To study the mechanism of T cell-mediated dominant immune regulation, we have developed a murine transplantation model in which B6 (H-2b) recipients given a single dose of CD154 mAb (MR1; 0.5mg at day 0) maintain Balb/c (H-2d) hearts/kidneys for >50d. Preliminary studies show that CD4+ Treg are critical for tolerance maintenance, and for keeping CD8+ T cell activation in check in this CD8-dominant rejection model. We propose the following specific aims: (1). To dissect mechanisms by which CD4+ Treg suppress alloreactive CD8+ T cells. Hypothesis: CD154- blockade induced Treg maintain immune tolerance by suppressing alloreactive naive/memory CD8+ T cells. Our data provides evidence that alloreactive CD8+ T cells: (i) are the major effectors in rejecting allografts; and (ii) are tightly controlled by CD4+ Treg (but not deletional) mechanism. AIIograft rejection and CD8+ T cell (CD44highCD62L/Iow) activation will be used as parameters of Treg function in vivo. First, we will identify operational Treg, and then examine the mechanisms by which CD4+ Treg suppress alloreactive CDS+ T cells. We will focus on the molecular nature of cell-cell interactions, and soluble mediators. As non-deletional means to control alloreactive memory/effector CD8+ T cells are lacking, we will explore the potential of CD4+ Treg to prevent their function. (2), To delineate systemic/local requirements for CD4+ Treg. Hypothesis: Treg generation/function can be finely tuned via TLR4 - STAT6 - HO-1 mechanisms. To study cross talk between innate/adaptive immunity under CD154 blockade, TLR4 KO/mutant and STAT4/6 KO mice will serve as transplant donors or recipients. By blocking or activating TLR4 on host T cells or donor APCs, we will study mechanism by which TLR4 signaling impacts Treg. STAT-6 dependence of operational Treg and control of alloreactive CD8+ T cells will be probed. The role of HO-1 on Treg will be addressed by using: (i) HO-1 Tg mice, (it) mice deficient of Bach1, an inducible repressor of the HO-1 gene, and (iii) Tat-HO-1, a chimeric HO-1 fusion protein that can enter CD4+ T cells. (3). To analyze TCR Vbeta gene repertoire usage by, and the clonality of Treg. Hypothesis: Intra-graft (GIL) clonal expansion with selective TCR Vbeta usage/CDR3 size restriction represents an important event in Treg generation, and potential novel marker to trace Treg. We will use TCR Vbeta RT-PCR/CDR3 spectrotyping/immunoscope to document the skewed Vbeta repertoire with restricted polymorphism in in vitro enriched CD4+ Treg from tolerant recipients, as compared with CD4+ T cells from naive/rejecting controls. We will differentiate between allo-Ag induced Treg (more likely to reveal restricted Ag-specificity/specific CDR3 sequences) vs. natural Treg. A kinetic study will screen for defined Treg-specific CDR3 sequences in CD4+ T cells in GILs, and secondary lymphoid organs (draining LN/spleen) to determine the time course and the site of their appearance. The functional significance of GIL vs. LN Treg generation will be addressed by using LN-deficient aly/aly mice.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1-SAT (90))
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Kehn, Patricia J
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University of California Los Angeles
Schools of Medicine
Los Angeles
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Shen, Xiu-Da; Ke, Bibo; Uchida, Yoichiro et al. (2011) Native macrophages genetically modified to express heme oxygenase 1 protect rat liver transplants from ischemia/reperfusion injury. Liver Transpl 17:201-10
Uchida, Yoichiro; Freitas, Maria Cecilia S; Zhao, Danyun et al. (2010) The protective function of neutrophil elastase inhibitor in liver ischemia/reperfusion injury. Transplantation 89:1050-6
Yamaura, K; Boenisch, O; Watanabe, T et al. (2010) Differential requirement of CD27 costimulatory signaling for naïve versus alloantigen-primed effector/memory CD8+ T cells. Am J Transplant 10:1210-20
Ke, Bibo; Shen, Xiu-Da; Gao, Feng et al. (2010) Adoptive transfer of ex vivo HO-1 modified bone marrow-derived macrophages prevents liver ischemia and reperfusion injury. Mol Ther 18:1019-25
Uchida, Yoichiro; Ke, Bibo; Freitas, Maria Cecilia S et al. (2010) The emerging role of T cell immunoglobulin mucin-1 in the mechanism of liver ischemia and reperfusion injury in the mouse. Hepatology 51:1363-72
Liu, Dahai; Shen, Xiu-Da; Zhai, Yuan et al. (2009) Intragraft selection of the T cell receptor repertoire by class I MHC sequences in tolerant recipients. PLoS One 4:e6076
Uchida, Yoichiro; Freitas, Maria Cecilia S; Zhao, Danyun et al. (2009) The inhibition of neutrophil elastase ameliorates mouse liver damage due to ischemia and reperfusion. Liver Transpl 15:939-47
Ke, Bibo; Shen, Xiu-Da; Gao, Feng et al. (2009) Small interfering RNA targeting heme oxygenase-1 (HO-1) reinforces liver apoptosis induced by ischemia-reperfusion injury in mice: HO-1 is necessary for cytoprotection. Hum Gene Ther 20:1133-42
Shen, Xiuda; Wang, Yue; Gao, Feng et al. (2009) CD4 T cells promote tissue inflammation via CD40 signaling without de novo activation in a murine model of liver ischemia/reperfusion injury. Hepatology 50:1537-46
Jindra, Peter T; Hsueh, Aileen; Hong, Longshen et al. (2008) Anti-MHC class I antibody activation of proliferation and survival signaling in murine cardiac allografts. J Immunol 180:2214-24

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