Immunoelectron microscopic localization of MHC structures in isolated pancreatic rat islets.

An immunogold-silver enhancement technique, which combines effective labeling of viable isolated islets with the ultrastructural resolution of cytological details, was applied in electron microscopy to identify major histocompatibility complex (MHC) structures on islet cells. Incubation of freshly isolated islets from CAP (RT1c) and LEW (RT1l) rats with OX18, an MHC class I antibody, showed strong positive reactivity in macrophages and/or dendritic-like cells (M0-DCs) and vascular endothelial cells (VEs) and a comparatively weaker reactivity in endocrine alpha-, beta-, and delta-cells. With MHC class II antibody OX6 (anti-I-A), M0-DCs were strongly labeled in both rat strains on the surface and on internal structures. Three of five particularly high titered batches of OX6 revealed MHC class II expression on VE and beta-cells. Four days of in vitro culture in combination with a high concentration of glucose and interferon-gamma induced strong enhancement of MHC class I structures and, to a lesser extent, class II structures on beta-cells.

Measurement of rat insulin. Enzyme-linked immunosorbent assay with increased sensitivity, high accuracy, and greater practicability than established radioimmunoassay.

Total immunoreactive insulin (IRI) is conventionally determined by radioimmunoassays. IRI measurement in rats can be made more sensitive, accurate, and practical, as demonstrated by a new modified enzyme-linked immunosorbent assay (ELISA). It is characterized by indirect binding of an anti-insulin antibody by an antiglobulin antibody and uses the principle of competitive saturation. In this ELISA, IRI can be determined in a wide range of concentrations, corresponding to the standards. The standard curve ranges from 100 to 0.049 ng/ml IRI (1 ng/ml approximately 23.4 microU/ml approximately 172 pM rat insulin). The statistical analysis shows between- and within-assay coefficients of variation of less than or equal to 15%.

New principle for large-scale preparation of purified human pancreas islets.

Because successful human islet transplantation requires large quantities of viable islets that must be separated from the highly immunogenic exocrine tissue and because handpicking is too time-consuming and laborious to be clinically relevant, a new approach for solving this problem has been established in rat models. It is based on the principle that magnetic microspheres (MMSs) coupled to lectins with binding specificity for the exocrine tissue portion are trapped in an electromagnetic field, thus providing effluent islets of a high degree of purity. In this study our aim was to adapt this principle to human islet preparations. In this context our prime interest was focused on a lectin suitable for human pancreatic tissue. Of 19 different lectins tested, only 1, Wisteria floribunda agglutinin (WFA), is suitable, as shown by immunofluorescence, MMS-lectin binding, and magnetic separation.

High lytic activity against human leukemia cells after activation of allogeneic NK cells by IL-12 and IL-2.

IL-12 is a novel cytokine with interesting features regarding its potential usefulness in peripheral blood stem cell transplantation and leukemia immunotherapy. We used cryopreserved leukemia cells of 18 patients with acute myelogenous (n= 14) or lymphocytic (n= 4) leukemia to investigate the effect of IL-12, alone or in combination with IL-2, on the cytolytic activity of NK cells against human leukemia targets. Effector cells were peripheral blood mononuclear cells from healthy donors which were depleted from CD3+ T cells by immunomagnetic separation. CD3-negative effector cells (mainly CD56+ NK cells) were treated for 24 h with various concentrations of IL-2 (100 U/ml to 1000 U/ml) and IL-12 (1 U/ml to 100 U/ml). Cytotoxicity was measured in a 4 h 51Cr-release assay. Whereas a two-fold enhancement of cytotoxic activity was observed after incubation with optimal doses of IL-2 or IL-12, the combination of both cytokines (500 U/ml IL-2, 100 U/ml IL-12) increased the lytic activity more than six-fold. This effect was accompanied by increased expression of cellular adhesion molecules (CD2, CD18) and CD25 on CD56+ effector cells. Of 18 leukemias investigated, five were completely resistant to lysis by effector cells activated with IL-2 or IL-12 alone. In three of these five cases, however, high cytolytic activity was observed after coincubation with IL-2 and IL-12. In comparison to allogeneic NK cells, autologous cells of three patients in remission demonstrated significantly lower cytotoxic activity. No killing of nonmalignant cells (PHA blasts) by allogeneic NK cells was observed. Our data demonstrate that IL-12 can enhance or even induce MHC-unrestricted cytotoxicity of IL-2-activated allogeneic natural killer cells. Since IL-12 has also been shown to have stem-cell mobilizing capacities, it could be used for the recruitment of both stem cells and antileukemic effector cells in the context of peripheral blood stem cell transplantation.

G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation: comparison of T cell depletion strategies using different CD34+ selection systems or CAMPATH-1.

Allogeneic transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) appears to be an attractive alternative to allogeneic bone marrow transplantation (BMT). However, because vast amounts of potentially graft-vs.-host-reactive T cells are transfused with PBPC grafts, the use of PBPC in the allogeneic setting may be associated with an increased incidence or severity of graft-vs.-host disease (GVHD). To evaluate strategies for prevention of GVHD after PBPC allografting, we have studied T cell depletion (TCD) of G-CSF-mobilized PBPC samples harvested from six healthy donors and from five patients scheduled for autologous PBPC transplantation. Three approaches (CAMPATH-1 plus autologous complement [C], immunomagnetic CD34+ cell selection, and biotin-avidin-mediated CD34+ cell selection) were compared. TCD of PBPC samples with the monoclonal antibody (MAb) CAMPATH-1 plus autologous C resulted in a median elimination of 2.16 log CD3+ T cells, whereas 39% of CD56+ natural killer (NK) cells and 56% of CD34+ progenitor cells were recovered. TCD by CD34+ cell selection with the Isolex (Baxter, Munich, Germany) or Ceprate (CellPro, Bothell, WA) devices achieved median depletions (Isolex vs. Ceprate) of 4.04 vs. 3.12 log T cells and > 5 vs. 3.27 log NK cells while allowing the recovery of 36 vs. 27% CD34+ cells. The median purity of CD34+ cells in the final product was 1.7 (CAMPATH-1), 94 (Isolex), and 65% (Ceprate). We conclude that all methods tested effectively deplete T cells from PBPC preparations harvested from healthy donors. Whereas immunomagnetic CD34+ selection is most effective in terms of elimination of T cells, the less intensive T and NK cell depletions achieved with CAMPATH-1 might be advantageous with regard to retaining engraftment potential and graft-vs.-leukemia (GVL) activity of PBPC allografts.

New parental cell lines for generating human hybridomas.

In contrast to the success achieved with the production of hybridomas in the mouse system, creating human hybridomas is problematic. The reason is believed to be a lack of suitable malignant human cell lines. The work presented here demonstrates the establishment of three human parent cell lines--two of which are of T cell origin--by installing hypoxanthine guanosine phosphoribosyl transferase (HGPRT) and/or thimidine kinase (TK) deficiencies into the leukemic cell lines REH, 1301 and SKW-3. In order to isolate true hybridomas, selection procedures must guarantee complete death of the enzyme-deficient malignant parent cells. In this respect sublines with a combined HGPRT and TK deficiency proved to be superior to those with only one enzyme deficiency, especially in combination with the newly developed hypoxanthine/aminopterine/thymidine/azaserine (HATA) selection medium. However, selection media should be of low nonspecific toxicity. This was shown to be a particular property of the thymidine-free azaserine/hypoxanthine (AH) selection medium. Preliminary data show an extraordinary ability of one subclone of the hypoxanthine guanosine phosphoribosyl transferase-negative T cell line SKW-3 to generate human T-T hybridomas. They are of a stable, nearly tetraploid karyotype and express new surface antigens, thus providing new possibilities for the investigation of human T lymphocyte function by means of hybridoma technology.

Magnetic albumin/protein A immunomicrospheres. I. Preparation, antibody binding capacity and chemical stability.

We describe a method of preparing small magnetic microspheres of albumin/protein A, uniform in size, at 200, 300 or 500 nm. It is shown that, independent of size, the microspheres always carry iron peripherally in their matrix and are thus magnetically responsive. A quantitative antibody binding capacity of 82 micrograms/mg microspheres was established for the 500 nm microspheres. The microspheres are stable in most commonly used buffers over a pH range of 2.5-9.2, but are appreciably unstable in such concentrated denaturing agents as 3 M TCN-, 6 M guanidine, or 8 M urea (loss of antibody binding capacity, 30% for TCN- and 70% for urea).

Increase of MLC sensitivity by elimination of a non-adherent responder cell subpopulation with anti-macrophage serum.

A specific rabbit anti-rat macrophage antiserum (SAM) was prepared with a cytotoxic reactivity pattern complementary to that of a specific anti-lymphocyte serum. This was used to characterise adherent and non-adherent spleen cell subpopulations in mixed lymphocyte cultures. Adherent SAM+ cells reacted as accessory cells whereas non-adherent SAM+ cells were suppressors. Selective elimination thus achieved resulted in a highly significant increase of MLC reactivity in certain strain combinations and in conversion from non-reactivity to reactivity in others.

Freezing of cells--replacement of serum by oxypolygelatine.

A commercially available plasma expander (Gelifundol) containing 5.5% oxypolygelatine in buffered saline was used instead of serum as a supplement for freezing several types of human cell and a mouse myeloma cell line in liquid nitrogen. Viability, recovery, proliferation and cytotoxic activity were compared after freezing with a plasma expander and after conventional freezing with human AB serum or fetal calf serum. The plasma expander proved to be equivalent or superior to AB serum by all parameters tested and acceptable even when compared with fetal calf serum. Furthermore, this preparation is cheap, sterile, free of BSE, viral and mycoplasmal contamination or antibodies and foreign serum proteins. We therefore recommend it for freezing of cells for culture of HLA typing.

Evidence that reduction of immunogenicity of T-depleted bone marrow depends on additional depletion of accessory cells. Implications for prevention of graft rejection.

An effective prophylaxis of graft-versus-host reaction in clinical bone marrow transplantation can be achieved by T depletion of the graft. However, this kind of graft manipulation is accompanied by a strongly increased incidence of graft rejection. In order to eliminate this hazardous complication we studied the possibility of preventing rejection of a BM graft by reducing its immunogenicity. In this report we demonstrate that the capacity of human BM cells to induce proliferation of allogeneic T cells is strongly reduced after treatment with the antilymphocyte/antimonocyte monoclonal antibody K31+ rabbit complement (C), indicating a reduced immunogenicity of the BM cells. On the other hand, the allostimulatory capacity of BM treated with clinically established T-depleting MoAbs, including the more broadly reactive CAMPATH-1, was not or only slightly reduced. MHC class II induction on BM cells with human recombinant gamma-interferon did not restore the allostimulatory capacity reduced by K31+C. Exogenous IL-1 partially inhibited the K31-mediated reduction of allostimulatory capacity. Reduction of the allostimulatory capacity of BM cells with K31+C correlated with a reduction of their capacity to support mitogen-induced proliferation of purified autologous T cells, indicating the absence of accessory functions. Addition of autologous peripheral adherent cells to K31+C treated BM restored its accessory function as well as its allostimulatory capacity. Labeling studies confirmed that accessory cells are critical for the described K31-mediated effects. We conclude that depletion of accessory cells in addition to lymphocytes with K31+C strongly reduces the immunogenicity of a BM graft, may help prevent graft rejection, and may serve as a model for preconditioning other organ grafts.

Inhibition of anti-HLA-B7 alloreactive CTL by affinity-purified soluble HLA.

The objective of this study was to elucidate the interaction of naturally occurring soluble MHC class I molecules with alloreactive CTL and to discuss its possible relevance to graft acceptance. An anti-HLA-B7 specific CTL-line, BV.B7, was generated in vitro. On phenotyping the cells after 6 weeks, 80% were found to be CD8+, 14% CD4+ and 6% CD8+CD4+. CD4+ CTL were depleted using immunomagnetic beads precoated with an anti-CD4 antibody. Of the recovered CTL greater than 96% were CD8+. A total of 12 HLA-B7 target cell lines and PHA blasts tested were specifically lysed in a 51Cr-release assay. Soluble HLA class I molecules were isolated on affinity chromatography columns using the anti-HLA-B7 ME 1 and the anti-heavy chain W6/32 monoclonal antibodies. Antigen purity was confirmed by analysis on SDS-PAGE gels. CTL were preincubated with 0.1-1.8 micrograms/ml soluble HLA for 30 min at 37 degrees C and subsequently tested for cytotoxicity in the 51Cr-release assay; 1.1 micrograms/ml HLA-B7 molecules reduced CTL cytotoxicity by 50% whereas non-B7 HLA had no effect. Further, CTL cytotoxicity was reduced by preincubation with anti-CD8, anti-TcR, and anti-CD3 antibodies. We anticipate a possible down-regulatory role of soluble HLA on CTL in allogeneic transplantation.

Studies on the antigenicity of vital allogeneic valve leaflet transplants in immunogenetically controlled strain combinations.

The use of defined inbred strains of rats enables reproducible experimentation on the antigenicity of heart valve leaflet transplantation. The inbred strains CAP, F344, and LEW were used as syngeneic, weakly allogeneic (RT-1-identical) and strongly allogeneic (RT-1-incompatible) strain combinations. After heart valve leaflet transplantation, humoral and cell-mediated immune responses were investigated. The results were: (1) Allogeneic heart valve leaflets are antigenic. (2) Just one heart valve leaflet, applied intravascularly induces sensitization of the recipient. (3) In the weakly allogeneic system, sensitization is only revealed by donor-specific skin transplants, while in the strongly allogeneic group, sensitization is demonstrated humorally as well. (4) The greater the immunogenetical difference, the sooner sensitization appears. In the strongly allogeneic system, skin transplants were rejected as "white grafts".

Immune reactivity after high-dose irradiation.

Immune reactivity after total-body irradiation was investigated in rats using skin graft rejection as the indicator system. After sublethal irradiation with 10.5 Gy (approximately 50% lethality/6 weeks) the rejection of major histocompatibility complex allogeneic skin grafts was delayed significantly compared with nonirradiated control animals (28 versus 6.5 days). In contrast, skin grafts were rejected after 7.5 days in sublethally irradiated animals and 7 days in lethally irradiated animals if additional skin donor type alloantigens--namely, irradiated bone marrow cells--were given i.v. either simultaneously or with a delay of not more than 24 hr after the above conditioning regimen. These reactions were alloantigen-specific. They were observed in six different strain combinations with varying donors and recipients. Starting on day 2 after irradiation, i.v. injection of bone marrow gradually lost its effectivity and skin grafts were no longer rejected with uniform rapidity; skin donor marrow given on days 4 or 8 did not accelerate skin graft rejection at all. These data show that for approximately 1-2 days after high-dose total-body irradiation rats are still capable of starting a vigorous immune reaction against i.v.-injected alloantigens. The phenomenon of impaired rejection of skin grafted immediately after high-dose irradiation appears to result from the poor accessibility of skin graft alloantigens during the early postirradiation phase when vascularization of the grafted skin is insufficient.

Functional characterization of prethymic T cells committed to alloreactivity.

The results of previous experiments on MHC fully allogenic bone marrow transplantation (BMT) in nonthymectomizd recipients indicated that anti-MHC alloreactivity starts to become irreversibly committed at the prethymic level. This is a matter of some controversy. Since it is possible that conflicting results depend on the methods chosen, we reexamined our previous results by applying two new approaches. Adult thymectomized (ATX) Balb/c mice received a syngeneic fetal thymus either 3 weeks before or 3 weeks after lethal irradiation and reconstitution with C57BL/6 BM incubated in antiserum. Since monoclonal antibodies such as anti-Thy 1 are of limited value for investigations of the above type (Thy 1 antigen crosses the prethymic/thymic border), we used two highly selective, excessively cytotoxic xenoantisera for incubation of the donor BM--either a specific anti-T cell serum (SAT) that eliminated only mature T cells, or a specific antilymphocyte serum (SAL) that reacted additionally with a subset of prethymic T cells (PTC). In both experimental approaches the results were similar: Recipients of SAT-BM developed antihost reactivity, in contrast to recipients of SAL-BM. SAT-BM recipients became immunodeficient, whereas SAL-BM chimeras were immunocompetent. Late mortality was observed only following SAT treatment. Preliminary morphological findings in the lymphoid tissue of BM recipients agree fully with the functional observations. We conclude that the data confirm our previous results in nonthymectomized BM recipients--i.e., PTCs initiate antihost reactivity in MHC fully allogeneic BMT--and PTC commitment is thymus/thymus factor independent. These conclusions are discussed with regard to the problems of MHC allogeneic clinical BMT.

Specific down-regulation of allograft reactivity at the cellular level: graft cells and responder T cells.

Local immune reactivity in an allograft is largely determined by (1) the immunogenicity of the grafted cells and (2) the presence of recipient allospecific T lymphocytes and, much more importantly, their capacity to react and initiate damage to the graft. Ad (1): It is still widely believed that allograft immunogenicity is sufficiently described by the cell surface expression of transplantation antigens, in particular MHC class I and II molecules. In extension of earlier, more complex models it will be demonstrated that transfected subclones of a macrophage cell line show unaltered, strong MHC expression but have become non-immunogenic and, most importantly, have acquired the capacity to render mature naive T cells anergic in a specific fashion. Ad (2): Starting from the above and from other observations, we have found in human organ graft recipients that cytotoxic T lymphocyte (CTL) frequencies may be specifically down-regulated but may regain their reactivity after cultivation under ex vivo conditions. In keeping with the latter we can now show quantitatively the variable presence and the functional potential of cell-released (soluble) human MHC (HLA class I) molecules: they are able to specifically inhibit human CTL reactivity in a dose-dependent fashion. Taken together, it may be concluded that studies at the cellular level, in particular the level of human cells, will increasingly help us to better understand and manipulate the immunological interplay between the local graft cell and its most important antagonist, the T lymphocyte.

Quantitative and biochemical characterization of soluble HLA class I antigens shed by cultured human cells.

sHLA has been described in human serum and other body fluids. In this study sHLA shed by cultivated human cells and their biochemical nature in solution were studied. EBV-transformed human B-lymphoblastoid cell lines (n = 4), permanent human lymphoblastoid tumor cell lines (n = 4), and PBLs from three donors were cultivated in vitro and sHLA measured in the supernatants. The Daudi cell line was used as a negative control in all experiments. Maximum expression of sHLA was measured after 8 hours, after which the concentrations gradually declined. The allospecificities A2 and B7 were also detectable in the ELISA. sHLA in the supernatants was further characterized by 1D-IEF. All bands representing the allotypes were detected, showing that cell supernatants can be used as antigen sources for biochemical tissue typing. These data show that sHLA expression is a characteristic of viable cells.

Covalent conjugates of monoclonal antibody and cobra venom factor mediate specific cytotoxicity via alternative pathway of human complement activation.

Because of lack of unspecific toxicity autologous serum is the optimal source of complement for in vitro cell depletion in human bone marrow transplantation. Covalent binding of cobra venom factor to an antibody leads to a specific complement activating conjugate with cytolytic potential independent of the capacity of the antibody to fix complement itself. Therefore this conjugation allows the use of various sera, including human as the source of complement. The kinetics of cobra venom factor-initiated cytolysis is compatible with in vitro treatment of bone marrow prior to transplantation.

Comparison of gamma-interferon and alloantigen-induced T cell factors in the induction of MHC class II antigen expression and in the modulation of the immunogenicity of lymphocyte-free rat bone marrow.

In a rat model, we compared the effects of various soluble products released by T cells, such as the unspecifically acting gamma-interferon or the newly detected alloantigen-induced factors that specifically act on nonlymphoid, hemopoietic bone marrow cells. We found two types of reactivity patterns with regard to the induction of MHC class II antigen expression on these cells. The very same patterns could be demonstrated when we investigated the modulation of their stimulatory capacity, i.e., their immunogenicity in a T proliferation assay. These findings are discussed in relation to the increasing incidence of immunologically mediated graft rejections in clinical bone marrow transplantation following T cell purging.

Porcine valves are reendothelialized by human recipient endothelium in vivo.

The degeneration of human allogeneic and porcine xenogeneic heart valves has not been clearly understood. The question is whether the observed loss of function and calcification is primarily an immunologic process or a mechanical process or is influenced by both factors. In the current study, we looked at explanted xenogeneic heart valves for the presence of recipient endothelium. Explanted valves were shock frozen and stored at -80 degrees C before use. They were subsequently examined by immunohistochemical staining with a variety of monoclonal antibodies. Xenogeneic valves showed clearly positive results for the human major histocompatibility complex class I and class II antigens and morphologically showed a thin layer of viable endothelium restricted to the annular region of the valve. Additionally, they were also positive for intercellular adhesion molecule-1 and the H-Y antigen. Although the xenogeneic valves were significantly degenerated, the endothelium was clearly defined and could be identified immunohistochemically as being of recipient origin. The grafts remained negative for endothelial cell-leukocyte adhesion molecule-1 and factor VIII. These data allow speculation on whether reendothelialization of valvular grafts with recipient endothelium is a normal repair mechanism in vivo.

Allogeneic mouse T cell lines distinguish three Balb/c leukemias from each other and from non-leukemic lymphocytes.

In order to separate graft-versus-leukemia (GVL) effect from graft-versus-host (GVH) reactions in allogeneic cell therapy, we established cytotoxic T cell lines (CTL) against irradiated A20, WEHI-3 and PU cells, ie cells from 3 hematopoietic tumors of Balb/c origin. Immunization with these cell lines did not lead to prolonged survival in Balb/c mice. The cytotoxic activity of the CTL was tested against the 3 leukemias. In all 9 combinations the highest specific lysis was achieved against the stimulating tumor. Cold target inhibition experiments showed that the activity against 51Cr-labelled leukemia cells could be almost completely inhibited by adding a sufficient amount of unlabelled target cells. On the other hand, when unlabelled concanavalin A-induced Balb/c blast cells were added, the inhibition was incomplete. By means of flow cytometry it was excluded that the different susceptibilities and inhibitory potentials of tumor cells and nonmalignant blasts are caused solely by differences in the MHC expression of the target cells. These findings confirm that allogeneic CTL are capable of discriminating malignant from nonmalignant cells, even if the tumor is nonimmunogenic in syngeneic animals.