The Characteristics And Roles Of MiR-155in T Cell Exhaustion

T lymphocytes are a crucial part of the immune system and are the major cellsinvolved in the adaptive immune response. Usually, T cells expand rapidly in response toantigenic stimulation, and then, as a result of antigen clearance, they die by apoptosis orbecome memeory T cells. However, in situations of chronic antigenic exposure, such asthose observed in cancer, chronic infections or autoimmunity diseases, antigen-specific Tcells may persist for extended periods of time and these conditions are often associated withfunctional exhaustion. Exhausted T cells are characterized by decreased cytokineexpression and dysfunction.MicroRNAs （miRNA） are small genome-encoded RNAs that regulate gene expressionby targeting complementary messenger RNAs （mRNA） and resulting in gene expressionsilencing through translational repression or target degradation. There are over1000miRNAs reported in humans, which may target about60%of mammalian genes. MiRNAsare associated with the pathogenesis and developement of many diseases, and miRNAs areinvolved in establishing and maintaining cell fate as well as regulating innate and adaptiveimmune systems.Among miRNAs the most present in immune cells, miR-155is shown to be critical forthe maturation and differentiation of several cell types, such as B cells, DCs and Th cells.MiR-155regulates CD4⁺T cell function by suppressing the expression of its target genes.MiR-155has been shown to promote Th cell-driven inflammation, however, the exactmolecular mechanism by which miR-155promotes Th cell-driven inflammation isunknown. The involvement of miR-155in the phenomenon of T cell exhaustion has yet tobe investigated.[Objectives]1. Study the function of CD4⁺T cells after exposure to persistent systemic antigen. 2. Identify the miRNAs which influence CD4⁺T cell exhaustion.3. Investigate the role of miR-155in the regulation of CD4⁺T cells subjected to apersistent antigenic stimulation.4. Explore the target of miR-155and its function in the CD4⁺T cell exhaustion model.[Methods]1. Purifed female Rag2^-/-Marilyn CD4⁺T cells, which are transgenic for a TCR specificfor the male H-Y peptide, were injected i.p into male Rag2^-/-IL-2Rγc^-/-recipient.Lymphocytes of spleen were collected21days after transfer. To identify the photype of thetransferred CD4⁺T cells, transferred CD4⁺T cells and na ve CD4⁺T cells were purified formicroarrays. IFN-γ secretion from culture supernatant of mixed splenocytes oranti-CD3/CD28stimulated CD4⁺T cells was evaluated by ELISA.2. MiR-155^-/-Rag2^-/-Marilyn mice were genenated by crossing breeding Rag2^-/-Marilynmice with miR-155^-/-B6mice. MiR-155^-/-Rag2^-/-Marilyn hemozygote mice were obtainedafter4generations. CD4⁺T cells from female miR-155⁺/⁺Rag2^-/-Marilyn andmiR-155^-/-Rag2^-/-Marilyn mice were i.p adoptive transferred into male Rag2^-/-IL-2Rγc^-/-recipient respectively. Lymphocytes of recipient spleen were collected21days aftertransfer. Antigen-specific CD4⁺T cell expansion and Th responses in spleen were detectedby FACS. IFN-γ secretion from culture supernant was evaluated by ELISA.Antigen-specific CD4⁺T cell infiltration in target organs were detected byimmunofluorescense. Moreover, CD4⁺T cells from the two goups were used formicroarrays. The potential miR-155target was further confirmed by real-time PCR,western blot and RNA immunopreciptation. In addition, a luciferase report plasmid whichcontained Hmox13’UTR was co-transfected with miR-155-Mimic or miR-Mimic controlinto HEK293T cells. The interation between Hmox1and miR-155was determined by dualluciferase assay.3. MiR-155^-/-Rag2^-/-Marilyn CD4⁺T cells were injected i.p into male Rag2^-/-IL-2Rγc^-/-recipient. Recipient mice were administrated i.p with HO-1inhibitor ZnPP every two daysfrom one day before the adoptive transfer. Mice injected with vehicle were used as controls.Spleen, heart, liver, lung, kidney and large intestine were collected21days post transfer.Antigen-specific CD4⁺T cells infiltration in target organs were evaluated by immunofluorecence. Antigen-specific CD4⁺T cells proliferation and Th cell responses inspleen were examined by FACS. Na ve miR-155^-/-CD4⁺T cells were activated byanti-CD3/anti-CD28simulation plus ZnPP or vehicle treatment for24h. IFN-γ and IL-2secretion from the supernant were evaluated by ELISA.4. B6and miR-155^-/-B6mice were immunized with MOG35-55peptide to induceexperimental autoimmune encephalomyelitis （EAE） respectively. Mice were injected i.pwith CoPP or ZnPP every two days from the immunization. Mice injected with vehiclewere used as controls. Spleen, draining lymph node, brain were collected at the indicatedtime post immunization. Infiltrated cell types and Th responses were detected and analyzedby FACS.[Results]1. Compared with na ve CD4⁺T cells, transferred CD4⁺T cells aquired a geneexpression profile similar to exhausted CD8⁺T cells. The exhausted CD4⁺T cells highlyexpressed Th1genes and inhibitory receptors, such as IFN-γ, STAT1, T-bet and PD-1.Blocking PD-1/PDL-1interation to the culture of splenocytes isolated from recipient micerestored T cell effector function, as assessed by IFN-γ production. Purified transferredCD4⁺T cells stimulated with anti-CD3/anti-CD28plus PD-1pathway engagement lowerIFN-γ production.2. Microarray results indicated that exhausted CD4⁺T cell expressed high level ofmiR-155. Transferred miR-155^-/-Marilyn CD4⁺T cells also expressed PD-1asmiR-155⁺/⁺counterpart, however, unlike exhausted miR-155⁺/⁺CD4⁺T cells, blockade ofPD-1/PD-L1interaction did not restore the function of exhausted miR-155^-/-CD4⁺T cells.Hmox1（The gene encodes HO-1） was one of the genes which were up-regulated inexhausted miR-155^-/-CD4⁺T cells. Compared with exhausted miR-155⁺/⁺CD4⁺T cells,Hmox1mRNA and HO-1protein level were highly expressed in exhaustedmiR-155^-/-CD4⁺T cells. RNA immunopreciptation results suggested that miR-155andhmox1mRNA could be coimmunopreciptated by anti-Argonaute2（anti-AGO2） antibody.Furthermore, in the dual luciferase assay experiment, cotransfected miR-155-Mimicrepressed expression of luciferase fused to Hmox13’UTR, while miR-Mimic control failedto repress luciferase expression. 3. ZnPP treatment enhanced the expansion of miR-155^-/-CD4⁺T cells transferred intomale recipients. Moreover, T cell infiltrates were also significantly increased in severalorgans after treatment with ZnPP. This revealed a role of HO-1for maintaining immuneunresponsiveness in exhausted miR-155^-/-CD4⁺T cells. HO-1inhibition with ZnPPincreased IL-2secretion from na ve miR-155^-/-CD4⁺T cells after in vitro stimulation withanti-CD3/CD28antibodies, however, HO-1inhibition did not improve the capacity ofexhausted miR-155^-/-CD4⁺T cells to produce IFN-γ. HO-1activity could account for T cellunresponsiveness in our system.4. B6with CoPP treatment lowered the clinal signs of EAE. ZnPP inhibition worsedthe clinical signs of EAE in miR-155^-/-mice with more CD3⁺T cell infiltrated into the brain.Th1and Th17cell responses in miR-155^-/-mice were lower than that in the other groups.[Conclusion]1. Exposure to persistent systemic antigen stimulation leads to T cell exhaustion,whose activity is controlled by PD-1.2. MiR-155is not required for T cell exhaustion, but it is indispensable for restoring Tcell function in the absence of PD-1inhibition.3. Hmox1is a direct target of miR-155. HO-1is responsible for maintaining immuneunresponsiveness in exhausted miR-155^-/-CD4⁺T cells.4. Inhibition of HO-1rescues susceptibility to EAE in miR-155^-/-mice.[Significance]1. We developed a CD4⁺T cell exhaustion model, which could be used for furtherstudy of T cell exhaustion.2. Our work has revealed one novel pathway in which miR-155, by targeting Hmox1,promotes T cell mediated inflammation.3. Inhibition HO-1could rescue the proliferative capacity of exhausted CD4⁺T cells,which could be essential for the development of innovative immunotherapies in chronicimmunological diseases.