Unknown,Transcriptomics,Genomics,Proteomics

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Kidney gene expression profile following the transplant-induced reactivation of MCMV

ABSTRACT: Reactivation of latent HCMV is a significant infectious complication of organ transplantation, and current therapies target viral replication once reactivation of transcriptionally silent, latent virus has already occurred. The specific molecular pathways that activate viral gene expression are not well understood. Our studies aim to identify these factors, with the goal of developing novel therapies that prevent transcriptional reactivation in transplant recipients. MCMV is a valuable model for studying latency and reactivation of CMV induced by organ transplantation. We previously demonstrated that transplantation of MCMV-latently infected kidneys into allogeneic recipients induces transcriptional reactivation of immediate early (IE) gene expression within 48 hr. We used microarrays to profile global mouse kidney gene expression associated with transcriptional reactivation of MCMV immediate early gene expression at 48 hr after transplant of MCMV latently infected kidneys into allogeneic recipients. We identified differentially expressed genes and pathways associated with both early acute rejection and reactivation of MCMV. control vs. allograft

ORGANISM(S): Mus musculus

SUBMITTER: Chunfa Jie

PROVIDER: E-GEOD-72392 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Reconstitution of cytomegalovirus (CMV)-specific immunity following transplant remains a primary clinical objective to prevent CMV disease, and adoptive immunotherapy of CMV-specific T cells can be an effective therapeutic approach. Due to the persistence of CMV, most CMV-specific CD8+ T cells become terminally differentiated effector cells (TEFF). However, a minor subset retains a memory phenotype (TM). Interestingly, recent studies suggest that CMV-specific CD8+ T cells with different phenotypes may have different abilities to reconstitute sustained immunity following transfer. The immunology of human CMV (HCMV) infections is reflected in the mouse model of MCMV infection. We found that HCMV- and MCMV-specific T cells displayed shared genetic programs, validating the MCMV model for studies of CMV-specific T cells in vivo. After transfer, the proliferative capacity of MCMV-specific TM cells was vastly superior to TEFF cells. Strikingly, TM cells expanded and established sustained and diverse T cell populations even after multiple challenges. Although both TEFF and TM cells could protect Rag-/- mice, only TM cells could consistently survive after transfer into immune replete, latently infected recipients and respond if recipient immunity was lost. These data show that CMV-specific TM cells retain memory function during persistent infection and can re-establish CMV immunity when necessary. C57BL/6 mice were infected intraperitoneally (i.p.) with MCMV strain MW97.01 between 6-16 weeks of age. Splenocytes were isolated from chronically-infected mice and co-stained with three PE-conjugated tetramers loaded with the antigenic peptides from M38, m139 and IE3, all of which promote memory inflation. Cells were then stained with fluorescently conjugated antibodies and sorted on a MoFlo cell sorter. Naïve CD8+ cells were identified as CD44lo. MCMV-specific T cells were identified as CD8+, CD44hi and tetramer binding and then further segregated into memory and effector cells subsets by their expression of KLRG1 and CD127.

Project description:Regulatory T (Treg) cells play an important role in the induction and maintenance of peripheral tolerance. Treg cells also suppress a variety of other immune responses, including anti-tumor and alloimmune responses. We have previously reported that tumor-activated Treg cells express granzyme B and that granzyme B is important for Treg cell-mediated suppression of anti-tumor immune responses (GSE13409). Here, we report that allogeneic mismatch also induces the expression of granzyme B. Granzyme B-deficient mice challenged with fully mismatched allogeneic P815 mastocytoma cells have markedly improved survival compared to WT and other granzyme- or perforin-deficient mice, suggesting an immunoregulatory role for granzyme B in this setting. Treg cells harvested from the tumor environment of P815-challenged mice express granzyme B. Treg cells also express granzyme B in vitro during mixed lymphocyte reactions and in vivo in a mouse model of graft-versus-host disease (GVHD). However, in contrast to findings from our previously published tumor model, granzyme B is not required for the suppression of effector T cell (Teff) proliferation in in vitro Treg suppression assays stimulated by either Concanavalin A or allogeneic antigen presenting cells. Additionally, in an ex vivo assay, sort-purified in vivo-activated CD4+Foxp3+ Treg cells from mice with active GVHD -- under conditions known to induce granzyme B expression in Treg cells -- suppressed Teff cell proliferation in a granzyme B-independent manner. Adoptive transfer of naive granzyme B-deficient CD4+CD25+ Treg cells into a mouse model of GVHD rescued hosts from lethatlity equivalently to naive wild-type Treg cells. Serum analysis of GVHD-associated cytokine production in these recipients also demonstrated that Treg cells suppressed production of IL-2, IL-4, IL-5, GM-CSF, and IFN-gamma in a granzyme B-independent manner. In order to determine whether the context in which Treg cells are activated alters the intrinsic properties of Treg cells, we used Foxp3 reporter mice to obtain gene expression profiles of CD4+Foxp3+ Treg cells purifed from naive resting spleens, spleens from mice with acute GVHD, and from ascites fluid of mice challenged intraperitoneally with allogeneic P815 tumor cells. Unsupervised analyses revealed distinct activation signatures of Treg cells among the 3 experimental groups. Taken together, these findings demonstrate that granzyme B is not required for Treg cell-mediated suppression of GVHD, which is in contrast to what we have previously reported for Treg cell function in the setting of tumor challenge. Cell intrinsic differences could partially account for these differential phenotypes. These data also suggest the therapeutic potential of targeting specific Treg cell suppressive functions in order to segregate GVHD and graft-versus-tumor effector functions. Experiment Overall Design: Six replicates of Naive CD4+Foxp3+ Treg cells were purified from resting spleens, five replicates of allogeneic tumor-activated Treg cells and three samples of GVHD-activated Treg cells. Experiment Overall Design: Naive reps 1-3 are controls for the GVHD-activated samples. Experiment Overall Design: Naive reps 4-6 are controls for the Allogeneic tumor-activated samples.

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Kidney gene expression profile following the transplant-induced reactivation of MCMV

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