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The generation of knockout mice with targeted gene disruption has provided a valuable tool for studying the immune response. Here we describe the use of CD4 and CD8 knockout mice to examine the role of CD4+ and CD8+ cells in initiating allotransplantation rejection. Pretreatment with a brief course of depletive anti-CD4 monoclonal antibody therapy allowed permanent survival of heart, but not skin, allografts transplanted across a major histocompatibility barrier. However, skin as well as heart grafts were permanently accepted in the CD4 knockout mice. Transfer of CD4+ cells into CD4 knockout recipient mice 1 d before skin engraftment reconstituted rejection, demonstrating that CD4+ cells are necessary for initiating rejection of allogeneic transplants. Major histocompatibility complex disparate heart and skin allografts transplanted into CD8 knockout recipients were rejected within 10 d. This study demonstrates that CD4+ but not CD8+ T cells are absolutely required to initiate allograft rejection.
Mice deficient in the gene encoding the peptide transporter associated with antigen processing (TAP1) have drastically reduced levels of surface expression of MHC class I molecules and few CD8+ T cells. Addition of class I binding peptides to cultured fetal thymi from TAP1 mutant mice invariably allowed the rescue of class I expression, while only some of these peptides promoted the positive selection of CD8+ T cells, which were polyclonal and specific for the peptide-MHC complex. A nonselecting peptide was converted to a mixture of selecting peptides when the residues involved in the interaction with TCRs were altered. A mixture of self-peptides derived from C57BL/6 thymi induced positive selection of CD8+ T cells at concentrations that gave relatively low class I surface expression. The implication of these observations is that self-peptides determine, in part, the repertoire of specificities exhibited by CD8+ T cells.
Immunocompetent cells in bone marrow allografts have been associated with a graft-versus-leukemia (GVL) effect. To further characterize effector mechanisms that may be involved in this GVL phenomenon, we have previously established an in vitro model to identify allogeneic T-cell clones that selectively mediate cytotoxicity against a patient's leukemic cells, but not against nonleukemic lymphocytes from the same patient. We have modified this in vitro model to test whether the Ph1 chromosome and the P210 fusion protein it controls have a detectable role in leukemia-specific recognition by allogeneic T-cell clones. In this report, T-cell lines reactive with allogeneic Ph1 chromosome-bearing (Ph1+) chronic myeloid leukemia (CML) cell lines were derived and selected to be minimally reactive with Ph1 negative (Ph1-) lymphoid lines from the same patient. However, after prolonged culture, these same T-cell lines also mediated significant destruction of the Ph1- target cells from the same patients. These T-cell lines specifically recognized cells from the allogeneic CML patient to which they were sensitized, and were not contaminated by an outgrowth of natural killer cells. Furthermore, subclones could be derived from these T-cell lines, and some of these subclones again showed selective killing of the allogeneic Ph1+ leukemia cell lines, and not of the Ph1- cell line from the same patient. Analyses of T-cell receptor (TCR) genes showed the alloreactive T-cell lines and the Ph1+ selective subclones derived from them to be of the same clonal origin. This suggests that the same T cells reacting with antigens expressed on the nonleukemic Ph1- targets can at times selectively and preferentially kill the allogeneic Ph1+ cells. As the same TCR that recognizes Ph1+ cells also can recognize the Ph1- targets, it appears that the Ph1+ chromosome does not play a detectable role in recognition by these allogeneic T-cell clones. This in vitro observation may provide a model for evaluating the relationship between GVL and graft-versus-host disease effects.
In epithelial cells integrins are segregated on discrete domains of the plasma membrane. Redistribution may also occur during migration or differentiation. However, little is known about the mechanisms that control such redistribution. Receptor internalization may be a part of one such mechanism. We developed a quantitative assay and measured internalization of two epithelial integrin heterodimers, alpha 6 beta 1 and alpha 6 beta 4, induced by cross-linking with specific antibodies. alpha 6 beta 1 is a receptor for EHS laminin, while alpha 6 beta 4 is a receptor for a component of the basement membrane. alpha 6 beta 4 plays an important role in the establishment of hemidesmosomes, and becomes redistributed on the epithelial cell surface when cells are in a migratory phase. We report that alpha 6 beta 4 is efficiently internalized in human keratinocytes. More than 25% of cell surface alpha 6 beta 4 was internalized at 30 minutes, after cross-linking with A9, an anti-beta 4 monoclonal antibody. alpha 6 beta 1 is also internalized, in melanoma and teratocarcinoma cells, with maximum values of 20% of total receptors expressed at the cell surface. No significant difference was observed between the alpha 6 isoforms A and B in these assays. To determine whether alpha 6 cytoplasmic domains could influence integrin endocytosis, we prepared chimeric constructs with the extracellular domain of a reporter protein (CD8), and the cytoplasmic domains of either alpha 6 A or alpha 6 B. Both alpha 6 cytoplasmic domains but not a control cytoplasmic domain promoted internalization of the chimeric proteins, after cross-linking with antibody. Internalization of alpha 6 integrins may have a role in redistributing these receptors at the cell surface. Furthermore, the cytoplasmic domains of alpha 6 may be involved in regulating integrin internalization.
The class II (Ia) MHC molecules are cell surface proteins that regulate the activation of T cells. B lymphocyte expression of class II molecules has been shown to be influenced by a number of external stimuli. It has been previously demonstrated that treatment of these cells with IL-4 leads to an increase in class II gene transcription at 18 h as well as to an increase in steady state class II mRNA. It has also been previously demonstrated that LPS treatment of splenic B cells from athymic mice results in a decrease in steady state mRNA encoding the A alpha class II protein. This decrease persists for at least 18 h. Nuclear run-on transcription assays now demonstrate that although steady state mRNA levels for A alpha are decreased by LPS treatment of athymic mouse lymphocytes, LPS does not decrease A alpha gene transcription, but rather modestly activates transcription of this class II gene. LPS and IL-4 have been demonstrated to be synergistic stimuli for a number of genes. Costimulation of splenic lymphocytes from athymic mice with IL-4 plus LPS leads to activation of transcription, but the increase in transcription is no more than that seen with IL-4 stimulation alone. However, in costimulated lymphocytes, steady state A alpha-encoding mRNA levels are intermediate between the increased levels seen with IL-4 stimulation and the decreased levels seen with LPS stimulation. Therefore, LPS and IL-4 act nonsynergistically in class II gene transcription and the effects of LPS in decreasing steady state mRNA are most likely posttranscriptional. An IL-4-inducible and an LPS-inducible DNA-binding protein have been previously identified in splenic lymphocytes from athymic mice. Both nuclear binding proteins form complexes with the same DNA fragments from a control region of the A alpha gene. These nucleoprotein complexes comigrate under nondenaturing conditions and display identical patterns of binding with a panel of oligonucleotide competitors. Oligonucleotides representing protein binding sites of the IL-4 and LPS-induced DNA-binding proteins cross-compete for protein binding. Therefore, the binding proteins induced by LPS and IL-4 are likely related, and may function at different efficiencies as activators of A alpha gene transcription.
We have studied the ligand specificity of a gamma delta T-cell receptor (TCR) derived from a mouse T-cell hybridoma (KN6). KN6 cells reacted with syngeneic (C57BL/6) cells from various origins (splenocytes, thymocytes, peritoneal exudate cells, etc.) and cells from many different mouse strains. KN6 reactivity against cells from a panel of congenic and recombinant mouse strains demonstrated that the ligand recognized by KN6 is controlled by an MHC-linked gene that most probably maps in the TL region. We cloned this gene and formally proved that it does map in the TL region. This gene turned out to be a novel class I gene (designated T22b) belonging to a hitherto unidentified cluster of TL region genes in strain C57BL/6. This gene was expressed in many different tissues and cell types. We also examined the tissue expression of several other TL genes. One of these, the structural gene (T3b) encoding the thymus leukemia (TL) antigen from C57BL/6 mice, was specifically expressed in the epithelium of the small intestine. Since the intestinal epithelium of the mouse is known to be the homing site for a subset of gamma delta T cells (i-IEL) bearing diverse TCR with V7 rearranged gamma chains, we propose that the T3b gene product is part of the ligand recognized by some of the i-IEL. Our data support the idea that gamma delta T cells might be specific for non-classical class I or class I-like molecules and suggest that gamma delta TCR and non-classical MHC co-evolved for the recognition of a conserved set of endogenous or foreign peptides.
The transporter associated with the antigen processing 1 (TAP1) gene encodes a subunit for a transporter, presumed to be involved in the delivery of peptides across the endoplasmic reticulum membrane to class I molecules. We have generated mice with a disrupted TAP1 gene using embryonic stem cell technology. TAP1-deficient mice are defective in the stable assembly and intracellular transport of class I molecules and consequently show severely reduced levels of surface class I molecules. These properties are strikingly similar to those described for the TAP2 mutant cell line RMA-S. Cells from the TAP1-deficient mice are unable to present cytosolic antigens to class I-restricted cytotoxic T cells. As predicted from the near absence of class I surface expression, TAP1-deficient mice lack CD4-8+ T cells.