Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice.

Inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome) is characterized by failure of immunoglobulin isotype switching and severe defects of cell-mediated immunity. To test the potential for gene transfer therapy to correct this disorder, we transduced murine bone marrow or thymic cells with a retroviral vector containing the cDNA for the murine CD40 ligand (CD40L) and injected them into CD40L-/- mice. Even low-level, constitutive expression of the transgene stimulated humoral and cellular immune functions in these mice. With extended follow-up, however, 12 of 19 treated mice developed T-lymphoproliferative disorders, ranging from polyclonal increases of lymphoblasts to overt monoclonal T-lymphoblastic lymphomas that involved multiple organs. Our findings show that constitutive (rather than tightly regulated), low-level expression of CD40L can produce abnormal proliferative responses in developing T lymphocytes, apparently through aberrant interaction between CD40L+ and TCRalphabeta+CD40+ thymocytes. Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades.

FL-160 proteins of Trypanosoma cruzi are expressed from a multigene family and contain two distinct epitopes that mimic nervous tissues.

The partial sequence of a gene encoding the COOH terminus of a protein of apparent molecular weight of 160 kD associated with the flagellum of trypomastigotes of Trypanosoma cruzi (FL-160 now renamed to FL-160-1) has been previously reported. The COOH terminus of FL-160-1 has an epitope, defined by 12 amino acids, which molecularly miMics a nervous tissue antigen of 48 kD found in myenteric plexus, sciatic nerve, and a subset of cells in the central nervous system. We now report that FL-160 is a family of highly related genes. The sequence has been determined for the entire open reading frame (ORF) of one of the members of the FL-160 gene family (FL-160-2) and three other partial ORFs. Sequence analysis reveals the various members of the FL-160 gene family to be approximately 80% homologous in the predicted amino acid sequence, but all retain the 12-amino acid molecular mimicry epitope on the COOH terminus. Comparison of the sequence of FL-160-2 to other sequences demonstrates amino acid homology to bacterial sialidase (27%), members of the SA85 gene family (25-30%) and the shed acute-phase antigen/neuraminidase/trans-sialidase gene family (25-30%). Quantitative hybridization at high stringency suggests 750 copies of FL-160 are present in the DNA of each parasite. Reverse transcription and sequence analysis demonstrates that at least five of the members of the FL-160 gene family are transcribed. The NH2 terminus of one of the FL-160 gene products was expressed and antibodies prepared. Antibodies directed to either the COOH or the NH2 terminus of FL-160 bind a 160-kD T. cruzi protein. Both antibodies bind the surface membrane in the flagellar pocket of the trypomastigote. Antibodies to the NH2 terminus bind epineurium and scattered linear densities in sciatic nerve in a pattern distinct from the pattern with antibodies to the COOH terminus. Thus, there are at least two distinct molecular mimicry epitopes on the FL-160 molecule and both mimic epitopes found in nervous tissues. FL-160 may be involved in the generation of autoimmunity to nervous tissues by molecular mimicry, observed in chronic Chagas' disease.

A role for endogenous transforming growth factor beta 1 in Langerhans cell biology: the skin of transforming growth factor beta 1 null mice is devoid of epidermal Langerhans cells.

Transforming growth factor beta 1 (TGF-beta 1) regulates leukocytes and epithelial cells. To determine whether the pleiotropic effects of TGF-beta 1, a cytokine that is produced by both keratinocytes and Langerhans cells (LC), extend to epidermal leukocytes, we characterized LC (the epidermal contingent of the dendritic cell [DC] lineage) and dendritic epidermal T cells (DETC) in TGF-beta 1 null (TGF-beta 1 -/-) mice. I-A+ LC were not detected in epidermal cell suspensions or epidermal sheets prepared from TGF-beta 1 -/- mice, and epidermal cell suspensions were devoid of allostimulatory activity. In contrast, TCR-gamma delta + DETC were normal in number and appearance in TGF-beta 1 -/- mice and, importantly, DETC represented the only leukocytes in the epidermis. Immunolocalization studies revealed CD11c+ DC in lymph nodes from TGF-beta 1 -/- mice, although gp40+ DC were absent. Treatment of TGF-beta 1 -/- mice with rapamycin abrogated the characteristic inflammatory wasting syndrome and prolonged survival indefinitely, but did not result in population of the epidermis with LC. Thus, the LC abnormality in TGF-beta 1 -/- mice is not a consequence of inflammation in skin or other organs, and LC development is not simply delayed in these animals. We conclude that endogenous TGF-beta 1 is essential for normal murine LC development or epidermal localization.

Early progression of thymocytes along the CD4/CD8 developmental pathway is regulated by a subset of thymic epithelial cells expressing transforming growth factor beta.

Precursor cells differentiate into mature CD4+ and CD8+ T cells in the inductive environment of the thymus by undergoing a series of distinct developmental steps marked by expression of the coreceptor molecules CD4 and CD8. Among the earliest cells to enter the CD4/CD8 developmental pathway are CD4-CD8lo precursors cells that differentiate into CD4+CD8+ thymocytes. Here we show that differentiation of precursor cells into CD4+CD8+ thymocytes requires at least one cell division and that their progression through a cell cycle is specifically retarded in the thymus by interaction with thymic epithelial cells that express transforming growth factor beta (TGF-beta) proteins. We also demonstrate that TGF-beta proteins, either in solution or bound to cell membranes, can regulate cell cycle progression and differentiation of CD4-CD8lo precursor cells into CD4+CD8+ thymocytes. The regulatory effect of TGF-beta is specific for CD4-CD8lo precursor cells as TGF-beta proteins do not regulate the earlier generation of CD4-CD8lo precursor cells from CD4-CD8- thymocytes. Finally, we demonstrate that TGF-beta proteins are expressed in vivo in the intact thymus on subcapsular and cortical thymic epithelium where they can contact developing CD4-CD8lo precursor cells. Thus, thymic epithelial cells expressing TGF-beta proteins can actively regulate the rate at which CD4+CD8+ thymocytes are generated from CD4-CD8lo precursor cells.

Characterization and cloning of a novel glycoprotein expressed by stromal cells in T-dependent areas of peripheral lymphoid tissues.

A novel glycoprotein (gp) expressed by stromal cells of peripheral lymphoid tissue has been characterized immunohistochemically, biochemically, and at the molecular level. This molecule, gp38, was identified with a monoclonal antibody (mAb) (clone 8.1.1) previously shown to react with a subpopulation of thymic epithelium. This mAb generated a reticular labeling pattern in medullary and paracortical areas of lymph nodes and in splenic white pulp. At the ultrastructural level, labeling by the 8.1.1 mAb was restricted to fibroblastic reticular stromal cells. Serial sections of lymph node and spleen labeled with anti-CD3, anti-B220, and 8.1.1 mAbs clearly showed that the 8.1.1+ cells were associated with T cell-dependent areas. In severe combined immunodeficiency (SCID) or Nu/Nu mice, splenic white pulp also exhibited reticular labeling with the 8.1.1 mAb in the absence of detectable numbers of T cells, indicating that the appearance of 8.1.1-reactive stromal cells in discrete areas of peripheral lymphoid tissue was T cell independent. The cDNA encoding this stromal cell molecule was cloned by direct expression in COS cells and found to encode a 172 amino acid sequence with the typical features of a type I integral membrane protein. COS cells transfected with the gp38 clone direct the expression of an approximately 38-kD protein that reacts with the 8.1.1 mAb but not with isotype-matched controls. Comparison of the predicted amino acid sequence of 8.1.1 mAb but not with isotype-matched controls. Comparison of the predicted amino acid sequence of 8.1.1 with proteins in the National Biomedical Research Foundation (NBRF) data base showed that gp38 is very closely related to the early response protein OTS-8 obtained from a cDNA library of tumor promoting agent (TPA)-induced murine osteoblastic cell line, MC3T3-E1.

Transgenic mice demonstrate that epithelial homing of gamma/delta T cells is determined by cell lineages independent of T cell receptor specificity.

gamma/delta T cells with different TCR repertoires are compartmentalized in different epithelia. This raises the possibility that the TCR-gamma/delta directs homing of T cells to these epithelia. Alternatively, the signals that induce TCR-gamma/delta expression in developing T cells may also induce homing properties in such cells, presumably in the form of cell surface receptors. We have examined this issue by studying the homing of gamma/delta T cells in transgenic mice constructed with specific pairs of rearranged gamma and delta genes. In such mice, most gamma/delta T cells express the transgene-encoded TCR. We find that homing to both skin and gut epithelia is a property of T cells and is not determined by the type of gamma and delta genes used to encode their TCR. We also studied the effect of TCR replacement on the expression of Thy-1 and CD8 proteins on the gamma/delta T cells associated with gut epithelia. Our results show that the expression of the appropriate type of TCR-gamma/delta is not required for the Thy-1 expression by these T cells, suggesting that Thy-1 is not an activation marker. In contrast, CD8 expression by gut gamma/delta T cells seems to depend on the expression of the appropriate type of TCR.

Cloning of the murine thymic stromal lymphopoietin (TSLP) receptor: Formation of a functional heteromeric complex requires interleukin 7 receptor.

The cellular receptor for murine thymic stromal lymphopoietin (TSLP) was detected in a variety of murine, but not human myelomonocytic cell lines by radioligand binding. cDNA clones encoding the receptor were isolated from a murine T helper cell cDNA library. TSLP receptor (TSLPR) is a member of the hematopoietin receptor family. Transfection of TSLPR cDNA resulted in only low affinity binding. Cotransfection of the interleukin 7 (IL-7)Ralpha chain cDNA resulted in conversion to high affinity binding. TSLP did not activate cells from IL-7Ralpha(-/)- mice, but did activate cells from gammac(-/)- mice. Thus, the functional TSLPR requires the IL-7Ralpha chain, but not the gammac chain for signaling.

Monoclonal antibodies to CD44 and their influence on hyaluronan recognition.

Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand.

Endocytosis and degradation of monoclonal antibodies targeting human B-cell malignancies.

Seven murine monoclonal antibodies (MoAbs) recognizing differentiation antigens present on B-lymphocytes were analyzed in preclinical studies for their potential use for antibody-targeted therapy of B-cell malignancies. MoAbs HD37 (anti-CD19), 1F5 (anti-CD20), HD6 (anti-CD22), MB-1 (anti-CD37), G28-5 (anti-CDw40), 7.2 (anti-class II), and DA4-4 (anti-IgM) were studied for their binding avidities, immunoreactivities, isotypes, endocytosis rates, degradation rates, and number of binding sites on Daudi cells. Lineweaver-Burke analyses of 125I-labeled MoAbs demonstrated immunoreactivities ranging from 59 to 92%. Scatchard analyses of 125I-MoAbs demonstrated that five of the antibodies had binding avidities in excess of 10(9) L/M, whereas MoAbs 1F5 and HD37 had avidities of 3-4 x 10(8) L/M. CD20, CD37, mu, and HLA Class II were found to be highly expressed (200,000-400,000 binding sites/cell) on Daudi cells whereas CD19, CD22, and CDw40 were less densely expressed (80,000-100,000 sites/cell). DA4-4 (mu), HD6 (CD22), and G28-5 (CDw40) were rapidly internalized by cells, HD37 (CD19) and MB-1 (CD37) underwent endocytosis at an intermediate rate, and 7.2 (class II) and 1F5 (CD20) were internalized slowly. Trichloroacetic acid precipitation and high-performance liquid chromatography revealed the following relative rates of 125I-MoAb degradation: DA4-4 (mu) greater than HD6 (CD22) greater than HD37 (CD19) greater than G28-5 (CDw40) greater than MB-1 (CD37) greater than 1F5 (CD20) greater than 7.2 (class II).

In situ ultrastructural demonstration of cells bearing Ia antigens in the murine pancreas.

Although an important role has been postulated for class II major histocompatibility gene products in the rejection of transplanted islets, the identity of the cells bearing these molecules within pancreatic tissue has been the topic of some controversy. To clearly define the cell population(s) within the murine pancreas that express Ia antigens, perfused murine pancreata were processed for the ultrastructural in situ demonstration of Ia antigens. It was found that Ia antigen expression was restricted to mononuclear cells adjacent to capillaries throughout the exocrine and endocrine portions of the pancreas. Vascular endothelium and ductular epithelium did not express detectable amounts of Ia antigens.

Expression of class II MHC antigens in murine pancreas after streptozocin-induced insulitis.

In this study, most class II antigen expression during streptozocin-induced insulitis was associated with the mononuclear cells infiltrating the pancreas. During the early stages of the insulitis response, phagocytic cells expressing class II antigens and containing beta-cell debris were often observed. Although there was de novo expression of class II antigens by ductular epithelium associated with the exocrine portion of the pancreas and transient expression by vascular endothelial cells, the endocrine cells of the islets remained devoid of class II antigens throughout the period of observation (42 days).

Oncostatin M transforms lymphoid tissue function in transgenic mice by stimulating lymph node T-cell development and thymus autoantibody production.

Oncostatin M (OM) is a member of the IL-6 subfamily of cytokines that is expressed in primary lymphoid tissues such as bone marrow and thymus, as well as in secondary lymphoid tissues and activated leukocytes. We produced transgenic mice that overexpressed the human, bovine, or mouse OM genes and compared their relative ability to modulate lymphopoiesis. Each species of cytokine induced a similar extrathymic pathway of T-cell development involving the accumulation of immature T cells within lymph nodes. Reconstitution experiments utilizing lethally irradiated athymic mice indicated that OM had caused hematopoietic precursors within fetal liver and bone marrow to initiate lymph node T-cell development in the absence of a thymic environment. Breeding experiments with IL6-/- and IL-7r(alpha)-/- deficient mice, indicated that induction of this extrathymic pathway by the OM transgene occurred in the absence of IL-6, but was strictly dependent on IL-7 receptor signaling. Separately, OM stimulated the accumulation of immature B cells within the transgenic thymus and caused the subcapsular regions of the thymus to expand with mature B and T cells. This thymus conversion to secondary lymphoid tissue was responsible for a lethal autoimmune-like disease marked by high titers of circulating autoantibodies, proteinuria, and glomerulonephritis. The conserved phenotypes elicited by these three forms of OM indicate that this potent hematopoietic cytokine can regulate lymphoid tissue function and morphogenesis.

Ontogeny of Langerhans cells in human embryonic and fetal skin: expression of HLA-DR and OKT-6 determinants.

Langerhans cells (LCs) have been identified in human skin by 10 weeks estimated gestational age (EGA), but it was not known when they first enter the epidermis or acquire HLA-DR, OKT-6, and ATPase reactivity. We assayed for LCs in human embryonic and fetal skin by using immunolabeling and histochemical techniques on epidermal sheets. HLA-DR+ and ATPase+ LCs were present in the epidermis by 6-7 weeks EGA, the youngest tissue examined. Most LCs were OKT-6- until about 12 weeks EGA when they underwent a dramatic increase in OKT-6 reactivity. Although LC densities between 50-100 days were statistically similar (100 cells/mm2 of epidermis), LCs early in development were smaller, less dendritic, and phenotypically heterogeneous. We conclude that LCs migrate into the epidermis during the first trimester and resemble the adult phenotype by the second trimester, long before the immune system is fully activated.

Thymic overexpression of CD40 ligand disrupts normal thymic epithelial organization.

We characterized the distribution of CD40 and CD40 ligand (CD40-L) in the adult and developing murine thymus. Before birth, CD40 was almost exclusively localized to scattered foci of medullary cells. By birth there was a dramatic upregulation of CD40 expression by cortical epithelial cells, which was accompanied by a consolidation of medullary epithelial foci. CD40-L+ thymocytes displayed a medullary location. Analysis of mice deficient in CD40-L expression indicated that CD40-L/CD40 interactions were not required for development of the medullary compartment. Overexpression of CD40-L targeted to thymocytes altered thymic architecture, as reflected by a dramatic loss of cortical epithelial cells, expansion of the medullary compartment, and extensive infiltration of the capsule with a mixture of CD3+ cells, B-cells, and macrophages/dendritic cells. Reconstitution of lethally irradiated normal mice with lck CD40-L bone marrow cells also resulted in loss of cortical epithelium and expansion of the medullary compartment. Disruption of the normal pattern of thymic architecture and epithelial differentiation as a consequence of increased intrathymic levels of CD40-L expression points to a role for CD40-L/CD40 interactions in the normal pattern of epithelial compartmentalization/differentiation within the thymic environment.

In situ localization of T cell receptor beta chain in the murine thymus: changes in the intrathymic distribution of thymocytes expressing beta chain during fetal development.

The expression of T cell receptor beta chain in the developing thymus was examined at the light and electron microscopic levels using the monoclonal antibody F23.1. Cells expressing cytoplasmic forms of beta chain were first observed at Day 16 of gestation, while thymocytes expressing cell surface beta chain were detected about a day later. Clustering of cortical F23.1+ cells was more pronounced in fetal thymus when compared to adult. The density of F23.1+ cells in the subcapsular areas of the thymus was initially lower than that in the rest of the cortex or the medulla. Within the subcapsular and cortical areas of the thymus there was an inverse relationship between the density of F23.1+ cells and cells labeled with the lectin from Dolichos bifloris, which binds to terminal alpha-linked N-acetylgalactosamine residues preferentially expressed by L3T4-/Lyt2- thymocytes. Although this pattern was less pronounced with increasing gestational age, it was still apparent at birth.