Regulation of transplantation immunity in vivo by monoclonal antibodies recognizing host class II restriction elements. II. Effects of anti-Ia immunotherapy on host T cell responses to graft alloantigens

Results of the preceding report demonstrated that in vivo treatment with monoclonal anti-I-A antibodies provided an effective means of prolonging the survival of murine tail skin allografts. The mechanism of antibody action was shown to include the activation of alloantigen-specific suppressor T cells (Ts), although the relationship between Ts expression and graft survival was not determined. This issue was addressed in the current studies through a kinetic analysis of suppressor and effector T cell responses in control and treated allograft recipients. Donor-specific delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses were detectable in untreated A/J recipients of B10.A allografts 8 days after transplantation, rising to near maximum levels by day 12. Rejection in these animals occurred by day 11. In contrast, the predominant cellular response of anti-I-A treated animals for 12 days after transplantation was that of transferable suppression, DTH and CTL reactivity not being evident until day 15, coincident with the decay of Ts activity. Rejection in these animals was observed approximately 19 days post-transplant. CTL responsiveness in the latter group could not be reconstituted by the addition of antigen-presenting cells to the secondary in vitro culture system, nor was the CTL deficit due to antibody carry-over. It is considered that the altered expression of effector cell responses to graft alloantigens is due at least in part to the in vivo inhibition of helper T cell activity by anti-I-A-induced Ts, and that rejection in the treated host results from an eventual decline in the functional expression of this regulatory T cell subset.     

Isolation of monoclonal antibodies recognizing rat bone-associated molecules in vitro and in vivo.

Knowledge of the number and kinds of differentiation steps that characterize cells of the osteoblast lineage is inadequate. To further analyze osteoblast differentiation, we generated a series of monoclonal antibodies (MAb) to osteogenic cells. Spleen cells from mice immunized with whole-cell populations enriched for expression of osteoblast-associated properties or bone formation in vitro were fused with the SP2/0 myeloma cell line. Supernatants from growing hybridomas were screened by indirect immunofluorescence on frozen sections of a portion of 21-day fetal rat heads that included the calvaria bone, periosteum, muscle, fibrous connective tissue, and skin. Six MAb were selected with bone-associated staining and limited ability to label other tissues. Either cell surface or cytoplasmic molecules were recognized by five of the MAb; one recognized a molecule detectable both in the cytoplasm, on the cell surface, and in the extracellular matrix. Of the antibodies selected, one identified both preosteoblasts and osteoblasts and has been found to be against alkaline phosphatase. The others recognized the mature osteoblasts, osteocytes, and chondrocytic cells. The pattern and distribution of the labeling in vivo extended to primary cells and cell lines in vivo. These results support earlier observations on molecules differentially expressed by cells at different stages of the osteoblast lineage and extend the available cell surface and cytoplasmic epitopes identifiable as marker molecules.     

Specific prolongation of MHC class II disparate skin allografts by in vivo administration of anti-IFN-gamma monoclonal antibody

In vivo rejection of MHC class II disparate skin allografts has been thought to involve IFN-gamma-induced expression of MHC class II alloantigens because less than 3% of skin epidermal cells express MHC class II alloantigens constitutively. In our study we directly tested this hypothesis by examining the effect of in vivo administered anti-IFN-gamma mAb on rejection of MHC class II disparate skin allografts, and comparing its effect on rejection of MHC class I disparate skin allografts placed on the same individual mice. We found that anti-IFN-gamma mAb blocked the rejection of MHC class II disparate skin allografts, but had no effect on the rejection of MHC class I disparate skin allografts. These results demonstrate that endogenously produced IFN-gamma is critical for rejection of MHC class II disparate skin allografts, but not for rejection of MHC class I disparate skin allografts. Thus, this study strongly supports the concept that MHC class II rejection responses require IFN-gamma induced MHC class II expression on keratinocytes of the allograft.     

Analysis of class I MHC antigens in the rat by monoclonal antibodies

Monoclonal antibodies (mAb) were made against class I MHC antigens of the i (mAb 42,70,39) and u (mAb 68-D) haplotypes in the rat by using specific strain combinations in order to obtain reagents for identifying the products of the RT1.An, RT1.Au, and RT1.Eu loci. These antibodies were hemagglutinating only; were IgG except for mAb 68-D3, which had a defective heavy chain; reacted identically with MHC-congenic strains and with their inbred donor strains; and precipitated class I MHC antigens. Strain distribution, sequential immunoprecipitation, and peptide mapping studies were used to define the specificities of the mAb, and the assignments were checked by comparing the specificities of the mAb with those of haplotype-specific alloantisera. The specificities were the following: mAb 42, An; mAb 68-D, Au; mAb 70, Eu; and mAb 39, an antigen encoded by a locus different from A and E. This new locus was designated RT1.F, and the allele detected by mAb 39, as Fa. The serologic data place RT1.F between RT1.A and RT1.D. The plasma membranes of DA.1I(BI) lymphocytes contain comparable amounts of An, Eu, and Fa antigens but express them on the cell surface in the order An much greater than Eu greater than Fa.     

In vivo administration of anti-CD3 monoclonal antibodies or immunotoxins in murine recipients of allogeneic T cell-depleted marrow for the promotion of engraftment.

The role of host anti-donor cells in rejection of fully allogeneic donor T cell-depleted marrow was investigated by using mAb or immunotoxins directed against T cell or NK cell determinants. Immunotoxins consisting of mAb conjugated to a low oligosaccharide-containing fraction of purified ricin toxin A chain (RTA) facilitated in vivo-depletion of target cell populations. BALB/c and DBA/1 donors were selected based upon their expression (BALB/c) or lack of (DBA/1) hemopoietic histocompatibility (Hh1) Ag, which may serve as targets for donor rejection in C57BL/6 hosts. When studies directed toward eliminating CD3+ cells were performed in both systems, injections of intact anti-CD3 mAb or anti-CD3-RTA reproducibly produced the highest engraftment values. The fact that engraftment values obtained with anti-CD3 or anti-CD3-RTA therapy in allogeneic systems were substantially higher than in syngeneic controls suggested that engraftment stimulatory proteins were released upon TCR engagement. Elevated levels of cytokines and a high mortality rate in allogeneic recipients confirmed that this was the case. Nonstimulatory preparations of anti-CD3F(ab')2 fragments and anti-CD3F(ab')2-RTA promoted engraftment of both types of allogeneic marrow, as measured by short term 125I-IUdR assays, suggesting that stimulation was not a prerequisite for engraftment. Recipients of anti-CD3F(ab')2 or anti-CD3F(ab')2-RTA showed a marked reduction of host CD3+ cells as measured by immunofluorescence and flow cytometry. In long term chimerism studies, recipients of Hh1-disparate marrow and anti-CD3F(ab')2 had a dramatic increase in donor cell engraftment as compared to controls, indicating that positive effects on engraftment were long lived. Studies further showed that BALB/c donor cells exhibiting an Hh1 disparity were rejected by host cells expressing NK1.1 or Ly-1 (NK cells and T cells). In contrast, DBA/1 donor cells that were not Hh1-disparate were rejected by cells expressing Ly-1, but not NK1.1 (T cells only). These studies provide definitive data that CD3+ cells participate in the rejection of either Hh1+ or Hh1null T cell-depleted allografts and offer new strategies for alloengraftment using regimens containing nonmitogenic anti-CD3.     

Regulation of the BALB/c anti-p-azophenylarsonate antibody response by monoclonal anti-idiotype. I. Anti-idiotope fine specificity

Five monoclonal anti-idiotype antibodies were prepared against the IgG1 monoclonal antibody, 5AF6, the prototype molecule representing the BALB/c 5AF6 idiotype family of antibodies specific for the p-azophenylarsonate (Ar) hapten. Three were of BALB/c origin and two were derived from allotype congenic strain CB.20. All five anti-idiotopes (id) reacted with the 5AF6 immunogen but not with four other BALB/c anti-Ar sharing other id with 5AF6. Four of the five showed some reactivity with three monoclonal anti-Ar derived from A strain mice that represent a minor component of the anti-Ar from that strain. Reactivity patterns of these anti-id indicated that all five reacted with different id on the 5AF6 molecule, yet all five were sufficiently close to the Ar-binding site for their binding to be blocked by the Ar hapten alone. Furthermore, all five anti-id could compete with each other for binding to 5AF6, indicating that the five id detected by these anti-id were in close proximity. Four of the five anti-id reacted with id produced by conformations requiring both the appropriate heavy and light chains. The fifth anti-id reacted with a heavy chain id stabilized by the presence of any light chain. The implications of such a diverse anti-id response against a single antibody molecule on anti-id network interactions are discussed.     

Role of class I and class II antigens in the allogenic stimulation: class I and class II recognition in allogenic stimulation; blocking of MLR by monoclonal antibodies and F(ab')2 fragments

Purified monomorphic monoclonal antibodies against Class I and Class II antigens in the inhibition of in vitro allogenic response were assayed. As expected, anti-Class II antibodies are highly inhibitory when used in concentrations greater than 5 micrograms/ml in MLRI and 50 micrograms/ml in MLRII. Surprisingly, anti-Class I monoclonal antibodies are as effective as anti-Class II in inhibiting primary MLR although they have no effect in MLRII. These results were confirmed by using F(ab')2 fragments. The inhibitory effect of anti-Class I has been shown to occur at the stimulator cell level. It is proposed that the allogenic stimulation is elicited after Class I and Class II recognition although only Class II differences are responsible for the proliferative response.     

Type II collagen-induced arthritis in mice. III. Suppression of arthritis by using monoclonal and polyclonal anti-Ia antisera

Pretreatment of mice genetically susceptible to type II collagen-induced arthritis (CIA) with monoclonal or polyclonal antisera specific for I region gene products (Ia antigens) suppressed or delayed the onset of CIA, whereas pretreatment with anti-Ia to an irrelevant haplotype was without effect. The humoral response to type II collagen was transiently depressed 14 days after immunization but antibody levels did not differ significantly after 28 days. The peak delayed-type hypersensitivity to type II collagen was unaffected by anti-Ia treatment. Monoclonal antibody of one anti-Ia specificity enhanced both the antibody response and the arthritis incidence in one mouse strain.     

Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.     

Evidence for involvement of clonal anergy in MHC class I and class II disparate skin allograft tolerance after the termination of intrathymic clonal deletion

Mechanisms of cyclophosphamide (CP)-induced tolerance to class I (D) and class II (IE) alloantigens were studied. Transplantation tolerance across H-2D plus IE Ag-barriers has been achieved when B10.Thy-1.1 (Kb,IAb,IE-,Db; Thy-1.1) mice were primed i.v. with 9 x 10(7) spleen cells plus 3 x 10(7) bone marrow cells from B10.A(5R) mice (5R; kb,IAb,IEb,Dd; Thy-1.2) and treated i.p. with 200 mg/kg of CP 2 days later. The tolerant state in the early and the late stage was confirmed by prolonged acceptance of donor-type skin grafts, and in vitro unresponsiveness to donor Ag. In the tolerant B10.Thy-1.1 mice treated with 5R cells 28 days earlier and followed by CP, intrathymic clonal deletion of V beta 11+ T cells reactive to IE-encoded antigens was observed in association with intrathymic mixed chimerism. 5R skin survived, however, even after the clonal deletion of V beta 11+ T cells terminated by 180 days after tolerance induction. V beta 11+ T cells, which reappeared in the periphery of the recipient B10.Thy-1.1 mice bearing 5R skin at this stage, were not capable of proliferating in response to receptor cross-linking with V beta 11-specific mAb. Furthermore, the CTL activity against class I (Dd) alloantigens of spleen cells from these tolerant mice was restored by the addition of IL-2 to MLC. Thus, our experiments provide direct evidence that tolerance to both class I (Dd) and class II (IEb) alloantigens by clonal allergy occurs during the termination of intrathymic clonal deletion. These results clearly show practical hierarchy of the mechanisms of transplantation tolerance.     

Inhibition by anti-HLA class II monoclonal antibodies of monoclonal antibody OKT3-induced T cell proliferation. Studies at the mRNA level

mAb to monomorphic determinants of HLA class II Ag have been shown to inhibit monocyte-dependent OKT3-induced T cell proliferation, indicating that MHC class II molecules play a regulatory role also in Ag nonrestricted, CD3-induced T cell proliferation. This effect involves several steps in the process of T cell activation and proliferation, including IL-1 beta, IL-6, and IL-2 secretion and IL-2R alpha expression. In the present study, we analyzed the effect of an anti-HLA class II mAb (Q5/6) on the mRNA expression of genes related to monocyte and T cell activation. mRNA levels for early (early c-myc, c-fos) and late (late c-myc, N-ras, c-myb) genes involved in T cell activation were determined as well as mRNA levels for IL-1 beta, IL-6, IFN-gamma, IL-2, and IL-2R alpha. The kinetics of mRNA induction for ICAM-1 was also investigated. The results show that in T lymphocytes the expression of c-fos and early c-myc mRNA was unaffected by mAb Q5/6, whereas the c-myb and N-ras mRNA levels were strongly diminished as well as those of IL-2, IL-2R alpha, and IFN-gamma mRNA. An early increase of ICAM-1 mRNA was partially inhibited. In monocytes, a marked reduction of IL-1 beta and IL-6 mRNA was found. It is concluded that the HLA class II determinant involved in the inhibition mechanism can be engaged in the control of IL-1 beta and IL-6 mRNA levels and constitute an accessory signal up-regulating IL-2 and IL-2R alpha gene activation, through a pathway not affecting c-myc and c-fos expression.     

Generation and specificity of monoclonal anti-idiotypic antibodies against human HIV-specific antibodies. I. Cross-reacting idiotopes are expressed in subpopulations of HIV-infected individuals

In this study we have generated monoclonal anti-idiotypic antibodies against human monoclonal and polyclonal anti-HIV antibodies in seropositive sera. A human anti-gp41 mAb (H2, IgM kappa) was used to immunize BALB/c mice and to prepare hybridoma anti-antibodies that react with H2 and not with normal human IgM. Similar monoclonal anti-antibodies were made in BALB/c mice immunized with Ig fraction prepared from a pool of HIV-seropositive sera. Both kinds of anti-idiotypic antibodies reacted with antibodies in pools of seropositive sera and with individual seropositive sera but not with normal human Ig or seronegative sera. The Id-positive Ig from single donors were isolated on two different anti-Id immunoabsorbents and shown to bind to p24 and gp120, respectively. The detection and isolation of idiotypically cross-reactive human anti-HIV antibodies from seropositive donors demonstrated, for the first time, the existence of shared Id expressed by antibodies against HIV Ag. The utility of cross-reacting anti-idiotypic antibodies as tools to dissect the network regulation of the anti-viral immunity in AIDS is discussed.     

T cell subsets in allograft rejection. In situ characterization of T cell subsets in human skin allografts by the use of monoclonal antibodies

We have provided evidence that both major T cell subsets, T4-positive (helper/inducer) and T8-positive (cytotoxic/suppressor), infiltrate human skin allografts. Overall, and in the graft dermis and graft bed, T4-positive cells were predominant (1.5 to 3 times more numerous than T8-positive cells). In contrast, T8-positive cells were relatively more numerous in the epidermis and hair follicles. Rejection probably proceeded by two apparently independent pathways: 1) direct contact killing of graft epithelial cells, presumably by immunologically specific T8-positive cytotoxic cells, and 2) injury of microvascular endothelium of both the graft and graft bed with secondary graft infarction. Although important in first set skin allograft rejection, the mechanism of the second type of killing is uncertain. T4-positive cells were probably involved, as evidenced by their greater numbers; furthermore, studies in mice have shown that transfused helper/inducer cells are able to effect first-set skin graft rejection. It remains to be determined whether T4-positive cells act alone or cooperate with other cells to destroy vessels and bring about graft rejection. Langerhans cells were recognized in epithelial and dermal compartments of both allografts and autografts by their reactivity with anti-T6 and anti-Ia antibodies. We could not determine whether such cells in allografts were of host or donor origin.     

Localized conformational changes induced in a class I major histocompatibility antigen by the binding of monoclonal antibodies

We describe two monoclonal antibodies, R3/47 and YR1/1, directed against different epitopes of the expressed rat class I major transplantation antigen RT1Aa, that interact with each other so that the binding of one antibody, YR1/1, is greatly enhanced by the binding of the other. The positive interaction between R3/47 and YR1/1 also occurs when using RT1Aa molecules solubilized from cell membranes in detergent. It is therefore unlikely that the molecular environment of the membrane contributes to the interaction. The ability of R3/47 to modify the YR1/1 determinant on the RT1Aa molecule is mediated without any significant loss of potency by highly purified monomeric Fab fragments. This result suggests that the binding of R3/47 to the RT1Aa molecule alters the YR1/1 determinant by initiating a propagated conformational change in the antigen.     

Functional abolition of monocyte HLA-DR by aldehyde treatment. A novel approach to studies of class II restriction elements in antigen presentation

The effect of low concentration aldehyde treatment (0.0012 to 0.005%) on the expression of HLA-DR Ag by human monocytes was investigated. This treatment was shown to selectively abolish the expression of HLA-DR determinants defined by a monomorphic mAb (YE2.36) in a rosette assay. The expression of class I MHC Ag and Fc gamma R remained unaffected. As a result, the presentation of the recall Ag tetanus toxoid and streptokinase-streptodornase (SK-SD) to freshly isolated autologous T cells and T cell clones was completely inhibited. Increasing the concentration of aldehyde to 0.05% consistently produced partial restoration of Ag-presenting capacity. Low dose aldehyde treatment did not affect monocyte viability or membrane turnover. Thus, aldehyde-treated monocytes produced a second generation of HLA-DR and expression was almost completely restored to normal after 24 h of culture. The presentation of monocyte class II Ag as alloantigens was also inhibited by low dose aldehyde treatment but inhibition was much less marked when monocytes were aldehyde treated at 2 h rather than at 24 h of culture. This is consistent with the reexpression of HLA-DR which occurred readily in the first 24 h of culture and much less readily thereafter. Low dose aldehyde treatment did not affect Ag uptake and processing. However, monocytes which had been pulsed with Ag, aldehyde-treated to abolish HLA-DR, and then cultured to allow regeneration of HLA-DR could present Ag only when given a second Ag pulse, suggesting that once the association between microbial Ag and HLA-DR had formed the Ag was not then free to reassociate with novel HLA-DR. Low dose aldehyde treatment did not affect monocyte IL-1 production, neither did it inhibit the detection of HLA-DR by soluble mAb in FACS analysis. These results are consistent with the view that low dose aldehyde treatment disrupts the tertiary structure of human Ia molecules such that allostimulatory determinants and restriction elements for exogenous Ag are rendered inaccessible to T lymphocyte receptors and to cell-bound anti-DR mAb in the rosette assay, although DR determinants may remain accessible to soluble mAb.     

Thymic transplantation in miniature swine. II. Induction of tolerance by transplantation of composite thymokidneys to thymectomized recipients

Previous studies in our laboratory have demonstrated that the presence of the thymus is essential for rapid and stable tolerance induction in allotransplant models. We now report an attempt to induce tolerance to kidney allografts by transplanting donor thymic grafts simultaneously with the kidney in thymectomized recipients. Recipients were thymectomized 3 wk before receiving an organ and/or tissues from a class I-mismatched donor. Recipients received 1) a kidney allograft alone, 2) a composite allogeneic thymokidney (kidney with vascularized autologous thymic tissue under its capsule), or 3) separate kidney and thymic grafts from the same donor. All recipients received a 12-day course of cyclosporine. Thymectomized animals receiving a kidney allograft alone or receiving separate thymic and kidney grafts had unstable renal function due to severe rejection with the persistence of anti-donor cytotoxic T cell reactivity. In contrast, recipients of composite thymokidney grafts had stable renal function with no evidence of rejection histologically and donor-specific unresponsiveness. By postoperative day 14, the thymic tissue in the thymokidney contained recipient-type dendritic cells. By postoperative day 60, recipient-type class I positive thymocytes appeared in the thymic medulla, indicating thymopoiesis. T cells were both recipient and donor MHC-restricted. These data demonstrate that the presence of vascularized-donor thymic tissue induces rapid and stable tolerance to class I-disparate kidney allografts in thymectomized recipients. To our knowledge, this is the first evidence of functional vascularized thymic grafts permitting transplantation tolerance to be induced in a large animal model.     

Regulation of MHC expression in vivo. Bacterial lipopolysaccharide induces class I and II MHC products in mouse tissues by a T cell-independent, cyclosporine-sensitive mechanism

The effect of injections of bacterial LPS on the expression of class I and II products of the MHC in mouse tissues was investigated. MHC products were assessed in tissue homogenates by radiolabeled antibody binding and in tissue sections by indirect immunoperoxidase (IIP) staining. In mice given two i.p. injections of LPS from Escherichia coli or Salmonella minnesota, there were increases in class I and II MHC products in kidney, liver, heart, lung, and pancreas. Focusing on the changes in kidney, we demonstrated that the increase in MHC expression occurred in tubules and, in the case of class I, in glomeruli. LPS treatment also increased the deposition of Ig in glomeruli. Expressed on a standard curve, the total kidney class I and II expression was elevated approximately 10-fold. Time course studies indicated that increased class I expression could be induced by a single LPS injection, whereas class II induction required a second injection. The induction was influenced by the LPS sensitivity of the mice, being much greater in LPS-sensitive C3H/HeSn mice than in LPS-resistant C3H/HeJ mice. LPS induced class I and II Ag in nude mice and in mice with severe combined immunodeficiency, indicating that T cells were not required. Nevertheless, the effect of LPS was inhibitable by cyclosporine and by a mAb against IFN-gamma indicating that IFN-gamma was required for the MHC induction. We conclude that LPS induces an increase in expression and a redistribution of MHC products in kidney and in other tissues by a T cell-independent, cyclosporine-sensitive pathway. These findings are probably related to the known ability of LPS to mediate release of IFN-gamma and other cytokines.     

Detection of new MHC mutations in mice by skin grafting, tumor transplantation and monoclonal antibodies: a comparison

Two mechanisms of major histocompatibility complex (MHC) mutations have been described in mice; gene conversion and homologous but unequal recombination. However, our knowledge of mutations in MHC is incomplete because studies have been limited almost exclusively to two haplotypes, H-2b and H-2d, while hundreds of haplotypes exist in nature; it has been biased by the use of only one procedure of screening for mutation, skin grafting. We used three procedures to screen for MHC mutations: (1) conventional techniques of skin grafting, (2) syngeneic tumor transplantation and (3) typing with monoclonal anti-MHC antibodies (mAbs) and complement. The faster technique of tumor transplantation detected mutants similar to those discovered by skin grafting technique. Screening with mAbs allowed us to detect both mutants that are capable of rejecting standard skin grafts and those that are silent in skin grafting tests, and which therefore resulted in a higher apparent mutation frequency. Two mutants of the H-2a haplotype were found that carry concomitant class I and class II antigenic alterations. Both MHC mutants silent in skin grafting tests and mutants carrying concomitant class I and class II alterations have never been studied before and are expected to reveal new mechanisms of generating MHC mutations. 1-Ethyl-1-nitrosourea (ENU) failed to induce de novo MHC mutations in male mice in our skin grafting series.