, which can rapidly eliminate of pathogens when the body re-encounter the same pathogen, but also it is the material basis for the vaccine effect. Immunological memory system consists of humoral Immunological memory and cellular Immunological memory , The memory B cells (memory B cells, Bm) which bear the memory function of humoral Immunological memory and the memory T cells (memory T cells, Tm) which bear the memory function of cellular Immunological memory. In human, the characteristic surface markers of Tm is the CD45RO⁺, but in mouse Tm include effect-type memory T cells (CD44⁺CD62L-CCR7-) and central-type memory T cells (CD44⁺CD62L⁺CCR7⁺) in according to its different homing parts and chemokine receptors. Memory T cell is born of a linear (or directional) differentiation and asymmetric division mode. The linear differentiation model: the initial T cells can be differentiated into effector cells, and then a very small part of them differentiate into memory cells. Another view is that Tm come from precursor cells arrivallig at the late stages of immune response last. Differentiation pattern of non-linear part that the initial cells directly differentiate into memory cells . Recent studies have shown that memory cells are more likely the source of the linear differentiation model.In vivo studies have shown that IL-7 played a crucial role in formation of CD4⁺Tm. L-7-/- mice and IL-7R mutant mice can not from CD4⁺Tm. Enhanced IL-7 signaling in the peak proliferation of CD4⁺T cells can increase the population of CD4⁺Tm in TCR transgenic mice by promoting the proliferation of effective CD4⁺T cell,increasing expression of Bcl-2 and preventing death in systole. In the time of T cell contraction to inject IL-2, IL-7 or IL-15 can slow CD8⁺T cell contraction. Inject IL-2 or IL-15 at systolic in vivo, the major accumulation of short-term survival effect and memory cells KLRG1^hiCD127^lo, whereas inject IL-7 the mainly major accumulation of long-term survival of memory cells KLRG1^loCD127^hi. The studies in vivo only show the effect of cytokines on the differentiation of CD4⁺Tm, but not show the molecular mechanism of differentiation induce CD4⁺Tm. So people isolate CD4⁺Tm, to study the molecular mechanism of the stability, re-stimulated survival and proliferation. Two types: spontaneous differentiation of phenotype memory T cell and antigen-specific T cells. Under normal physiological conditions, two types of CD4⁺Tm self-stability require IL-7 and IL-15.The rapid proliferation of the phenotype memory CD4⁺Tm need MHC-II molecules, whereas the rapid proliferation of the antigen-specific CD4⁺Tm main dependent IL-7. IL-7R(CD127) is high expression in the resting T cells, whereas is down quickly in the activated T cells. Only a few can be transformed into Tm cells and expressed IL-7Ra again. It has proven that the expression of IL-7Ra can be used to detection of memory cell precursors in the CD8 ⁺ T cells.Given the current differentiation of CD4⁺Tm mainly studies in vivo. In order to further study the mechanism of differentiation of CD4⁺Tm, we first establish model of CD4⁺Tm which stimulated with IL-7 in vitro and intend the expression of CD44 and CD62L as an indicator by flow cytometry, Then use different combinations of cytokines or cytokine stimulate to observe the capacity of generation CD4⁺Tm; and then through the antigen re-stimulation and CFSE labeling to verify CD4⁺Tm by proliferation; Finally, to investigate the molecular mechanism of CD4⁺Tm by real time quantitative RT-PCR and Western -blotting.Part I: The establishment of CD4⁺Tm generated in vitroObjective: First consider the use of CD4⁺T cells from OT-II mice to create the model. However, due to OT-II mice feeding difficulties, insufficient numbers, so we intend to use FBS as antigen, with CD4⁺T cells from wild-type C57BL/6 mice to create the model, simultaneously, with OT-II CD4⁺T cells compared to prove the usefulness of C57BL/6 mice model. DC treated with antigen and IL-7 stimulate initial CD4⁺T cells in vitro, detect the expression of CD44 and CD62L at different times and re-proliferation condition after antigen stimulation again, the establishment CD4⁺Tm generated model to lay the foundation for investigatig the conditions of induction CD4⁺Tm generating in vitro. Methods: Take the femur and tibia from C57BL/6 mouse under sterile conditions, extraction of bone marrow cells treated with FBS or OVA stimulation in vitro, by adding IL-4 (1ng/ml) and GM-CSF (10ng/ml) induced bone marrow-derived mature DC (mature dendritic cell, OVA-DC or FBS-DC). Take spleen from OT-II and C57BL/6 mouse, isolate mononuclear cells and separate CD4⁺T cells by immunomagnetic beas under sterile conditions; the OVA-DC and OT-II CD4⁺T cells mixed 1:10, FBS-DC and C57BL/6 CD4⁺T cells mixed in the same proportion, cultured in medium containing IL-7 (1ng/ml) of 10% FBS-1640. chang half of the medium every 3 days, check the expression of CD44, CD62L by flow cytometry in the 5d, 10d, 15d, 20d, 25d, 30d respectively; At the same time on the 30d, collect cells labeled with CFSE, then stimulate with OVA-DC or FBS-DC for 72 hours, detect cell proliferation by flow cytometry. At the same time, with the CFSE labeled naive OT-II and C57BL/6 CD4⁺T cells, then OVA-DC or FBS-DC stimulate for 72 hours, then detect the proliferation by flow cytometry as a control. Results: The naive CD4⁺T cells (0d) mostly express CD44^loCD62L^hi, as the culture time, the expression of CD44 gradually increased, CD62L decreased; in the OVA-DC stimulate CD4⁺T cells from OT-II mice group the ratio of CD44^hiCD62L^lo to 80% at last. The expression pattern of CD4⁺Tm is similar in two groups. Proliferation experiments show cultured 30d cells fast response to the same antigen, mostly gather in the third,fourth,fifth or more generates. The results suggest that the phenotype and function of induced cells by this method is in accordance with CD4⁺Tm characteristics. Conclusion: In vitro, IL-7 can induce CD4⁺ Tm generation. The expression pattern and function of two groups CD4⁺Tm are similar, so that, wild-type C57BL/6 mice can instead of OT-II mouse to study the mechanism of CD4⁺Tm formation .Part II: The influence of different cytokines on induction of CD4⁺Tm in vitroObjective: To explore the relationship between a variety of cytokines and CD4⁺Tm , we use cytokine IL-2, IL-4, IL-7, IL-15, IFN-γ, TGF-βand Flt-3L to induce of the initial CD4⁺T cells in vitro, check the expression of CD44 and CD62L and proliferation re-antigens stimulate. Methods: Take the femur and tibia from C57BL/6 mouse under sterile conditions, extraction of bone marrow cells treated with FBS stimulation in vitro, by adding IL-4 (1ng/ml) and GM-CSF (10ng/ml) induced FBS-DC. Take spleen from C57BL/6, isolate mononuclear cells and separate CD4⁺T cells by immunomagnetic beas under sterile conditions; the FBS-DC and C57BL/6 CD4⁺T cells mixed 1:10, respectively, with IL-2 (50U/ml), IL-7 (1ng/ml), IL-15 (5ng/ml), IL-4 (10ng/ml), IFN-γ(10ng/ml), Flt-3L (5ng/ml),TGF-β(1ng/ml), IL-7+IL-2 and IL-7+IL-15 of 10% FBS-1640 culture medium, change half of the medium every 3 days, detect the expression of CD44, CD62L by flow cytometry in the 5d, 10d, 15d, 20d, 25d, 30d respectively; At the same time on the 30th day, collect cells labeled with CFSE, then stimulate with FBS-DC for 72 hours, detect cell proliferation by flow cytometry. Results: Respectively in IL-4, IL-15, Flt-3L, IFN-γand TGF-βculture system alone, cell death of less than 20d, while IL-2, IL-7, IL-7⁺IL-2, IL-7⁺IL-15 cell culture system survive; as the culture time, CD44 expression gradually increase, CD62L expression decrease; Another cytokine IL-2 and IL-7⁺IL-2 training system group the time of its CD44^hiCD62L^lo cell generation in about 10 days, but the proportion is much higher than other groups. Compared with other groups, proliferation experiments show that IL-2 and IL-7+IL-2 groups of cell proliferation are significantly reduced, no significant difference between them; IL-7+IL-15 group compared with IL-7 alone is no significant difference. The results suggest that IL-2 only can effectively activate CD4⁺T cells, which induce the same phenotype as CD4⁺Tm cell, but they do not have the capability of rapid proliferation; while in IL-7 co-culture system, it can inhibit the function of IL-7. Conclusion: IL-15 do not induce generation of CD4⁺Tm alone in vitro, and can not influence the capability of IL-7. IL-2 do not induce CD4⁺Tm, but inhibit the function of IL-7. Literature review, no reports about IL-2 can inhibit IL-7 the induction of CD4⁺Tm generated. Because IL-15 also uses IL-2R andγc as part of receptor, and IL-15 do not inhibit IL-7 induced CD4⁺Tm generation, indicating that the inhibitory effect of IL-2 may not be through a competitiveγc, but their signal condition.Part III: The mechanisms of CD4⁺Tm generated in vitroObjective: To preliminary study the mechanism of CD4⁺Tm induced by IL-7 in vitro, detect related signaling molecules, transcription molecules and the apoptosis-related protein of CD4⁺Tm induced by IL-7. Methods: Take the femur and tibia from C57BL/6 mouse under sterile conditions, extraction of bone marrow cells treated with FBS stimulation in vitro, by adding IL-4 (1ng/ml) and GM-CSF (10ng/ml) induced FBS-DC. Take spleen from C57BL/6, isolate mononuclear cells and separate CD4⁺T cells by immunomagnetic beas under sterile conditions; the FBS-DC and C57BL/6 CD4⁺T cells mixed 1:10, respectively, with IL-7 (1ng/ml), IL-7+IL-2 of 10% FBS-1640 culture medium, changed half of the medium every 3days, detect of IL-4, IFN-γ, Bcl-2, Bax by Real-time quantitative RT-PCR in the 5d, 10d, 15d, 20d, 25d, 30d respectively and STAT5 /p-STAT5(Y694), AKT/p-AKT, pro-Caspase-3/Active Caspase-3, Bcl-2 by Western -blotting after 30 days. Results: Compared with the naive CD4⁺T cells group, IL-7 culture system, IL-4, IFN-γ, p-STAT5, Bcl-2 are significantly higher, Active Caspase-3 is lower. IL-7 may prompt anti-apoptotic protein Bcl-2 through the JAK/STAT. Conclusion: IL-7 plays important role in the activation of naive CD4⁺T cells, increasing the expression of Bcl-2 by JAK/STAT5, thereby inhibit the activation of caspase-3, so that, part of the activated CD4⁺T cells survive for long term, then formation of CD4⁺Tm.