Innate Recognition of Allogeneic Non-Self Induces Monocyte Differentiation to Mature Dendritic Cells In Vivo

Abstract

after is a response to non-physiological cell death (‚danger‘) rather than to allogeneic non-self. we tested the alternate hypothesis that the innate immune system distinguishes between self and allogeneic non-self, causing DC maturation and activation of the adaptive immune response. Methods: T, B and NK cell-deficient B6 Rag -/- gc -/- or WT CX3CR1- GFP +/- reporter mice in monocyte lineage cells express GFP syngeneic (B6) or allogeneic (Balb/c) vascularized heart grafts. Graft-infiltrating cells analyzed 1, 3, 5, 10, 21 and 42 days after transplantation (Tx) and the function of host-derived DC tested ex and in vivo Results: in the graft by day after were host monocytes (mono-DC). numbers (2-5 fold) in allo than syn grafts at early time points (days 1-10) and at later time points (days 21 42) in allografts. Mono-DC allografts higher proportion of mature cells (MHCII + CD80 + ), IL-12p40, allogeneic T cells in the MLR, endotoxin-free antigen (OVA) to TCR-tg CD4 + (OT-II) and CD8 + (OT-I) T cells early and late after Tx. In vivo depletion of monocytes with clodronate liposomes abrogated the proliferation of alloreactive 1H3.1 TCR-tg CD4 + T cells transferred to allograft recipients. Depletion of host CD11b + cells by diphtheria toxin (DT) CD11b-DTR bone marrow chimeras dramatically reduced T cell accumulation in heart allografts examined on day 15 after Tx while DT-treated WT bone marrow chimeras rejected their allografts by day 10 with evidence of heavy T cell infiltration. self and allogeneic Multimers of soluble peptide-major histocompatibilty complex (pMHC) molecules are used in basic as well as in clinic immunology. They allow a specific visualization, phenotype characterization and isolation of antigen- specific T cells from ex vivo samples. Adoptive transfer of antigen-specific T cells sorted by pMHC multimers is an effective therapeutic strategy for treatment of patients with malignancies or infectious diseases after transplantation. A new reversible pMHC multimer technology, called pMHC Histamer technology , was developed enabling a specific detection and isolation of antiviral T cells from peripheral blood mononuclear cells. The HLA-A02/CMVpp65 and HLA-A01/ADV5_Hexon Histamers were generated by coupling 6xHis-tagged pMHC molecules onto cobalt-magnetic beads. The specificity and sensitivity of the magnetic bead-based Histamers was evaluated by flow cytometry. Sorting of antiviral CD8 + cytotoxic T cells (CTLs) was performed by magnetic cell separation, followed by the monomerization of the pMHC Histamers in the presence of L-histidine. Sorted T cells were analyzed in phenotypical and functional assays. The reversible Histamers showed superior specificity and sensitivity (up to 99.5%). Antigen-specific T cells isolated by this technology gave an excellent purity of up to 99.6%. A rapid and complete disassembly of the T-cell surface-bound pMHC Histamers followed by the subsequent dissociation of the pMHC