Biomedical subjects
H J Winn
Publications and source records attributed to H J Winn.
CD4+ lymphocytes play a dominant role in murine xenograft rejection.
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Surface antigens of murine natural killer cells.
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Prevention of alloantibody formation after skin grafting without prolongation of graft survival by anti-L3T4 in vivo.
Treatment of mice in vivo with monoclonal antibodies against the L3T4 antigen (CD4 in human beings) has been shown to suppress the humoral response to several foreign antigens and to prolong the survival of allografts in some cases. Experiments were therefore performed to test whether anti-L3T4 antibody treatment would suppress alloantibody production after skin transplantation. Monoclonal anti-L3T4 antibody (GK1.5) was administered to C57BL/6 (B6) mice prior to BALB/c skin grafting. The production of B6 anti-BALB/c alloantibody was then tested after graft rejection. The results showed that: (1) graft survival of BALB/c skin on B6 mice was not substantially prolonged by anti-L3T4 treatment; (2) graft survival was significantly prolonged if mice were treated with both anti-L3T4 and anti-Lyt2 antibody; (3) the production of alloantibody following grafting was decreased by anti-L3T4 treatment and was completely eliminated if thymectomy was also performed; (4) thymectomy prolonged the effectiveness of the anti-L3T4 treatment; (5) tolerance to alloantigens presented at the time of anti-L3T4 treatment was not achieved; and (6) well-established cytotoxic antibody titers rose to higher levels after secondary grafting even with concurrent anti-L3T4 treatment, while weak antibody titers remained stable or decreased. These results indicate that L3T4+ cells are essential in providing the "help" necessary for generating humoral responses to alloantigens but that elimination of these L3T4+ cells still allows the generation of help for cell-mediated immunity. The data also suggest that class I antigens must be presented on class II molecules in order to elicit an antibody response.
Immunohistochemical analyses of skin graft rejection in mice. Kinetics of lymphocyte infiltration in grafts of limited immunogenetic disparity.
We have examined the nature and kinetics of T cell infiltration into murine first-set skin allografts across a variety of histocompatibility differences. Both Ly 1+2- and Ly 2+ cells migrate into grafts that differ with respect to discrete class I, class II, or minor H barriers. The lymphoid cells in the allograft did not express MEL 14 or the B cell antigen B220. The cells in the graft progress from the vascular anastomosis region into the dermis with apparent homing and clustering about hair follicles and epithelial cells at the dermoepidermal junction. Microscopic necroses appear in a patchy distribution in these areas. The intensity of the infiltrate is related to the nature of the alloantigen recognized but it does not correlate with the tempo of rejection. Class II-disparate allografts evoke a peak response (mean 62 +/- 34 T cells/0.1 mm2) that is 2-6-fold greater than class I-disparate allografts or syngeneic control peak responses (mean 14 +/- 2.4 T cells/0.1 mm2). Although a difference at a minor H antigen evoked an intense peak T cell response (192 +/- 33 T cells/0.1 mm2), the mean survival time of the allografts was significantly longer than that observed with class I or class II differences. The IL-2 receptor+ subset of activated T cells is extremely rare in syngeneic grafts, but ranges from 0 to 56% of infiltrating T cells in allografts. We conclude that all major T cell subsets contribute to the in situ response to all classes of alloantigens, but at the apparent exclusion of the MEL 14+ T cell pool and B cells.
Failure to induce chimerism or tolerance in mice with monoclonal anti-T cell antibodies plus allogeneic bone marrow infusion.
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Monoclonal antibody therapy. Anti-idiotypic and non-anti-idiotypic antibodies to OKT3 arising despite intense immunosuppression.
The frequency, timing, and specificity of the humoral antibody response to a murine monoclonal antibody (OKT3, IgG2a) were measured in 21 consecutive renal allograft recipients. These patients received i.v. OKT3, 1-5 mg/day for 10-20 days as treatment for acute graft rejection. Maintenance immunosuppression consisted of azathioprine and corticosteroids. Using three different assays, an antibody response was detected in 75% of the 20 patients with adequate samples. The ELISA assay of the overall IgM and IgG reactivity to OKT3 revealed that IgM anti-OKT3 appeared in 65% and IgG anti-OKT3 in 50% of the patients, reaching a peak 20-33 days after the last dose of OKT3. The IgM preceeded the IgG in most cases (P less than 0.02) and in 8 cases was detected during therapy. One patient had high levels of IgM anti-OKT3 before therapy, yet responded normally to OKT3. Interference with the therapeutic effectiveness was evident in one patient who developed IgG antibodies during therapy. His serum blocked the binding of F-OKT3 to normal lymphocytes in the presence of normal BALB/c serum. The blocking assay, done by flow cytometry, measured anti-idiotypic (Id) reactivity since the sera did not affect the binding of OKT8 (another IgG2a) or anti-Leu4 (another anti-T3), and the blocking activity remained after affinity absorption with normal mouse IgG. Using this assay, 60% of the patients made an anti-Id response. One made only anti-Id, and several had anti-Id at times when other reactivities were undetectable. Antibodies to non-idiotypic, presumably isotypic, determinants represented on OKT8 occurred in only 44%, while other reactivity (OKT4; IgG2bK) was less common (12%) and weaker. While no adverse allergic reactions occurred in this group of patients, the anti-Id antibodies, which are a prominent feature of the immune response to this and probably other monoclonal antibodies, can block their therapeutic effectiveness and can arise despite intense immunosuppression. This response may require the use of different idiotypes for prolonged or repeated courses of therapy and may be the major obstacle to the use of human monoclonal antibodies.
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.
Lysis of antibody-coated cells by platelets.
Antibody-coated erythrocytes are lysed by murine C5- whole blood but not by plasma separated from such blood. The lytic activity has been shown to derive from platelets that attach to sensitized cells probably through membrane receptors for C3b. Whole blood or platelet-rich plasma (prp) obtained from mice that have been treated with purified cobra venom factor has little or no activity unless it is fortified with fresh C5- plasma. Lysis is observed only if the reactants are incubated at 37 degrees C and mechanical shaking is practiced, at least intermittently, throughout the period of incubation. Adherence of platelets and subsequent lysis are mediated by antibodies of a variety of immunoglobulin classes, including those that fail to mediate complement-dependent lysis. Platelet-mediated lysis is limited to cells to which the platelets adhere; 51Cr labeled, unsensitized cells that are mixed with prp and sensitized, unlabeled cells do not release 51Cr. Normal murine lymphoid cells and ascites tumor cells of mice, rats, and guinea pigs were apparently unaffected by sensitization and incubation with prp. However, because adherence of platelets to these sensitized cells was not observed, it is not clear whether the cells are resistant to the lytic action of platelets or whether the conditions of incubation were unfavorable for the attachment of platelets to the surfaces of nucleated cells. The significance of the lytic reaction described here is not known but may lie in antibody mediated release of microbicidal substances from platelets.
Fibronectin is produced by blood vessels in response to injury.
During the time of tissue repair that ensues subsequent to tissue injury, blood vessel wall fibronectin increases concomitantly with endothelial proliferation and angiogenesis. However, the source of this blood vessel fibronectin had not been delineated. In this report we have demonstrated that microvascular fibronectin is produced in situ by the proliferating vessels surrounding excisional wounds. 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 injured skin sites at various stages during the healing process, and staining the specimens with reciprocal species-specific anti-fibronectin. The proliferating donor vessels that surrounded the wounded graft had increased fluorescence staining with FITC conjugated mouse anti-rat fibronectin and no staining with rat anti-mouse fibronectin. This finding was taken as direct evidence that the fibronectin was produced in situ by the rat vessels and not derived from circulating mouse plasma.
An isotopic antiglobulin test for quantitation of anti-sheep red blood cell antibodies of individual Ig classes in mice bearing different IgCH haplotypes.
An isotopic antiglobulin test (IAT) for anti-sheep red blood cell (SRBC) antibodies of different immunoglobulin (Ig) classes was developed and used to measure levels of IgM, IgA, IgG, IgG2a, IgG2b, and IgG3 anti-SRBC antibody in sera from 12 inbred strains of mice. Characterization of the Ig class specific heteroantisera prepared for use in this assay revealed substantial amounts of antibodies reactive with idiotypic and allotypic determinants of IgG1, IgG2a, IgG2b and IgA. Accordingly, class specific antibodies which do not discriminate among Ig molecules of different IgCH phenotypes were isolated from each heteroantiserum by absorption and elution from Ig's of selected strains. Levels of anti-SRBC antibody are expressed as units of antibody/ml relative to an anti-SRBC antiserum reference standard. The use of conversion factors (estimates of the relative reactivity of each anti-Ig reagent) allows quantitative comparisons within the between IgG subclasses. This method allows measurements of nanogram amounts of specific anti-SRBC antibodies of a single Ig class, and simultaneous measurements of antibody levels in 6 Ig classes from a single 30 microliter serum sample. Analysis of the responses of several strains of inbred mice indicates striking variations (5-31-fold) in the amounts and relative proportions of different Ig classes. The IgG subclass distributions observed appear to be associated with the IgCH but not the H-2 haplotypes of the strains studied.
The vascular bed as the primary target in the destruction of skin grafts by antiserum. I. Resistance of freshly placed xenografts of skin to antiserum.
Rat skin grafted onto immunosuppressed mice is resistant to mouse anti-rat serum during the first 7-10 d after transplantation. It gradually acquires susceptibility, reaching a peak of sensitivity at 14-16 d after grafting. The grafts remain sensitive to antiserum, though at decreasing levels for an additional 3 wk, and grafts that persist beyond that time are resistant to antiserum for as long as they survive. In the study reported here, it is shown that the initial period of resistance to antiserum is due to factors acting locally within the graft and is entirely uninfluenced by the regimen of immunosuppression or the protective dressings that are used. After administration of antiserum, deposits of the injected immunoglobulin and of endogenous C3 are found on the luminal surfaces of graft vessels, although no significant tissue damage is observed. Rat skin that has become highly sensitive to antiserum 14-16 d after transplantation loses that sensitivity if it is regrafted to a new recipient, and then regains it 8-10 d later. Thus, the resistance of freshly grafted skin to antisera is associated with the process of healing into place, a conclusion that is supported by the observation that the intracutaneous administration of antisera to rats causes intense local inflammation and necrosis. The skin is therefore sensitive just before it is removed for grafting, but temporarily loses sensitivity thereafter. Resistance to antiserum during the first 3 or 4 d after transplantation is probably attributable to the fact that at that time grafts are vascularized poorly if at all. The state of resistance extends for several days after vascularization of the graft takes place and is then only gradually lost, a phenomenon that seems to be associated with the resistance of newly formed and regenerating blood vessels to vasoactive substances. This view is in accord with and, indeed, supports the idea that the induction of vascular injury is an essential step in antisera-mediated damage to tissue grafts.
The vascular bed as the primary target in the destruction of skin grafts by antiserum. II. Loss of sensitivity to antiserum in long-term xenografts of skin.
Rat skin that survives for long periods of time on immunosuppressed mice becomes resistant to anti-graft serum and remains so for as long as it survives. When long-standing grafts are removed and placed on new immunosuppressed mice, they remain resistant to antiserum for as long as they survive. The acquired resistance to antiserum seems, therefore, to be due to changes in the grafts rather than to changes in their hosts. Furthermore, it was found that the acquisition of resistance is correlated with replacement of graft endothelium by host cells, as demonstrated by the use of immunofluorescent techniques in conjunction with mouse anti-rat serum and rat anti-mouse serum. Evidently, humoral antibodies are able to cause acute damage to skin grafts, and presumably to grafts to other organized tissues, only if they react with antigens of graft endothelium. Long-term grafts that are retransplanted to their original donors or to rats syngeneic with those donors are in most cases rejected, whereas 14-d-old grafts similarly regrafted are in no case rejected. Apparently, the responses of the secondary recipients to the mouse endothelial antigens in long-term grafts lead to destruction of the entire grafts. When long-standing rat skin xenografts are removed and placed on untreated mice syngeneic with the primary hosts, they are in every case rejected, although they survive slightly longer than skin taken directly from rat donors. Rejection is accompanied by a mononuclear infiltrate and is qualitatively indistinguishable from the rejection of freshly prepared rat skin. Clearly, sensitized cells are more efficient than humoral antibody in destroying grafted tissues.
Genetic control of the expression of immunoglobulin isotypes in the responses of mice to sheep red blood cells.
Studies have been carried out on the genetic control of the expression of individual Ig classes in the responses of A/J (A) and C57BL/6J (B6) mice to sheep red blood cells (SRBC). An isotopic anti-immunoglobulin test employing Ig class-specific reagents was used to measure levels of IgM, IgA, IgG1, IgG2a, IgG2b and IgG3 anti-SRBC antibodies in sera of hyperimmunized mice. Compared with B6 mice, A mice are low IgM and IgA responders but high responders with respect to all IgG subclasses. In (B6 X A)F1 mice low responsiveness was dominant for all Ig classes indicating that IgM and IgA responses are controlled by genes different from those which control IgG subclass responses. Analysis of the responses of [(B6 X A)F1 X A] and [(B10.A X A)F1 X A] backcross mice indicates multigenic control of IgG subclass responses. Expression of IgG2a and IgG3 responses appear to be regulated largely by single genes whose action is modified by genes at other loci; control of IgG1 and IgG2b is, clearly, polygenic with no evidence for major control by any single gene. The magnitude of all IgG subclass responses appears to be regulated by a gene(s) associated with or linked to the IgCH locus. Analysis of the responses of H-2-congenic mice shows that major histocompatibility genes do not have a strong influence on the patterns on IgM and IgG responses in A or B6 mice. The overall results indicate that while there is some common control of all IgG classes, there is also separate polygenic control of individual Ig classes.
Natural killing and antibody-dependent cellular cytotoxicity of tumor targets are mediated by the same effector cell.
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Studies on natural killer (NK) cells. II. Serologic evidence for heterogeneity of murine NK cells.
NK cells are divided into 2 subclasses on the basis of cell surface antigens: NKA cells, which preferentially lyse lymphoma cells and which are readily destroyed by treatment with anti-H-2, rabbit anti-mouse, or anti-NK specific antibodies in the presence of C; and NKB cells, which preferentially lyse nonlymphoma tumor cells and which are resistant to anti-NK specific antibodies, partially sensitive to anti-H-2 specific antibodies, and only fully sensitive to rabbit anti-mouse serum and C. Thus, suspensions of spleen cells that were treated with anti-NK-1.2 antibodies and C lost virtually all lytic activity for 6 lymphoma cell lines, whereas lytic activity for 2 nonlymphoma cell lines remained largely intact. It must be noted, however, that unlike NK-1.2 expression, target cell preference is not an absolute indicator of NK cell subclass. Cytotoxic T cells were readily distinguished from NKA and NKB cells on the basis of their sensitivity to monoclonal anti-Thy-1.2 antibodies and resistance to anti-NK-1.2 antiserum and C.
Immunologic monitoring with monoclonal antibodies to human T-cell subsets.
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Studies on natural killer (NK) cells. I. NK cell specific antibodies in CE anti-CBA serum.
An antiserum prepared by injecting CE mice with CBA spleen cells has been shown to contain antibodies specifically reactive with NK cells. The serum also contains antibodies detectable in C-dependent dye exclusion tests, but in a study of (CE X CBA)F1 X CE mice reactivity in this test was found to segregate independently of sensitivity of NK cells to inactivation. Moreover, abrogation of NK activity was observed with concentrations of antiserum and C that were considerably lower than those required for detectable cytotoxicity as measured by dye uptake. Thus, it is possible to use this antiserum for the selective removal of NK activity. Analysis of the strain distribution of NK cell sensitivity to CE anti-CBA serum suggests that this reagent may detect an allele of the NK-1 system, and we have tentatively designated the allelic product as NK-1.2, and the corresponding antibodies as anti-NK-1.2. However, the possibility that CE anti-CBA serum detects a new system of NK-specific antigen has not been excluded.