Downregulation of T cell receptor expression by CD8(+) lymphocytes in kidney allografts.

Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses.     

Characterization of the murine H2–M3wt-restricted CD8 response against a hydrophobic, protease-resistant, phospholipid-associated antigen from Listeria monocytogenes

Summary: Mice infected with Listeria monocytogenes (LM) generate protective CD8 cells of varying specificity. One subset, unlike conventional LM-immune CDS cells, can respond to antigen-presenting cells (APC) treated with heat-killed LM (HKLM), These cells proved to have surprisingly uniform specificity, recognizing a product we designated HKLM-associated antigen (HAA) presented by the non-classical dass Ib product H2–M3'. HAA proved to be extremely hydrophobic and the bioactive portion of the molecule was highly protease-resistant, leading us initially to speculate that it might be a non-peptide. Recent studies, however, identify HAA as a complex containing lemA, a listerial protein bearing the immunogenic amino terminal peptide sequence fMIGWII, tightly associated with bacterial cardiolipin. A variety of cell types can process and present exogenous HAA/lemA. and the phospholipid component appears essential for this processing. Endosomal acidification and proteolysis are required for processing, but the site where antigen binds to H2–M3^wt within APC remains uncertain. HAA/lemA-immune effectors are unusually cross-reactive. We could readily detect H2–M3^wt-restricted responses to APC incubated with unrelated N -formylated peptides, and bacteria, HAA-like products represent an intriguing new set of bacterial antigens recognizable by immune CD8 cells.     

The binding and processing of monoclonal human IgG1 by cells of a human macrophage-like cell line (U937)

The goal of these experiments was to assess the relationship between the binding and processing of IgG by Fc-receptor-bearing cells. Cells of the U937 human macrophage-like cell line were incubated with 125I- labeled monomers, dimers, oligomers (composed of 2-4 IgG1 subunits), and HP (heavy polymers composed of 5 or more subunits per polymer) of monoclonal human IgG1 in vitro. Binding was assessed by spinning cells through a layer of phthalate oils. Internalization of IgG1 was assessed by quantitating residual binding to cells after surface-bound IgG was removed by a brief treatment with a solution containing 0.25 M acetic acid and 0.5 M sodium chloride. Catabolism was assessed by measuring the release of radioactive fragments of IgG1, which were not precipitated by 10% trichloroacetic acid. Unstimulated U937 bound about 10,000 molecules per cell of IgG1 monomer, with an equilibrium binding constant (Ka) of 5 X 10(8) M-1. After stimulation with a conditioned medium in vitro, binding per cell was increased 3-7--fold, and the Ka was decreased 2-4--fold. Both unstimulated and stimulated cells internalized and catabolized labeled IgG1 HP, but stimulated cells internalized and digested much more IgG1 HP per cell than unstimulated cells. Both monomers and dimers of IgG1 were internalized and degraded very slowly by stimulated cells, even though both preparations readily bound to cells. In contrast, oligomers and (to an even greater extent) IgG1 HP were internalized and degraded much more rapidly. Internalization of IgG1 HP was markedly inhibited by incubation at 4 degrees C, but not by incubation with a variety of metabolic inhibitors. Catabolism was inhibited by chloroquine and monensin (inhibitors of lysosomal acidification) and by cytochalasin (an inhibitor of microfilament polymerization). Binding to the surface of cells was not markedly inhibited by any agent tested. The capacity of cells to bind labeled IgG1 was markedly reduced by prior incubation in the presence of unlabeled IgG1. This reduction was in part due to the steric blockade of receptors caused by the avid, but reversible, binding of IgG1. In addition, IgG1 oligomers or HP (but not IgG1 monomers or dimers) also caused an irreversible reduction in the number of Fc receptors by a process analogous to receptor down-regulation, as observed in other receptor--ligand systems.     

Measurement of chimerism in cynomolgus monkeys using human-specific short tandem repeat-based assay

Preclinical testing of a mixed chimerism mediated organ transplant tolerance strategy, in a cynomolgus macaque model, would be facilitated by the establishment of a reliable technique for quantitative assessment of chimerism. Among various techniques used for measurement of chimerism in humans, microsatellite DNA profiling has been considered the most versatile one that can discriminate between two individuals. We adopted a commercially available short tandem repeat profiling methodology to cynomolgus monkeys using two human specific alleles, TPOX and CSF1PO. Polymerase chain reaction (PCR) was used to amplify these alleles, and the analysis of the PCR products was performed by capillary electrophoresis. Of 54 cynomolgus macaques investigated, only one pair with the same ABO blood type demonstrated identity at both alleles. This implies that this technique should interfere minimally with the assignment of donor-recipient pairs based upon molecular tissue typing or mixed lymphocyte cultures.     

Comparison of intravenous gamma globulin and a monoclonal anti-Fc receptor antibody as inhibitors of immune clearance in vivo in mice.

Fc-receptor-mediated clearance and nonspecific phagocytic clearance were assessed after the infusion of monomeric human IgG, heat-aggregated human IgG, and a monoclonal anti-mouse macrophage FcII receptor antibody (2.4G2) into normal mice. Each agent blocked Fc-receptor function in vivo, but 2.4G2 was much more potent per microgram than the other agents. Monomeric IgG in blocking doses did not affect other aspects of immune function. In contrast, aggregated IgG, and to a lesser extent, 2.4G2 reduced serum complement levels. In addition, these agents also caused moderate reductions in nonspecific phagocytic function. Monoclonal anti-mouse macrophage C3bi receptor antibody (Mac-1), another monoclonal antibody which binds to macrophage CR3 receptors without interfering with Fc-receptor function, also reduced serum complement and inhibited nonspecific phagocytic function. Complement depletion alone (produced by infusion of cobra venom factor) could not account for the observed changes in Fc receptor or nonspecific phagocytic function. We conclude that both monomeric IgG and anti-Fc-receptor antibodies can markedly inhibit Fc-receptor function in vivo; however, the pattern of physiologic changes produced by these agents differs.     

The selection of M3-restricted T cells is dependent on M3 expression and presentation of N-formylated peptides in the thymus

The major histocompatibility complex (MHC) class Ib molecule H2-M3 binds N-formylated peptides from mitochondria and bacteria. To explore the role of M3 expression and peptide supply in positive and negative selection, we generated transgenic mice expressing an M3-restricted TCR-alpha/beta from a CD8(+) T cell hybridoma (D7) specific for a listerial peptide (LemA). Development of M3-restricted transgenic T cells is impaired in both beta2-microglobulin-deficient and transporter associated with antigen processing (TAP)-deficient mice, but is not diminished by changes in the H-2 haplotype. Maturation of M3/LemA-specific CD8(+) single positive cells in fetal thymic organ culture was sensitive to M3 expression levels as determined by antibody blocking and use of the castaneus mutant allele of M3. Positive selection was rescued in TAP(-/-) lobes by nonagonist mitochondrial and bacterial peptides, whereas LemA and a partial agonist variant caused negative selection. Thus, M3-restricted CD8(+) T cells are positively and negatively selected by M3, with no contribution from the more abundant class Ia molecules. These results demonstrate that class Ib molecules can function in thymic education like class Ia molecules, despite limited ligand diversity and low levels of expression.