Flow cytometric analysis of T cells: diagnostic applications in transplantation.
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Antibodies, Monoclonal↗
Biomedical subjects
R B Colvin
Publications and source records attributed to R B Colvin.
Monoclonal antibodies for the diagnosis and treatment of transplant rejection.
Extensive experience with antibodies to lymphocytes for immunosuppression suggested the need for better means of evaluating alterations of peripheral blood lymphocytes during treatment. Improved immunosuppression was also anticipated if more precise control of selected lymphocyte populations could be achieved. The advent of monoclonal antibodies has permitted progress in both of these areas. For evaluation of the immune status of patients, the ratio between the numbers of the subclass of T lymphocytes having helper/inducer properties and those having suppressor/cytotoxic activity (OKT4+/OKT8+ cells) has been explored. Transplant rejection is more common in the presence of normal cell ratios than when ratios are reversed. In the former situation, however, when rejection occurs it is much more readily reversed than in the latter. Monoclonal antibodies have been used to treat transplant rejection in both nonhuman primates and in patients. Patient trials have so far been confined to the use of the pan-T-cell antibody OKT3. This has proved to be the most effective agent available for the reversal of acute cellular rejection in human kidney transplants. The problems of recurrent rejection activity after treatment and of antibody formation to the monoclonal antibody itself are being examined.
Animals↗
Fibronectin deposition in delayed-type hypersensitivity. Reactions of normals and a patient with afibrinogenemia.
During development of delayed hypersensitivity (DH) skin reactions, fibronectin accumulates in two distinct sites: (a) the dermal interstitium in a pattern similar to fibrin and with a time course similar to that of fibrin deposition and mononuclear cell infiltration, and (b) blood vessel walls in a pattern suggestive of basement membrane staining and with a time course similar to that of endothelial cell proliferation. In vitro fibronectin can bind to monocytes or endothelial cells and simultaneously bind to fibrin or collagen matrices; by such interaction in vivo it may affect cell migration or proliferation. Thus, fibronectin deposition in DH reactions may facilitate cell-matrix interactions; however, the possibility exists that extravascular fibronectin accumulation may be only secondary to interstitial fibrin clot formation, and that blood vessel-associated fibronectin may be only a function of adsorption onto basement membrane (type IV) collagen. To address these possibilities, we investigated the association of fibronectin with fibrin, type IV collagen, and mononuclear cell infiltrates in DH reactions. Skin sites of DH reactions in normal volunteers were biopsied at 24, 48, and 72 h after intradermal challenge and examined by immunofluorescence technique. At all time points most of the interstitial fibronectin coincided with fibrin; however, some interstitial fibronectin was coincident with mononuclear cells positive for HLA-DR or monocyte-specific antigen. The coincidence of fibronectin with mononuclear cells was more apparent in a 48-h DH reaction from a patient with congenital afibrinogenemia. Vessel wall fibronectin was increased by 48 h after challenge and appeared as a fine linear band on the luminal side of a much thicker band of type IV collagen. Thus, the coincidence of extravascular fibronectin with mononuclear cells, its appearance without fibrin in the site from a patient with afibrinogenemia, and incomplete correspondence of vessel wall fibronectin with type IV collagen suggest that fibronectin localization in DH reactions involves endothelial cell and mononuclear cell binding as well as adsorption to fibrin and/or type IV collagen.
Afibrinogenemia↗
Fibronectin in developing rabbit cornea.
Fibronectin is believed to be important in tissue morphogenesis. We examined the distribution of fibronectin in developing rabbit cornea by immunohistofluorescence. Cryostat sections of cornea from 13, 15, and 20-day-old fetuses, 3-day neonates, and adults were incubated with affinity-purified fluoresceinated guinea pig anti-rabbit fibronectin antiserum (aFN). aFN bound to components within the presumptive stromal region and along the basal surfaces of corneal and lens epithelia during early stages of mesenchymal invasion. At 15 days of gestation, fluorescence was associated with the stromal extracellular matrix of the cornea, the subepithelial zone, and the lens capsule. In the 20-day fetus an intense aFN fluorescence was present along the inner corneal stromal border coincident with the formation of Descemet's membrane. Fluorescence within the corneal stroma appeared as fine lines, restricted to the collagen lamellae, remaining through birth and disappearing in the adult. Although stromal fluorescence disappeared in the adult, Descemet's membrane continued to fluoresce, albeit to a lesser extent. The results of our studies indicate the presence of fibronectin in developing rabbit cornea. Because fibronectin is important to cell adhesion in vitro, and because intercellular and cell-extracellular matrix interactions, including adhesion, are necessary for tissue morphogenesis, our observation suggests that fibronectin plays an important role in corneal morphogenesis.
Animals↗
Lymphocyte activation antigens. I. A monoclonal antibody, anti-Act I, defines a new late lymphocyte activation antigen.
Lymphocyte activation entails a sequence of events identified by analyzing the time course of expression of various distinctive cell surface molecules on lymphocytes that appear early (before initiation of DNA synthesis), parallel with DNA synthesis and cellular proliferation, or late (after peak proliferation). In this study we present identification of a novel late lymphocyte activation antigen, Act I, utilizing a murine monoclonal antibody. Anti-Act I was identified in a fusion of NS1 with BALB/c spleen cells immunized with a human tetanus toxoid-reactive T lymphoblast line. Flow cytometry analysis shows that Act I antigen is present in markedly greater amounts on activated T and B lymphocytes than on resting, small peripheral blood lymphocytes. Act I expression by these lymphocytes is promoted by PHA, tetanus toxoid, or alloantigens and lags behind maximal thymidine incorporation by 1 to 2 days. Thymocytes can be triggered to express Act I antigen during maturation induced by PHA/T cell growth factor stimulation. In vitro, anti-Act I does not affect antigen- or lectin-stimulated T cell proliferation, T cell-mediated lymphocytotoxicity, or T cell growth factor-induced proliferation of T lymphoblasts. By immunoperoxidase analysis, Act I antigen is restricted to lymphoid tissue, staining many lymphocytes in the paracortex, germinal centers, and mantle zones of lymph nodes, tonsil, and spleen. By immunoprecipitation, the Act I antigen is a single band of 63,000 m.w. on reduced or nonreduced SDS gels. The distribution, size, time course, and functional correlates indicate that Act I is different from other known T cell activation markers detected with anti-Tac, OKT9, B3/25, OKT10, 4F2, CBL1, and anti-Ia-like antibodies. Although the function of Act I is still undetermined, it may serve as a useful marker of a late stage of activation in vitro and in vivo.
Animals↗
Pre-inflammatory expression of fibronectin (Fn) and Ia in acute experimental allergic encephalomyelitis (EAE): modulation of endothelial cells (EC) in the immune response detected by quantitative immunoperoxidase studies using monoclonal antibodies (MAb).
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Animals↗
The immunopathology of experimental allergic encephalomyelitis. I. Quantitative analysis of inflammatory cells in situ.
Acute experimental allergic encephalomyelitis (EAE) is a T cell-mediated, neurologic disease that is under immunogenetic control. We systematically analyzed the quantity and distribution of T cells, B cells, and macrophages in the central nervous system (CNS) of susceptible and resistant guinea (GP) with a panel of seven monoclonal antibodies by using the avidin-biotin complex (ABC) immunoperoxidase technique and alpha-naphthyl-butyrate esterase (ANBE) staining. Adult EAE-susceptible strain 13 GP immunized with isogeneic spinal cord homogenate (SC) or with myelin basic protein (MBP) developed clinical signs (paralysis, weight loss, etc.) in 2 to 3 wk. T cells were present in all CNS inflammatory foci and comprised 44% of the perivascular mononuclear cells. T cells diffusely infiltrated the neuropil away from inflammatory cell aggregates. These T cells were judged to be extravascular by the lack of an associated identifiable vessel in counter-stained sections, and by their persistence following exhaustive perfusion of the brains. In routine sections, mononuclear cells could be detected only in perivascular aggregates. IgM+ B cells comprised 9% of the perivascular infiltrates and did not diffusely infiltrate the parenchyma. ANBE+ macrophages comprised the remaining 47% of the identified perivascular cells. SC- and MBP-immunized GP showed equivalent numbers of inflammatory foci, T cells, and macrophages, but SC-immunized GP had more IgM+ cells in the meninges and choroid plexus (p less than 0.001, p less than 0.02, respectively). Virtually all cells in perivascular locations were Ia+. Ia+ mononuclear cells were also present in the neuropil. EAE-resistant strain 2 GP immunized with SC developed no clinical signs. These GP had fewer perivascular foci than strain 13 GP but, when present, the cellular composition, including the density of diffuse parenchymal T cell infiltrates, was indistinguishable. Significantly fewer parenchymal mononuclear cells in the strain 2 GP, however, displayed Ia, both in perivascular and diffuse infiltrates (p less than 0.001). We conclude that T cell migration into the CNS parenchyma is a characteristic feature of acute EAE in the GP, but that T cells can occur in this pattern without clinical signs of disease. The two features that distinguish susceptible and resistant strains were the frequency of perivascular infiltrates and the expression of Ia on parenchymal mononuclear cells, which probably reflects their enhanced immunologic activation in situ.
Acute Disease↗
Immunologic monitoring of monoclonal antibody therapy: comparison of five antibodies as immunosuppressants of renal allograft rejection.
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Animals↗
Immunologic monitoring of antithymocyte globulin treatment for acute allograft rejection.
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Adult↗
The immunopathology of experimental allergic encephalomyelitis. II. Endothelial cell Ia increases prior to inflammatory cell infiltration.
Experimental allergic encephalomyelitis (EAE) is a T cell-mediated neuroimmunologic disease model characterized by meningeal and parenchymal mononuclear cell infiltrates (see preceding companion paper). Here we report enhanced staining for Ia in the central nervous system (CNS) microvasculature endothelium in acute EAE in adult strain 13 guinea pigs (GP) sensitized with GP spinal cord homogenate (SC) or with GP myelin basic protein (MBP) in complete Freund's adjuvant (CFA). Cryostat sections of CNS and other tissues were stained with two monoclonal antibodies, 5S2 and 22C4, to GP Ia determinants, and with polyclonal antibody to factor VIII-related antigen (VIII-RA) as an endothelial cell marker. Morphometric techniques were employed on immunoperoxidase counterstained and coded sections to determine the frequency of Ia+ vessels and cells. Rare (approximately 10% of VIII-RA+) vascular endothelial cells were Ia+ in the CNS of normal and CFA-sensitized controls. SC- or MBP-sensitized strain 13 GP sacrificed on day 7, before the onset of neurologic signs (pre-clinical), had no detectable CNS mononuclear cell infiltrates, but had increased (approximately 30% of VIII-RA+) endothelial cell Ia staining over controls (p less than 0.001). The endothelial Ia staining persisted (approximately 35% of VIII-RA+) in vessels as the animals developed paralysis. There were no differences in endothelial cell Ia between SC- and MBP-induced disease. EAE-resistant strain 2 GP sensitized with SC/CFA had no neurologic signs, and had fewer inflammatory foci than strain 13 GP with EAE, but had similar numbers of Ia+ endothelial cells. No differences in endothelial cell Ia staining were found in non-CNS tissues among any GP groups. In EAE, increased endothelial cell Ia is a pre-inflammatory, target organ-specific alteration that persists during inflammation. The findings suggest that in vivo modulation of endothelial cell Ia may be part of the local immune response. Endothelial cells may play a significant role, in antigen presentation or in promoting T cell migration, in the in situ immune response in the CNS.
Animals↗
Effects of long-term lithium administration on renal structure and function in rats. A distinctive tubular lesion.
Because lithium salts are widely used for long-term therapy of affective disorders and have been recently implicated as a cause of tubulointerstitial renal disease, we have undertaken experiments designed to establish the site of the early and late pathologic lesions and to determine their correlation with the lithium-induced concentrating defect. Male Wistar rats given a semisynthetic diet that contained lithium carbonate, 90 mEq/kg dry weight, developed serum lithium levels in the human therapeutic range; pair-fed controls received sodium carbonate. Within 3 weeks, treated rats developed marked polyuria, with elevation of free water clearance and vasopressin-resistant diabetes insipidus. Early morphologic changes were confined to the cortical collecting tubules and, possibly, contiguous portions of distal tubules. The tubules were dilated and irregularly lined with cells that had bulging or thinned basophilic cytoplasm, enlarged nuclei, sometimes basal vacuolization, and a few mitoses. These changes were evident at 3 weeks and progressed through the end of the observation period at 18 weeks. The proliferative component of the lesion was demonstrated by the finding of a significant and specific increase in 3H-thymidine uptake by nuclei of collecting/distal tubules of lithium-treated rats. The lesion, but not the increased thymidine uptake, extended into the medullary collecting ducts at 9 and 18 weeks. Although occasional intratubular mononuclear cells were seen at 9 and 18 weeks, no interstitial inflammation or fibrosis was seen. These tubular epithelial lesions were not seen in the kidneys of Brattleboro rats or glucose-treated Wistar rats despite comparable polyuria. We suggest that this early, persistent, and reproducible lesion, characterized by reactive and proliferating tubular cells in the cortical collecting tubules, predisposes the kidney to injury from otherwise mild or insignificant insults and may explain the sporadic occurrence of serious tubulointerstitial disease in patients on long-term lithium therapy.
Animals↗
Fibrinogen-fibrin interactions with fibroblasts and macrophages.
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Epithelium↗
Association of herpesvirus infections with T-lymphocyte-subset alterations, glomerulopathy, and opportunistic infections after renal transplantation.
We studied the interrelation among herpes-virus infections, T-lymphocyte subsets, opportunistic infections, and renal histopathology in 28 recipients of renal allografts. All primary or reactivated herpesvirus infections occurring in the first three months after transplantation in recipients of cadaveric grafts accompanied persistent inversions in the ratio of OKT4 (helper/inducer) to OKT8 (cytotoxic/suppressor) lymphocytes. In the less heavily immunosuppressed recipients of organs of living related donors, these inversions were seen only in association with clinically apparent cytomegalovirus infections. Five of seven opportunistic infections occurred in patients with OKT4/OKT8 ratios of less than 1.0. Biopsy specimens from patients with renal dysfunction occurring in association with a low OKT4/OKT8 ratio frequently revealed glomerular damage rather than acute cellular rejection. Monitoring of T-lymphocyte subsets provides early evidence of herpesvirus infections and identifies patients at increased risk for opportunistic infection after renal transplantation.
Biopsy↗
Hepatitis B immune complex glomerulonephritis: simultaneous glomerular deposition of hepatitis B surface and e antigens.
A case of a 29-year-old homosexual male with chronic hepatitis B infection, cryoglobulinemia, and glomerulonephritis is described. The glomerular lesions were not readily classified, but exhibited features consistent with immune complex deposits, in both peripheral loop (predominantly epimembranous) and mesangial sites. Immunofluorescence studies, which included the use of a monoclonal antibody, showed that HBsAg was present in mesangial deposits but not in peripheral loop deposits. Immunoperoxidase studies employing HRP-conjugated anti-HBc and anti-HBe provided evidence that the epimembranous deposits contained HBe. Studies of the patient's serum revealed high titers of HBsAg, low titers of HBeAg, as well as anti-HBe and anti-HBc. The cryoglobulins contained small amounts of HBsAg, HBeAg, and anti-HBc, but anti-HBe and anti-HBs antibodies were not found. The possible mechanisms accounting for accumulation of HBsAg and HBeAg in glomeruli are discussed. In addition, the literature concerning the nature of hepatitis B antigens found in glomerular deposits is reviewed.
Adult↗
Immunopathologic characterization of a monoclonal antibody that recognizes common surface antigens of human ovarian tumors of serous, endometrioid, and clear cell types.
A murine monoclonal antibody, OC125, reacts with a surface component of ovarian tumor cells from humans, but fails to react with normal adult ovarian cells. The spectrum of reactivity of OC125 in ovarian tumors from humans was defined by testing cryostat tissue sections from 60 selected ovarian tumors by indirect immunofluorescence. OC125 stained 7/7 benign, and borderline serous ovarian tumors, 19/23 (83%) serous adenocarcinomas, 2/2 mixed serous and endometrioid carcinomas, 2/3 endometrioid carcinomas, 1/4 clear cell carcinomas, and 2/2 undifferentiated carcinomas. No reactivity was found in eight mucinous ovarian tumors or any of the other 11 epithelial sex cord, germ cell, on hematopoietic tumors tested. In neoplastic cysts, papillae, and glands, the staining was most intense on the luminal surface or in subjacent cytoplasm. Cells in solid sheets also showed peripheral staining. Within a reactive tumor, both negative and positive cells could be found, intimately intermixed. There were no differences in these staining patterns between tissues from primary and metastatic sites. The expression of the OC125 antigen was not related to the degree of malignancy as judged by pathologic criteria. Although mucinous tumors lacked reactivity with OC125, seven of eight mucinous adenomas and adenocarcinomas bound a monoclonal antibody against carcinoembryonic antigen (CEA). Thus, OC125 recognizes a common antigen in some but not all ovarian tumors of serous, clear cell, endometrioid, or undifferentiated type. OC125 may prove useful in the pathologic and cytologic identification of certain types of ovarian tumor cells. Its lack of reactivity with mucinous tumors suggests these belong in a distinct subgroup of ovarian epithelial tumors.
Adenocarcinoma↗
Tissue distribution of a coelomic-epithelium-related antigen recognized by the monoclonal antibody OC125.
OC125, a murine monoclonal antibody, recognizes an antigenic determinant (CA125) that is associated with greater than 80% of epithelial ovarian neoplasms of serous, endometrioid, clear cell, and undifferentiated types. In the present report, a sensitive biotin-avidin immunoperoxidase technique was used to determine reactivity of OC125 with normal adult and fetal tissues, as well as with neoplasms of nonovarian origin. In fetal tissues, the antibody reacted with amnion and with derivatives of the coelomic epithelium, i.e., the müllerian epithelium and the lining cells of the peritoneum, pleura, and pericardium. Among adult tissues, OC125 reacted with the epithelium of fallopian tubes, endometrium, and endocervix. The CA125 determinant was also detected on mesothelial cells in the adult pleura, pericardium, and peritoneum, particularly in areas of inflammation and adhesion. Curiously, the surface epithelium of normal fetal and adult ovaries, thought to be derived from coelomic epithelium, did not express the determinant, except in inclusion cysts, areas of metaplasia, and papillary excrescences. Of neoplastic tissues of nonovarian origin, OC125 reacted consistently only with adenocarcinomas of the endocervix, endometrium, and fallopian tube, and with mesotheliomas. Only seven of 64 nongynecological tumors tested reacted with OC125. Thus, OC125 detects a differentiation antigen shared by fetal coelomic epithelium and its derivatives in the fetus and the adult. Apparently, this antigen disappears early in the course of formation of the ovarian epithelium and is reexpressed in certain reactive and neoplastic lesions, a process that could be termed "molecular metaplasia."
Adenocarcinoma↗
Modulation of surface antigens of a human monocyte cell line, U937, during incubation with T lymphocyte-conditioned medium: detection of T4 antigen and its presence on normal blood monocytes.
The human monocyte line, U937, derived from an individual with histiocytic lymphoma, undergoes morphological and functional changes when incubated with medium conditioned by lectin-stimulated cloned human T lymphocytes. Using monoclonal antibodies and flow cytometry, we therefore analyzed alterations in surface components that might accompany these morphological changes, in comparison with components present on normal blood monocytes. The U937 cells possess three surface antigens in common with blood monocytes, detected with OKM1, 4F2, and anti-monocyte.2 (the last monocyte specific). DR antigen was not detectable on U937 cells with three anti-DR framework antibodies but was detected on blood monocytes. Unexpectedly, OKT4, a monoclonal antibody to T4 antigen previously believed to be restricted to helper T lymphocytes, also reacted with U937 cells. Six monoclonal antibodies to other epitopes on T4 also reacted with U937 cells. None of these could be inhibited by blocking of Fc receptors. T4 with its various epitopes were also expressed on normal human blood monocytes. Other lymphocyte surface markers (T3, T8, T6) and fibronectin were not detectable on U937 cells or monocytes. An individual, whose lymphocytes lacked the epitope detected with OKT4 but had epitopes detected with OKT4 A, B, C, and D, had monocytes with identical reactivity, evidence that the T4 on monocytes and lymphocytes are products of the same structural gene. Stimulation of U937 cells for 24 hours with supernatants from Con A-stimulated T lymphocyte clones caused an increase in expression of OKM1 and Fc receptor activity and a decrease in expression of T4, consistent with a more mature phenotype of blood monocytes. Although the function of the T4 molecule is unknown, it is notable that it is displayed by two cells of distinct lineage which interact in the response to soluble antigens.
Animals↗
Fibronectin beneath reepithelializing epidermis in vivo: sources and significance.
Fibronectin and fibrinogen occur under the migrating epidermal tongue during reepithelialization of an excisional wound, and fibronectin increases in conjunction with capillary and fibroblast ingrowth during wound healing. Although we have previously shown that fibronectin is produced by proliferating blood vessels, the source of fibronectin associated with reepithelialization and fibroblast ingrowth has not been determined. In this report we demonstrate that subepidermal fibronectin derives mostly from plasma early in reepithelialization of an excisional wound and comes from both plasma and in situ production late in reepithelialization. This finding was established by extirpating 3 mm of skin from the center of a well-healed rat xenograph on the flanks of immunosuppressed mice, harvesting the open wound sites at 2, 4, 7, and 10 days after injury, and staining the specimens with reciprocal species-specific anti-fibronectin antibodies conjugated with fluorescein. In the first 4 days after wounding, newly forming rat epidermis migrated mainly over mouse fibronectin. In contrast, by 7 days after excision, the rat epidermis transits over a matrix containing both mouse and rat fibronectin, or rat fibronectin alone, indicating that a major component of the fibronectin is produced in situ. Although the biologic significance of these observations has not been fully elucidated, fibronectin may be part of a provisional matrix that functions to support, if not actively participate in, cell recruitment to sites of inflammation or wound healing.
Animals↗