Early induction of MHC antigens in human liver grafts. An immunohistologic study.

The present study documents major histocompatibility complex (MHC) Class I and II expression during early acute rejection of human liver grafts. Serial graft biopsies (pretransplant, time zero, and 1 week) were studied. Ten patients received azathioprine (AZA) and prednisone; the other six patients were treated with quadruple therapy (azathioprine, cyclosporine A, prednisone, and cyclophosphamide). To study the specificity of changes in MHC antigen expression, biopsies of six patients with minor or no morphologic abnormalities served as controls. In addition, phenotypes of inflammatory cells present during rejection were analyzed using a panel of monoclonal antibodies. The results show that during acute rejection expression of MHC Class I and II antigens increased significantly in the AZA-treated patients, in a pattern similar to that seen in the patients treated with quadruple therapy, showing enhanced MHC Class I expression on hepatocytes, bile duct epithelium, and sinusoidal endothelium, and Class II antigen on Kupffer cells and sinusoidal endothelium. Bile duct epithelium was consistently positive for Class II antigen; no significant difference with the nonrejection group was observed. T cells are the predominant inflammatory cells during rejection with equal quantities of CD4+ and CD8+ cells. A majority of the infiltrating T cells show expression of Class II antigen but do not react with anti-interleukin-2 receptor antibody. This may be the result of immunosuppressive therapy or a simple reflection of the temporary expression of interleukin-2 receptors during lymphocyte activation. The authors hypothesize that the induction of MHC antigens on bile duct epithelium leads to rejection whereas the expression on hepatocytes represents an epiphenomenon.     

Tissue distribution of restricted leukocyte common antigens. A comprehensive study with protein- and carbohydrate-specific CD45R antibodies

A panel of monoclonal antibodies, specific for either protein or carbohydrate components of the leukocyte common antigen (LCA), were used to characterize the expression pattern of different variants of this antigen complex. Immunohistochemical and flow cytometric analysis was carried out on human lymphoid tissues from different anatomical compartments. We found that variations in terminal sialic acid or O-linked sugar moieties were associated with drastic differences in reactivity with some of the CD45R antibodies. For example, the carbohydrate determinants recognized by MT2 (CD45RA) and 2B11 (CD45) were not or were very weakly expressed on germinal center cells, whereas the carbohydrate determinant recognized by MT3 (CD45RB) was not expressed on the majority of mantle zone B cells. On the other hand, KiB3 (CD45RA) reacted with a protein determinant that is normally only exposed on mantle zone B cells but can be uncovered on marginal zone B cells and the CD45RA-positive T-cell subpopulation by enzymatic treatment. The findings suggest that differential biosynthesis and expression of the carbohydrate components of LCA are important mechanisms to generate additional LCA heterogeneity. Similar to the differential usage of the variable region, the exact carbohydrate composition is also linked to the stage of cell development. Heterogeneity in carbohydrate composition of the extracellular domain of LCA appears to enable interactions with different ligands, thereby holding the key to the regulation of LCA cytoplasmic tyrosine phosphatase activity.     

B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage-associated antigens

Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced. The mAb, designated HIS14 (IgG1), HIS22 (IgM) and HIS24 (IgG2b), were characterized for binding to lymphoid and nonlymphoid tissues by immunoperoxidase staining of frozen sections and by (double-) immunofluorescence staining of single cell suspensions from lymphoid organs. HIS14 recognized a pan B cell determinant: it reacted with virtually all cells of each anatomic B cell compartment and with about 95% of surface (s)Ig+ cells in thoracic duct lymph and in suspensions of spleen and lymph nodes. HIS22 and HIS24 detected B lineage-associated antigens expressed by major subpopulations of B cells. HIS22 predominantly stained the lymphocyte corona, but not (or weakly) the germinal centers and splenic marginal zones, whereas HIS24 reacted with both corona and germinal center and not (or weakly) with marginal zone. In accordance with this, substantial proportions of sIg+ cells in spleen cell suspensions did not express HIS22 or HIS24 determinants (20% and 27%, respectively). In bone marrow the vast majority of cytomplasmic mu+ pre-B cells were HIS14+ and HIS24+, and up to one third also HIS22+, indicating an appearance of the determinants early in B lymphocytopoiesis. The antigens recognized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentiation: none of the mAb bound to plasma cells. As far as detectable, neither cells of myeloid and erythroid lineages in bone marrow nor thymocytes were stained by HIS14, HIS22, or HIS24. In suspensions of peripheral lymphoid organs (spleen and lymph nodes) but not in thoracic duct lymph, HIS14 and HIS24 labeled a small proportion (12% and 14%, respectively) of Ig- cells. HIS22 did not bind to Ig- peripheral lymphocytes. Reactivity of HIS14, HIS22 and HIS24 with nonlymphoid tissues was virtually absent; HIS22 stained the high endothelial venules in lymph nodes and Peyer's patches. As determined by immunoblotting, the antigenic determinants on lymph node cells recognized by HIS14, HIS22 and HIS24 were present on molecules with an apparent molecular mass of 205 kDa, 210 (and 175) kDa and 205 kDa, respectively, which is similar to the molecular mass of the B cell form of the rat leukocyte common antigen. In addition, the antigens recognized by HIS14, HIS22 and HIS24 co-capped with the leukocyte common antigen. This suggests that each of the three mAb recognize determinants present on the B cell form of the leukocyte common antigen.     

Splenic marginal zone lymphomas presenting with splenomegaly and typical immunophenotype are characterized by allelic loss in 7q31-32.

Splenic marginal zone lymphoma (SMZL) is a rare non-Hodgkin's lymphoma that recently has been recognized as an entity. The first goal of this study was to identify potential chromosomal aberrations in this entity by cytogenetic analysis and comparative genomic hybridization (CGH). The second goal was to assess the frequency of 7q31-32 allelic imbalances in SMZL with primary involvement of the spleen and the typical immunophenotype (IgM+; IgD(dim); and CD5-, CD10-, and CD23-). We applied CGH and cytogenetics to 13 cases of SMZL with primary splenic involvement. By CGH, we found DNA copy number changes in 11 of 13 cases. Overall chromosomal gains were more frequent than chromosomal losses. Gains were most frequently detected for chromosome X, chromosome 3, and chromosome 18. Losses commonly involved chromosome 7 and chromosome 6.CGH and cytogenetic analysis showed a deletion in chromosome 7q31 in 4 cases. Loss of heterozygosity (LOH) analysis using three microsatellite markers located at 7q31 revealed LOH in 9 cases. Remarkably, 2 of the 4 cases that lacked a 7q31 deletion had an atypical immunophenotype because they were partially CD23 positive. The other 2 cases were not informative. The findings indicate that SMZL with primary splenic presentation and the typical IgM+, IgDdim, CD5-, CD10-, CD23- immunophenotype is characterized by the presence of deletions in chromosome 7q31-32.     

Inhibition of alloreactivity in vitro by monoclonal antibodies directed against restricted isoforms of the leukocyte-common antigen (CD45).

The leukocyte common antigen (LCA) is a protein tyrosine phosphatase and is identified by the CD45 cluster of monoclonal antibodies. CD45 is expressed in high-density on cells of hematopoietic lineage and generally is immunoprecipitated as 4 bands (220, 205, 190, and 180 KDa). Genetic studies have shown that a single gene produces additional forms of the molecule by alternate splicing including CD45RA (220, 205), CD45RO (180), and CD45RB (220, 205, 190). We have prepared a CD45RB Mab termed "MT3" that binds to a sialic acid dependent epitope. Since the LCA is one of the major targets of antilymphocyte globulin, we assessed a panel of CD45 and CD45R Mab for their ability to inhibit alloreactivity in vitro. MT3 (CD45RB) inhibits the allogeneic MLR and inhibits CD4+ T cells from expressing interleukin 2 receptors, and prevents CD4+ CD45RA- T cells from entering the proliferative phase of the cell cycle. Mem 93 (CD45RA) inhibits the generation of cytotoxic T cells. These data suggest that the CD45RB and CD45RA isoforms of the LCA may be appropriate targets for in vivo immunotherapy.     

Fetal and neonatal development of human spleen: an immunohistological study.

Localization and immunophenotype of lymphocyte subsets in fetal human spleens were studied by employing a panel of monoclonal antibodies (McAb) in an immunoperoxidase staining procedure on frozen tissue sections. Spleens varied from 15 weeks of gestational age (gestational weeks, gw) to newborn. The white pulp consisted of intermediate-sized lymphocytes; no separate compartments could be discerned. Germinal centre development was not observed. Dendritic cells stained for B2, HB5, aC3bR, anti-DRC and OKIa, but in most cases not for immunoglobulin. Although low cellular immunity is observed in neonates, T cells showed adult phenotypes in proportions comparable to the adult situation; immature OKT6(+) lymphocytes were rarely seen. Very few cells stained with anti-NK cell antibody Leu7. B cells all expressed B1, Leu14, aC3bR, T10 and OKIa, were strongly positive for BA1, and mostly stained very weakly for B2 and HB5. Almost all B cells expressed IgM and IgD simultaneously, and very few expressed IgG. IgA-positive cells were absent. At 15 gw a considerable number of IgM(+) B cells showed Leu1 staining, but this decreased during development. These cells may represent the normal counterpart of Leu1(+) IgM(+) cells observed in B-CLL and immunocytic and centrocytic malignant lymphomas. After 25 gw only very few Leu1(+) IgM(+) cells were seen. Altogether, the morphology and immunophenotype of white pulp B cells were different from the predominating adult B-cell subsets, at least until birth. These 'immature' splenic B cells may be precursors for adult splenic B-cell subsets. Considering the presumed role of the marginal zone in the immunity against TI-2 antigens, the absence of a marginal zone at birth may be a main factor in the defective immunity against these antigens in neonates.     

HLA-G protein expression as a potential immune escape mechanism in classical Hodgkin's lymphoma

Classical Hodgkin's lymphoma (cHL) is characterized by the presence of an abundant reactive infiltrate, lacking effective cytotoxic responses. Especially in Epstein–Barr virus (EBV)-negative cHL, the neoplastic Hodgkin–Reed–Sternberg (HRS) cells have lost protein expression of major histocompatibility complex (MHC) class I, enabling escape from cytotoxic T lymphocyte (CTL) responses. However, downregulation of MHC class I generally induces natural killer (NK) cell activation. The paucity of NK cells in the reactive infiltrate of cHL and the systemic NK cell deficiency observed in cHL patients led us to investigate the expression of human leukocyte antigen (HLA)-G, which is known to inhibit NK-cell- and CTL-mediated cytotoxicity. By immunohistochemistry, HLA-G protein was expressed by HRS cells in 54% (95/175) of cHL cases. This expression was associated with absence of MHC class I on the cell surface of HRS cells ( P  < 0.001) and EBV-negative status ( P  < 0.001). Previously, genetic markers located in the proximity of the HLA-A and HLA-G genes had been shown to be associated with susceptibility to EBV-positive cHL. In the present study, these markers associated with MHC class I protein expression but not with presence of HLA-G. Our results suggest that induction of HLA-G protein expression in HRS cells contributes to the modulation of immune responses observed in cHL.