Study On The Effects Of Conditional Deletion Of Smad4on The Activation Of T Cells

Transforming growth factor-β (TGF-β) superfamily has been demonstrated toexert a series of effects on T lymphocytes, such as modulating the development,activation, proliferation, and differentiation of T cells. There are three TGFβ isoformsin mammals: TGFβ1, TGFβ2and TGFβ3, which signal through a heteromericcomplex of type I and type II transmembrane receptor serine/threonine kinases thatact in series. Among the three TGFβ isoforms, TGFβ1is predominantly expressed inthe immune system. The major substrates of the TGF-β receptor complex are Smad(Sma and Mad Homologue) proteins, which have eight members i.e., two TGF-βR-Smads (Smad2and3), three bone morphogenetic protein (BMP) R-Smads (Smad1,5and8), one Co-Smad (Smad4) and two I-Smads (Smad6and7).Smad4is one of the most important intracellular mediators of TGF-β signaling.After the receptor-induced phosphorylation of Smad2and Smad3, they formcomplexes with Smad4and are translocated to the nucleus. Numerous papers in theliterature are available for the formation of Smad2/Smad2/Smad4, Smad3/Smad3/Smad4and Smad2/Smad3/Smad4complexes after TGF-β stimulation and Smad4appears to be an obligatory partner in these complexes. In the nucleus, the polymericcomplexes in cooperation with other transcription factors, co-activators andco-repressors, regulate the transcription of specific genes. However, in the year of2006, some scientists found another protein-transcription intermediary factor1-γ(TIF1-γ), which can play a competition role with Smad4protein in combination withSmad2or Smad3and mediate the complex translocated to the nucleus. Besides theTGF-β/Smad pathway, TGF-β also can activate the non-Smad pathways, includingthe extracellular-signal-regulated kinase (Erk), Jun N-terminal kinase (JNK) and p38mitogen-activated protein (MAP) kinase pathways.Although Smad4is an important component of the TGF-β/Smad pathway andTGF-β has profound influence on T cells immunity balance, there are few studies onrelationship between Smad4absence and T cells. Our study is to investigate whetherSmad4null mice have the same effects as TGF-β on T cell proliferation, T cellactivation and effector T cell differentiation. Since it has been revealed that loss of Smad4results in lethality at embryonic (E) days6–7due to impaired extraembroynicmembrane formation and decreased epiblast proliferation, we used the animal modelwith a Smad4conditional knockout allele using the Cre-loxP approach.1. Purpose and contents of the research1) The influence of conditional deletion of Smad4on T cell development andproliferationOne of the earliest understood effects of TGF-β was its ability to arrest the cellcycle at the early G1phase and thus inhibit the growth of various cell types, includingthe T lymphocytes in the immune system. Previous studies believed that Smadsproteins are of central importance for TGF signaling on the aspect of cell cycle arrest.In the nucleus, R-Smad/co-Smad complexes can regulate target genes, lead to thesuppression of mitogenic transcriptional signals and the induction of cell cycleinhibitory signals. Therefore, we want to know whether Smad4can plays a key role inthe procedure of T cell development and proliferation, and whether our Smad4geneconditional deletion on T cells animal models may lead to impaired T celldevelopment and inhibit the proliferation of T cells, either in the thymus or in theperipheral tissues.2) The influence of conditional deletion of Smad4on CD4⁺T cell activation anddifferentiationPrevious studies have showed that TGF-β exerts suppressive effects on CD4⁺Tcells. TGF-β can inhibit CD4⁺T cell proliferation, CD4⁺T cell activation and effectorCD4⁺T cell differentiation including Th1/Th2/Th17cell differentiation. TGF-β mayalso mediate its immunosuppressive effects on T cells through promoting thegeneration of CD4⁺CD25⁺regulatory T cells, which suppress other immune cellsactivation and differentiation by secreting various soluble factors and cell-cell contact.Our study is to define the effects of Smad4on the T cell activation and effector T celldifferentiation.3) The influence of conditional deletion of Smad4on CD8⁺T cells activation andproliferationIn our study, we are surprised to find that the old mice with Smad4conditionaldeletion showed defective CD8⁺T cells activation. We therefore established an animal model of Listeria monocytogenes (LM) infection as a clinically relevant modelof cell-mediated immunity and compared the proportions of CD8⁺CD44hiT cells inthe Smad4conditional deletion mice (Smad4^Cre/Co/Co) to those in the control genetargeting mice(Smad4Co/Co)0,5, or7days after LM infection.4) The influence of conditional deletion of Smad4on effector CD8⁺T celldifferentiationAlthough the evaluation of effector CD8⁺T cell differentiation under thecondition of Smad4absence is of potential value, direct studies of the effects ofconditional deletion of Smad4specifically in T cells on the levels of antigen-specificCD8⁺T cell responses have been lacking. The early CD8⁺T cell response to LM hasbeen shown to be critical for the development of sterilizing immunity. We want toknow the influence of conditional deletion of Smad4in T cells brings to effectorCD8⁺T cell differentiation and the underlying mechanism. Previously studies haveshown that TGF-β can inhibit IL-2–dependent proliferation of T cells by blockingIL-2production. We presume that the abnormal level of intracellular cytokine IL-2orthe member receptor of IL-2expressed on the CD8⁺T cell attribute to the drawback ofCD8⁺effector CTL activation in Smad4null mouse. Besides, now that CD40Ligand(CD40L, CD154) play an important role in the process of induced the activation ofpre-CTL to effector CTL, the express level of CD40L is one of the object of ourstudy.2. Methods of the research2) Smad4excision PCRThe genome DNA was got from tail samples, and the conditions of excision PCRare as follows:94°C,1min；68°C,1min20s；72°C,30s；94°C,2min；94°C,30s,60°C,30s,72°C,1min,30,repeat×30times；72°C,8min；4°C, preserved.3) Immunoblotting AnalysisLysates of cells prepared in M2lysis buffer and1×SDS-PAGE loading bufferwere separated by SDS–polyacrylamide gel electrophoresis, transferred ontopolyvinylidene difluoride membranes, incubated with specific primary antibodies ofSmad4and horseradish-peroxidase-linked secondary antibodies. Immunoreactivebands were visualized by the ECL Chemiluminescence Kit. 4) Flow CytometryCells from spleens, lymph nodes, or thymus were depleted of erythrocytes byhypotonic lysis and were washed3times by PBS. Cells were incubated with specificantibodies for30min on ice and keeping in dark to prevent the fluorescencequenching. All samples were analyzed with FACS Calibur (Becton Dickinson) andWinMDI3.0software.For intracellular cytokine staining, single-cell suspensions of spleens or lymphnodes were stimulated with50ng/ml phorbol12-myristate13-acetate (PMA),10mg/ml Brefeldin A (BFA) and1mM Ionomycin for4hours. After stimulation, cellswere first stained with surface marker such as CD4, CD8et al, then fixed andpermeabilized with a cytofix/cytoperm kit, and finally stained with intracellularcytokine antibody such as IL-4, IL-10and IL-17et al.5) Statistical AnalysesStudent's t test was used to calculate statistical significance for difference in aparticular measurement between groups. P value less than0.05%was consideredstatistically significant.6) OthersTo analyze apoptosis and proliferation, cells were dispersed and stained withFITC-labeled Annexin V or CFSE according to the manufacturer's instructions.3. Results and conclusions of the research1) The result of genotypingIn our study, we used several methods to detect the genotypes of the mice.Cre-mediated excision of exon8of the Smad4gene was detected by PCR.Immunoblotting analysis showed complete loss of Smad4protein expression in thethymocytes of Smad4co/co;Lck-Cre mice compared with their littermate controls(wild-type and heterozygous littermates).2) The effect of conditional deletion of Smad4on the development of T cellsWe used several means to observe the influence of conditional deletion of Smad4brings to the development of T cells. The results show that conditional deletion ofSmad4has little influence on the development of CD4⁺T cells, CD8⁺T cells, regulatory T cells, γδT cells and natural killer T cells in the thymus. Smad4is notessential for TGF-β-mediated growth arrest in T cells.Our results also show that Smad4conditional deletion on T cells has no effect onthe differentiation of Th1/Th2/Th17cells. We considered that there is abundant effectbetween TIF1-γ and Smad4, it is not strange that Smad4null mice have no different indevelopment and differentiation of T cells, while TGF-β play an inhibit role in thisprocess.3) The influence of conditional deletion of Smad4on T cells proliferationThe influence of conditional deletion of Smad4put on the proliferation of T cellsis partial, because that there is no effect on the total number of spleen either in vivo orvitro and the number of CD4⁺T cells either in early or old ages, but the number ofCD8⁺T cells has significant difference between Smad4Co/Coand Smad4^Cre/Co/Comicein their old ages (52weeks of age).4) The influence of conditional deletion of Smad4on T cells activationOur findings show that the rate of CD4⁺CD44hiT cells in aged mice's spleen andlymph node in Smad4conditional mutagenesis models have no difference with theWT mice. Interestingly, we find that the in aged mice's spleen CD8⁺CD44hiT cellshave decreased in the absence of Smad4. To investigate the impact conditionaldeletion of Smad4put on the activation process of CD8⁺T cells, we determine toestablish an infection model used tail vein injection of LM.5) The influence of conditional deletion of Smad4in gastrointestinal epithelialThe previously study using Lck-Cre; Smad4fl/flmice reported the development oftumors at multiple sites in the gut. In the year2011, another research reported thatconditional deletion of floxed Smad4during T cell development using either theLck-Cre or CD4-Cre systems led to the development of gastrointestinal epithelialtumors but the tumors were at a single site, the sub-pyloric duodenum,different fromthe previously study report. While in our study, we have not found any tumors ingastrointestinal system. We presume that the discordance between these researchesare likely attributable to environmental (e.g., microbiotal) factors. However,differences in genetic backgrounds of the mice remain a formal possibility, ourSmad4conditional deletion mouse(Smad4^Cre/Co/Co) mice were on the background of gene targeting mouse(Smad4^Co/Co).6) The influence of conditional deletion of Smad4on CD8⁺effector CTLIn our study, We observed the rate of the spleen CD8⁺CD44hiT cells on0day,5day and7day after the tail vein injection of2×105LM.We find that the rates ofspleen CD8⁺CD44hiT cells have significant difference between Smad4Co/CoandSmad4^Cre/Co/Comice on5days after infection of LM (2×105CFU iv). We consider thatconditional deletion of Smad4may bring delay of activation of effector CD8⁺CTL.To further investigate the influence Smad4conditional deletion put on the activationand killing capacity of CD8⁺effector CTL, we observed the specific marker ofactivated CD8⁺effector CTL.The level of granzyme B and IFN-γ and the rate ofshort-lived effector CD127lowKLRG1hicells in spleen of Smad4^Cre/Co/Comice isdistinguished lower than Smad4Co/Co.In a conclusion, we consider that conditionaldeletion of Smad4also have impact on the killing capacity of CD8⁺effector CTL.7) The mechanism of abnormal activation of CD8⁺effector CTL in Smad4null miceNow that IL-2family of cytokines is essential for homeostatic control of Tlymphocytes in the periphery and IL-2augments the proliferation of activated T cellsat the initiation of the immune response, we presume the phenomenon that delayactivation of CD8⁺effector CTL in Smad4null mice may be caused by abnormalexpress level of IL-2or IL-2R. But to our disappointed that the level of intracellularcytokine IL-2in CD8⁺T cells or chain of IL-2Rα (CD25), chain of IL-2Rβ (CD122),chain of IL-2Rγ (CD132) have no significant difference between Smad4Co/CoandSmad4^Cre/Co/Comice. Further investigate have proved that the abnormal activation ofCD8⁺effector CTL in Smad4conditional mutagenesis models may attribute todecreased express of CD40L which play an important role in the stimulate process inactivation of pre-CTL. Besides that, we find the lower rate of spleen CD43⁺CD27⁺cells may be another potential reason for the delay activation of CD8⁺effector CTL inSmad4conditional mutagenesis models.4. Innovation of the researchAlthough the study of the effect of Smad4in the immune system is becoming thehot spot, there is little acknowledge about the relationship between the conditionaldeletion of Smad4and the activation of Antigen-specific CD8⁺effector CTL. Ourstudy have found that,in the absence of the Smad4,the activation of Antigen-specific CD8⁺effector CTL was delayed and the secretion of granzyme B and IFN-γ which arethe attributes of effector CTL was decreased. We preliminary view that themechanism of abnormal activation of CD8⁺effector CTL is laid on the lowerexpresses of CD40L on the CD4⁺T cells surface. CD40L interact with CD40expressed on the surface of the dendrite cell (DC) may induced much more expressionof MHC and co-stimulate molecular CD80/CD86and then induced the activation ofpre-CTL. We assume that the reduced expression of CD40L on CD4⁺T cells surfaceinfluence the activation of CD8⁺effector CTL in the Smad4null mouse.