2016 Comprehensive Update of the Banff Working Group on Liver Allograft Pathology: Introduction of Antibody-Mediated Rejection.

The Banff Working Group on Liver Allograft Pathology reviewed and discussed literature evidence regarding antibody-mediated liver allograft rejection at the 11th (Paris, France, June 5-10, 2011), 12th (Comandatuba, Brazil, August 19-23, 2013), and 13th (Vancouver, British Columbia, Canada, October 5-10, 2015) meetings of the Banff Conference on Allograft Pathology. Discussion continued online. The primary goal was to introduce guidelines and consensus criteria for the diagnosis of liver allograft antibody-mediated rejection and provide a comprehensive update of all Banff Schema recommendations. Included are new recommendations for complement component 4d tissue staining and interpretation, staging liver allograft fibrosis, and findings related to immunosuppression minimization. In an effort to create a single reference document, previous unchanged criteria are also included.

Omega-3 fatty acids prevent early-life antibiotic exposure-induced gut microbiota dysbiosis and later-life obesity.

Early-life antibiotic exposure can disrupt the founding intestinal microbial community and lead to obesity later in life. Recent studies show that omega-3 fatty acids can reduce body weight gain and chronic inflammation through modulation of the gut microbiota. We hypothesize that increased tissue levels of omega-3 fatty acids may prevent antibiotic-induced alteration of gut microbiota and obesity later in life. Here, we utilize the fat-1 transgenic mouse model, which can endogenously produce omega-3 fatty acids and thereby eliminates confounding factors of diet, to show that elevated tissue levels of omega-3 fatty acids significantly reduce body weight gain and the severity of insulin resistance, fatty liver and dyslipidemia resulting from early-life exposure to azithromycin. These effects were associated with a reversal of antibiotic-induced dysbiosis of gut microbiota in fat-1 mice. These results demonstrate the beneficial effects of omega-3 fatty acids on antibiotic-induced gut dysbiosis and obesity, and suggest the potential utility of omega-3 supplementation as a safe and effective means for the prevention of obesity in children who are exposed to antibiotics.

Prevention of antibiotic-associated metabolic syndrome in mice by intestinal alkaline phosphatase.

AIMS: To examine whether co-administration of intestinal alkaline phosphatase (IAP) with antibiotics early in life may have a preventive role against metabolic syndrome (MetS) in mice. METHODS: A total of 50 mice were allocated to four treatment groups after weaning. Mice were treated with azithromycin (AZT) ± IAP, or with no AZT ± IAP, for three intermittent 7-day cycles. After the last treatment course, the mice were administered a regular chow diet for 5 weeks and subsequently a high-fat diet for 5 weeks. Body weight, food intake, water intake, serum lipids, glucose levels and liver lipids were compared. 16S rRNA gene pyrosequencing was used to determine the differences in microbiome composition. RESULTS: Exposure to AZT early in life rendered mice susceptible to MetS in adulthood. Co-administration of IAP with AZT completely prevented this susceptibility by decreasing total body weight, serum lipids, glucose levels and liver lipids to the levels of control mice. These effects of IAP probably occur as a result of changes in the composition of specific bacterial taxa at the genus and species levels (e.g. members of Anaeroplasma and Parabacteroides). CONCLUSIONS: Co-administration of IAP with AZT early in life prevents mice from susceptibility to the later development of MetS. This effect is associated with alterations in the composition of the gut microbiota. IAP may represent a novel treatment against MetS in humans.

Silencing TNF-α in macrophages and dendritic cells for arthritis treatment.

OBJECTIVES: Tumour necrosis factor (TNF)-α secreted by macrophages and dendritic cells (DCs) plays a predominant role in arthritis. Our previous studies suggest that a small peptide, RVG-9R (29-aa peptide derived from the rabies virus glycoprotein, fused to 9R residues), can deliver small interfering RNA (siRNA) to macrophages and DCs. We therefore tested whether knockdown of TNF-α expression in macrophages and DCs by RVG-9R/bound siRNA targeting TNF-α reduces the severity of collagen antibody-induced arthritis (CAIA) in mice. METHOD: Arthritis was induced in mice by injecting a combination of antibodies to collagen followed by lipopolysaccharide (LPS) treatment. Mice were also injected with TNF-α siRNA complexed with RVG-9R peptide or an irrelevant peptide RVMAT-9R on days 1, 3, 5, and 7. As a positive control, dexamethasone was injected intravenously. Paw thickness was measured every 2 days and the mice were killed on day 10 for testing synovial TNF-α levels and histological analysis of joints. RESULTS: In control mice, arthritis developed on day 4 and reached its peak between day 7 and day 9. Treatment with siTNF-α bound to RVG-9R, but not to RVMAT-9R, resulted in reducing paw thickness scores to the same level as dexamethasone treatment, associated with reduced TNF-α level in synovial fluid. Histological analysis of joints in the control RVMAT-9R/TNF-α siRNA-treated mice showed marked pannus formation and destruction of cartilage and subchondrial bone, as well as severe infiltration of inflammatory cells into the synovium. By contrast, the joint pathology was markedly reduced in RVG-9R/TNF-α siRNA-treated mice resembling the dexamethasone-treated mice. CONCLUSIONS: Suppression of TNF-α expression in macrophages and DCs by RVG-9R-mediated siRNA delivery could potentially be a clinically viable strategy for treatment of arthritis.

Role of appendix in the development of inflammatory bowel disease in TCR-alpha mutant mice.

T cell receptor-alpha mutant mice (TCR-alpha-/-), created by gene targeting of the TCR-alpha gene in embryonic stem cells, spontaneously develop inflammatory bowel disease (IBD) resembling human ulcerative colitis. Since gut-associated lymphoid tissue is likely to play an important role in the development of chronic intestinal inflammation, we examined the changes in the appendix lymphoid follicle (ALF) and Peyer's patches (PP) in these mice. We found the structure of the ALF to be remarkably similar to that of the PP in the small intestine; in both instances, lymphoid follicles covered by surface epithelium (dome-formation) were found. The amount of proliferation in the lymphoid follicles of the appendix estimated by in vivo incorporation of 5-bromo-2'deoxyuridine was more than two times that of PP in TCR-alpha-/- mice. ELISPOT assay showed an increase of IgA, IgG1, and IgG2a, but not IgM-secreting B cells in ALF of TCR-alpha-/- mice compared to TCR-alpha+/- control mice. Furthermore, TCR-alpha-/- mice revealed an increase of autoantibody-producing B cells against the cytoskeletal protein tropomyosin in ALF as compared to PP. When TCR-alpha-/- mice underwent appendectomy at a young age (3-5 wk), the number of mesenteric lymph nodes cells at 6-7 mo were markedly less than in the sham-operated TCR-alpha-/- mice. Furthermore, appendectomy at 1 mo of age suppressed the development of IBD, with only 3.3% of these mice developing IBD in the 6-7-mo period of observation. In contrast, approximately 80% of controls, including the sham-operated TCR-alpha-/- mice, developed IBD during this period. These results suggest that ALF, rather than PP, is the priming site of cells involved in the disease process and plays an important role in the development of IBD in TCR-alpha-/- mice.

T-cell migration into allografts.

The ability of T and B lymphocytes to migrate into skin allografts undergoing rejection was studied in mice. Spleen cells from CBA/J mice sensitized to transplantation antigens of A/J or C57BL/6 mice were separated on immunabsorbent columns into purified populations of T and B cells, labeled in vitro with 3H-uridine and injected intravenously into CBA/J mice with 7-day old skin iso and allografts (A/J or C57BL/6). The mice were sacrificed 24 h later and studied by autoradiography. After transfer of either unfractionated spleen cells or T cells, large numbers of labeled cells were found in the cellular infiltrate of allografts, whereas extremely few were seen in isografts. In contrast, after transfer of B cells, almost no labeled cells were detected either in the allografts or the isografts, although they, like T cells, homed normally to lymphoid tissue.

Suppressive role of B cells in chronic colitis of T cell receptor alpha mutant mice.

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-alpha-/- mice was explored by creating double mutant mice (TCR-alpha-/- x immunoglobulin (Ig)mu-/-), which lack B cells. TCR-alpha-/- x Ig mu-/- mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-alpha-/- mice. Colitis was induced in recombination-activating gene-1 (RAG-1-/-) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-alpha-/- x Ig mu-/- mice. When purified B cells from TCR-alpha-/- mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1-/- mice. Administration of the purified Ig from TCR-alpha-/- mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-alpha-/- x Ig mu-/- mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-alpha-/- x Ig mu-/- mice as compared to Ig mu-/- mice and TCR-alpha-/- mice. Administration of the purified Ig from TCR-alpha-/- mice into TCR-alpha-/- x Ig mu-/- mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.

A cell-mediated reaction against glomerular-bound immune complexes.

Lewis rats were given a single i.v. injection of soluble immune complexes containing human serum albumin (HSA) and rabbit anti-HSA antibodies, prepared in antigen excess. This resulted in localization of HSA and rabbit gamma globulin (RGG) in glomerular mesangial regions without producing definite histologic changes. 24 h after the injection of immune complexes, groups of these rats received lymph node cells or T-cell preparations from syngeneic donors sensitized to RGG, HSA, or ovalbumin; another group received no cells. All of these groups and a group of normal control rats were given injections of [3H]thymidine at 18, 27, and 44 h. The animals were killed 48 h after the time of cell transfer. In histologic sections, glomerular abnormalities were found only in some of the animals that had received immune complexes and lymph node cells or T-cell populations from donors sensitized to HSA or RGG; the lesions were characterized by focal and segmental increase in cells in mesangial regions. Autoradiographs revealed significantly greater numbers of labeled cells in mesangial regions and glomerular capillaries in the groups that had received immune complexes and cells from HSA- or RGG-sensitized donors than in any of the other groups. Electronmicroscopic studies suggested that the increase in cellularity in mesangial regions resulted from an influx of mononuclear phagocytes. The findings indicate that cell-mediated reactions can be initiated by the interaction between sensitized T lymphocytes and antigens present in immune complexes within mesangial regions.

Evidence for a pathogenic role of a cell-mediated immune mechanism in experimental glomerulonephritis.

Lewis rats were injected intravenously with rabbit anti-rat glomerular basement membrane (GBM) antisera in doses that were sufficient to cause glomerular fixation of rabbit gamma globulin (RGG) detectable by immunofluorescence, but which failed to induce histologically detectable lesions. 24 h later, groups of rats received lymph node cells or serum from syngeneic donors that had been immunized with either RGG or ovalbumin; they were injected with [3H]thymidine three times during the next 2 days, and sacrificed 48 or 96 h after transfer. Only the rats given anti-GBM antiserum plus lymph node cells from donors sensitized to RGG showed histological glomerular lesions, in the form of segmental hypercellularly and necrosis. Autoradiographs revealed the greatest number of labeled cells in glomeruli in the same group. In analogous experiments, it was shown that T-cell-enriched populations could induce hypercellular glomerular reactions. On the basis of electronmicroscopic and autoradiographic observations, it appears that the glomerular hypercellularity resulted from both infiltration of mononuclear cells and proliferation of endothelial cells. The findings indicate that interaction of specifically sensitized lymphocytes with glomerular-bound antigen can induce a cell-mediated (delayed-type) reaction in glomeruli.

Location of T cell and major histocompatibility complex antigens in the human thymus.

A series of monoclonal antibodies were used to study the intrathymic distribution of T cell-specific antigens, Ia antigens, and beta 2-microglobulin in frozen sections of human thymus by immunofluorescence and immunoperoxidase techniques. Most of the cortical thymocytes reacted with anti-T4, anti-T5, anti-T6, anti-T8, and anti-T10 antibodies, thus indicating coexpression of multiple antigens on cortical lymphocytes. The staining of cells in the medulla was most satisfactorily judged in sections stained with the immunoperoxidase technique. Many medullary cells reacted with anti-T4--and a smaller fraction with anti-T5, anti-T6, anti-T8, and anti-T10 antibodies. In addition, T1 and T3 antibodies, which react with all peripheral T cells, stained a majority of medullary cells. The medullary cells were also more intensely stained with antibodies directed against beta 2-microglobulin than the majority of cortical cells. Hence, the staining profile of medulla approximates the staining pattern of peripheral T cells, with large numbers of cells bearing T1+, T3+, and T4+ antigens (helper/inducer cells) and a small number of cells bearing T1+, T3+, and T5+/T8+ antigens (suppressor/cytotoxic cells). This supports the conclusion that mature cells present in the medulla are derived from immature cells in the cortex. However, a small number of cells scattered throughout the cortex stained with T1 and T3 antibodies, which suggests that maturation of thymocytes can also occur in the cortex. Antibody directed against Ia antigens resulted in a characteristic patchy pattern of staining in the cortex and in diffuse staining in the medulla, which was interpreted as resulting from staining of epithelial reticulum. The majority of thymocytes did not stain. The staining pattern suggests a close relationship between epithelial cells and thymocytes.

Distribution of T cell subsets in human lymph nodes.

A series of T cell-specific monoclonal antibodies was used to determine the location of T lymphocyte subpopulations in frozen sections of human lymph nodes by means of an immunoperoxidase technique. The majority of cells in the paracortical regions were reactive with anti-T1 and anti-T3 antibodies, which define all mature peripheral T cells. In contrast, the majority of cells within primary follicles were unreactive with anti-T1 and anti-T3 antibodies, but were reactive with anti-Ia and anti-IgM antibodies. In addition, a substantial number of T1+, T3+ cells were found in the germinal centers of secondary follicles on the capsular side. The vast majority of T1+, T3+ cells in the paracortex and the follicles were reactive with anti-T4 antibody, which defines inducer/helper T cells. Only a minority of cells in these areas were reactive with anti-T5 and anti-T8 antibodies, which define cytotoxic/suppressor cells. No lymphocytes were stained with anti-T6 antibody, which reacts with a majority of thymocytes but not with peripheral T cells. Scattered cells in the paracortex showed staining for Ia antigen in an irregular dendritic pattern. The findings demonstrate that the major T cell population found within human lymph node bears the mature T1+, T3+, T4+ phenotype characteristic of inducer T cells. Moreover, the location of this population indicates that they play a role in the induction of B cell differentiation in vivo.

Stages of B cell differentiation in human lymphoid tissue.

Monoclonal antibodies reactive with B cell-specific differentiation and other antigens were used to investigate stages of B cell maturation in human lymphoid tissue, using an immunoperoxidase technique on frozen tissue sections. Lymphoid follicles, which represent the major anatomic compartment of B cells, demonstrated cellular antigenic expressions that appear to reflect differentiation of B cells. The majority of cells in the primary follicles and the mantle zones of secondary follicles expressed surface antigens similar to those of circulating B cells, namely IgM, IgD, Ia, B1, and B2. In contrast, the germinal center cells of secondary follicles stained for IgM, IgG, B1, B2, and Ia antigens, but not for IgD, and furthermore, acquired the T10 antigen. The germinal centers stained much more intensely than mantle zones with anti-B2, whereas no such striking difference in the staining intensity was observed with anti B1. Plasma cells, which represent the end stage of B cell differentiation, showed intense cytoplasmic staining with the anti-T10 antibody. The results indicate that the generation of germinal center cells in primary lymphoid follicles involves phenotype changes that correspond largely to those previously observed after both antigenic and mitogenic activation of B lymphocytes.

T cell-mediated pathology in two models of experimental colitis depends predominantly on the interleukin 12/Signal transducer and activator of transcription (Stat)-4 pathway, but is not conditional on interferon gamma expression by T cells.

The requirements for interleukin (IL)-12/signal transducer and activator of transcription (Stat)-4 signaling and induction of T cell-specific interferon (IFN)-gamma expression in the development of T helper cell (Th)1-type pathology were examined in two different models of experimental colitis. In each model, abnormal reconstitution of the T cell compartment in immunodeficient mice by adoptive cell transfer leads to a wasting syndrome and inflammation of the colon, induced by IFN-gamma and tumor necrosis factor (TNF)-alpha-producing T cells. We show here that treatment with anti-IL-12 antibodies in one of the models, or reconstitution with T cells from Stat-4-deficient (Stat-4(null)) mice in both models resulted in a milder disease in the majority of recipient animals, compared with those that were left untreated or that had been reconstituted with wt cells. Protected mice in each group also harbored lower frequencies of IFN-gamma-producing T cells than did diseased mice, suggesting that effects on wasting and colitis resulted from the attenuation of IFN-gamma expression by T cells. To test whether the development of pathogenic T cells in the two colitis models was directly dependent on T cell-specific IFN-gamma expression, IFN-gammanull donors were used for T cell reconstitution in each system. Surprisingly, large numbers of IFN-gammanull-reconstituted mice developed wasting and colitis, which in many cases was of comparable severity to that seen in animals reconstituted with wt cells. Furthermore, T cells from these animals expressed TNF-alpha, demonstrating that they had retained the ability to produce another proinflammatory cytokine. Taken together, these results demonstrate that in some forms of chronic experimental colitis the development of pathogenic T cells is influenced predominantly, though not exclusively, by IL-12 via the actions of Stat-4 proteins. Furthermore, our data suggest that in the models of colitis studied here the effects of IL-12/Stat-4 or other Th1 promoting pathways are not limited to the induction of IFN-gamma gene expression in T lymphocytes.

Cytokine imbalance and autoantibody production in T cell receptor-alpha mutant mice with inflammatory bowel disease.

Spontaneous inflammatory bowel disease (IBD) resembling human ulcerative colitis develops in mice mutant for the T cell receptor alpha gene (TCR-alpha-/-). TCR-alpha-/- mice lack TCR-alpha/beta+ cells but contain TCR-gamma/delta+ cells and a small population of a unique CD4+, TCR-alpha-/beta+(low) cells. Since all the immunoglobulin (Ig) classes are present in these mice, help to B cells must be provided by cells other than TCR-alpha/beta+ cells. In the present study, we found serum levels of IgG1 and IgG2 to be markedly increased in TCR-alpha-/- mice with IBD as compared to TCR-alpha-/- mice without IBD or TCR-alpha+/- controls. An increase in IgG1-, IgG2a- and IgA- but not IgM-secreting mesenteric lymph node (MLN) B cells was detected in TCR-alpha-/- mutant mice. There was also a marked increase in MLN B cells secreting autoantibody (IgG) to tropomyosin, a cytoskeletal protein. Examination of the hyperplastic MLN showed a marked increase in the number of B, TCR-delta+, and CD4+ TCR-alpha-/beta+ cells, similar to the cell population observed at the site of colonic inflammation. Analysis of spontaneous cytokine production by MLN cells using an enzyme-linked immunospot assay, immunohistochemistry, and reverse transcription/polymerase chain reaction showed a decrease of interleukin 2 (IL-2) but a marked increase of IL-4 and interferon gamma (IFN-gamma) production in TCR-alpha-/- mice with IBD as compared to TCR-alpha-/- mice without IBD and TCR alpha+/- control mice. Both TCR-alpha-/beta+ and TCR-delta+ cells were found to be capable of producing IL-4; IFN-gamma was produced mostly by non-T cells, many of which were shown to be CD3- NK 1.1+ cells. We propose that the cytokine imbalance present in these mice results in expansion of B cells, production and switching of autoantibodies to IgG2 subclass, and development of IBD. It is possible that the unusual CD4+ TCR-alpha-/beta+ population and expanded TCR-gamma/delta+ population present in TCR-alpha-/- mice plays a central role in this abnormal immune response.

Oligoclonality of human intestinal intraepithelial T cells.

T cells bearing the T cell receptor alpha/beta (TCR-alpha/beta) are the predominant lymphocyte population in the human intestinal epithelium. To examine if normal intestinal intraepithelial lymphocytes (IEL) have a TCR repertoire distinct from the TCR-alpha/beta repertoire in peripheral blood lymphocytes (PBL), comparative analysis of relative V beta gene usage in IEL and PBL was performed by quantitative polymerase chain reaction. In each of the six individuals examined, one to three V beta families made up more than 40% of the total V beta transcripts detected in the IEL, whereas there was a more even distribution of V beta gene usage in the paired PBL. The predominant V beta families, especially V beta 1, V beta 2, V beta 3, and V beta 6, were frequently shared among IEL of different individuals. PCR cloning and sequence analysis of the predominant V beta 6 family in two individuals revealed an identical V-D-J-C sequence in 13 of 21 clones obtained from one donor, and a different repeated sequence in 18 of 27 clones examined in the second donor. These data suggest that the V beta skewing in IEL is due to an oligoclonal T cell expansion and may reflect the response of the intestinal mucosal immune system to a restricted set of as yet undefined antigens present in the gut.

Human lymphocytes bearing T cell receptor gamma/delta are phenotypically diverse and evenly distributed throughout the lymphoid system.

A direct quantitative and phenotypic cytofluorographic analysis of TCR-gamma/delta+ lymphocytes as well as an immunohistologic study of their tissue distribution and microanatomy was made possible by the availability of two mAbs (anti-TCR-delta 1 and anti-C gamma M1) specific for framework determinants on human TCR gamma and delta chains, respectively. TCR-gamma/delta+ lymphocytes, ranging between greater than 0.5 and 16% of CD3+ cells, were found in fetal and postnatal thymus, fetal and adult peripheral lymphoid organs, and adult peripheral blood. While TCR-gamma/delta+ lymphocytes comprised a small subpopulation of T cells (mean, approximately 4%) occasionally greater than 10-16% of CD3+ cells expressed TCR-gamma/delta. Virtually all TCR-gamma/delta+ thymocytes/lymphocytes expressed CD7, CD2, and CD5 but were heterogeneous with respect to their expression of CD1, CD4, CD8, CD28, CD11b, CD16, and Leu-7. Human TCR-gamma/delta+ cells populate both organized lymphoid tissues (thymus, tonsil, lymphnode, and spleen) as well as the gut- and skin-associated lymphoid systems at similar frequencies without obvious tropism for epithelial microenvironments. TCR-gamma/delta+ lymphocytes tend to be located within a given organ wherever TCR-alpha/beta+ lymphocytes are found. This study shows that TCR-gamma/delta+ lymphocytes constitute a small but numerically important, phenotypically diverse T cell population distributed throughout the body. These results support the concept that TCR-gamma/delta+ cells comprise a distinct, functionally heterogeneous, mature T cell sublineage that may substantially broaden the T cell repertoire at all immunologically relevant sites.

Gliadin-primed CD4+CD45RBlowCD25- T cells drive gluten-dependent small intestinal damage after adoptive transfer into lymphopenic mice.

BACKGROUND AND AIMS: Coeliac disease is a common small intestinal inflammatory disorder that results from a breach of intestinal tolerance to dietary gluten proteins, driven by gluten-reactive effector T cells. We aimed to assess the pathogenic role of gluten-reactive T cells and to generate a model of gluten-induced enteropathy. METHODS: CD4+CD25- T cell fractions were adoptively transferred into lymphopenic mice, leading to "baseline" small intestinal inflammation. RESULTS: Rag1-/- recipients of gliadin-presensitised CD4+CD45RBlowCD25- T cells, but not CD4+CD45RBhigh naive T cells, gained less weight and suffered from more severe duodenitis when challenged with oral gluten than recipients on gluten-free diet, or recipients of control (ovalbumin)-presensitised T cells. This was accompanied by deterioration of mucosal histological features characteristic of coeliac disease, and increased Th1/Th17 cell polarisation in the duodenum and the periphery. Interestingly, reintroduction of a gluten-free diet led to weight gain, improvement of histological duodenitis, and a decrease in duodenal interferon gamma and interleukin 17 transcripts. Moreover, B cell-competent nude recipients of gliadin-presensitised CD4+CD45RBlowCD25- T cells produced high levels of serum anti-gliadin immunoglobulin A (IgA) and IgG1/IgG2c only when challenged with oral gluten. CONCLUSIONS: CD4+ T cell immunity to gluten leads to a breach of oral gluten tolerance and small intestinal pathology in lymphopenic mice, similar to human coeliac disease. This model will be useful for the study of coeliac disease pathogenesis, and also for testing novel non-dietary therapies for coeliac disease.

Monoallelic expression of the interleukin-2 locus.

The lymphokine interleukin-2 (IL-2) is responsible for autocrine cell cycle progression and regulation of immune responses. Uncontrolled secretion of IL-2 results in adverse reactions ranging from anergy, to aberrant T cell activation, to autoimmunity. With the use of fluorescent in situ hybridization and single-cell polymerase chain reaction in cells with different IL-2 alleles, IL-2 expression in mature thymocytes and T cells was found to be tightly controlled by monoallelic expression. Because IL-2 is encoded at a nonimprinted autosomal locus, this result represents an unusual regulatory mode for controlling the precise expression of a single gene.

Influences of gamma interferon on synovial fibroblast-like cells. Ia induction and inhibition of collagen synthesis.

The shape and function of adherent cells cultured from rheumatoid synovial membranes are influenced by immune cells, and their products. The synovial cells produce collagenase and prostaglandin E2 (PGE2), the levels of which are increased when the cells are incubated with the monokine, mononuclear cell factor/interleukin 1. The majority of adherent synovial cells are fibroblastlike in appearance and synthesize collagens and fibronectin; the synthesis of collagens and fibronectins are also increased by a monocyte factor. In the present study we found that the fibroblastlike cells expressed major histocompatibility complex class II (Ia-like) antigens after initial dispersion from the synovial membrane. Monocyte lineage antigens were detected on some round cells in early passage, but no T lymphocytes were identified in established cultures. There was loss of Ia expression on the fibroblastlike cells with age and passage in culture. The addition of the lymphokine, gamma interferon (recombinant), induced class II antigen (DR and DS/DQ) expression in early or late passage cells in a time- and dose-dependent manner and required protein synthesis. Furthermore, the adherent synovial fibroblastlike cells continued to be Ia-positive when examined as long as 10 d after the removal of gamma interferon. Ia expression was also induced by gamma interferon in normal skin fibroblasts. Synovial cells that could be induced to express Ia also bound a monoclonal antibody to type III collagen (a fibroblast marker). Gamma interferon, while inducing Ia expression, decreased the binding of type III collagen antibody on unstimulated as well as monokine-stimulated cells. Analysis of [3H]proline-labeled medium by SDS polyacrylamide gel electrophoresis showed that gamma interferon decreased the synthesis of type I and III collagens and fibronectin by adherent synovial cells in a dose-dependent manner. These findings suggest that Ia expression by synovial tissue cells is not cell-specific, but reflects one or several related events, such as the degree of T lymphocyte infiltration, the presence of factors that stimulate gamma interferon release, or an increased sensitivity of the cells to gamma interferon. Whereas the synthesis of class II antigens is enhanced by the lymphokine gamma interferon, and a monocyte factor(s) stimulates collagen, collagenase and PGE2 synthesis by the same cells, gamma interferon inhibits basal and monokine-induced collagen synthesis. Thus, lymphokines and monokines may influence the extent of fibrosis as contrasted to matrix destruction at various stages of the rheumatoid lesion by affecting the function of fibroblastlike synovial cells.

Human giant cell tumors of bone identification and characterization of cell types.

Cells cultured from human giant cell tumors of bone were characterized on the basis of morphological features, proliferative capacity, presence of granulocyte-monocyte antigens, receptors for skeletal hormones, and soluble cell products. Three major cell types were identified. One population consisted of mononuclear cells with fibroblastic morphology, which proliferated in culture and most likely represent the neoplastic element of the tumor. Phenotypically they resembled a connective tissue stromal cell. A second population of mononuclear cells lacked receptors for skeletal hormones and did not persist in culture. These cells were likely of monocyte-macrophage lineage. A third population of cells consisted of large multinucleated giant cells. These cells possessed phenotypic features of osteoclasts including receptors for calcitonin. Human giant cell tumors of bone are most likely a neoplasm of connective tissue stromal cells, which have the capacity to recruit and interact with multinucleated giant cells that exhibit phenotypic features of osteoclasts.