Effects Of Epigenetic Modifications On The Functionality Of Human CD8~+ Memory T Cells

Memory CD8~+T cells(CD8~+Tm)acquire enhanced effector function and are capable of providing superior protective immunity to host compared to their na?ve counterparts(CD8~+Tn).When encountered with a previously experienced antigen,CD8~+Tm will generate a much more robust immune response by immediate effector cytokine(IFN-?,granzyme B,perforin,etc.)production and rapid proliferation,leading to much faster clearance of pathogen.However,the underlying molecular mechanisms of enhanced functionality of CD8~+Tm remain largely unclear.In the present study,we sorted human CD8~+Tn and Tm from peripheral blood of healthy donors,activated them with anti-CD3/CD28 coupled microbeads for 4h and24h to mimic early and late phase of T cell activation triggered by TCR/co-stimulatory signals.Then we determined the genome-wide transcription profiles of CD8~+Tn and Tm at rest and after activation.The results showed that regardless of stimulation 5-10%of total genes were differentially expressed between CD8~+Tn and Tm(expression fold difference?3).Among all these differentially expressed genes,almost 200 genes were only dramatically increased in CD8~+Tm in the presence of stimulation,but not in CD8~+Tn;these genes showed most significant enrichment in different aspects of immune responses by gene ontology analysis,and they were not just limited to common effector molecules expressed by CTLs(such as Gzmb,Prf1 and Ifng),many characteristic genes expressed by Th subtypes,such as Il4,Il5,Il9,Il17f,Il21,Il22 etc.,were also included in this set.Among these genes involved in immune responses,we chose those with most dramatic differences in expression between CD8~+Tm and Tn,and then confirmed their expression by quantative RT-PCR,ELISA and flow-cytometry.Il2,which was similarly expressed between CD8~+Tm and Tn,was taken as control.All the differential genes detected by qRT-PCR,such as Ifng,Il10,Il9 and Il17f etc.,showed a higher level of mRNA expression in activated CD8~+Tm than Tn,and in parallel,their protein expression were also higher in CD8~+Tm,as determined by ELISA and flow-cytometry analysis.We then detected histone H3 K9K14 acetylation at important gene loci.CD8~+Tm showed a significantly higher level of H3 acetylation at Ifng promoter proximal to TSS compared to Tn prior and following activation,on the other hand,no differences in H3 acetylation was observed at Il2 locus.Furthermore,we found that recruitment of p300 at Ifng locus was higher in CD8~+Tm,but was similar between CD8~+Tm and Tn at the Il2 locus following activation.In further experiment,deletion of p300binding sites within Ifng promoter significantly reduced transcription activity of Ifng.We have also determined global histone H3 acetylation levels in human CD8~+Tm and Tn by flow-cytometry on per-cell basis.The results showed that global histone H3 acetylation of CD8~+Tn was higher than that of CD8~+Tm at rest.However,per-cell histone acetylation was inceased in both CD8~+T cell subsets following activation,and the increase was much more robust for CD8~+Tm compared to that of Tn,resulting in similar per-cell histone acetylation between these two subsets.Consistent with this,expression of almost all histone acetyltransferases(HATs)and histone deacetylases(HDACs)were similar between CD8~+Tm and Tn.Taken together,we can summarize that:1)Transcription signature of CD8~+Tm is different from that of Tn,and many genes involved in immune responses were expressed to a higher level in CD8~+Tm;2)Histone H3 acetylation correlates with the expression of important genes in CD8~+Tm;3)Recruitment of histone acetyltransferase p300,but not the expression of HATs or HDACs,might be essential for regulating histone H3 acetylation and gene transcription in CD8~+Tm;4)Global histone H3 acetylation may have a distinct role in regulating the functionality of human CD8~+Tm.Our data thus might improve our knowledge of the transcription profiling and epigenetic mechanisms underlying enhanced functionality of CD8~+Tm,and provide new insights for targeting histone acetylation to modulate the magnitude of immune responses.