The use of chromosomal satellites as donor cell markers in human bone-marrow transplantation is described. The recipient was a 17 year old boy with aplastic anemia, an HLA-A, B, C and D as well as AB0 and Rh compatible brother served as the donor. No antigenic markers could be recognized. Donor cells had two distinct satellites on one of the chromosomes No. 21, while no such satellites were found in the recipient cells. After transplantation the recipient's bone marrow was replaced with cells containing the satellites, and the satellite marker was also found in PHA stimulated peripheral lymphocytes. This finding provided direct evidence of successful engraftment.

Using antisera against the alpha, the beta or the complex of both chains of HLA-DR antigens, we have studied the role of individual chains of HLA-DR antigens in activation of T cells in autologous mixed lymphocyte reaction (AMLR). Alpha chain-specific antibody, not anti-beta chain serum prevented T cells from acquiring responsiveness to interleukin-2 (IL-2), suppressed the production of 1L-2, and inhibited the T cell proliferative response in both primary and secondary AMLR cultures. However, proliferation of already activated IL-2 reactive T cells supported by IL-2 was not affected by any of the four different types of anti-DR sera used. Fifty to sixty percent of T cells activated by AMLR or by PHA possessed DR antigens and functioned well as stimulator cells in secondary AMLR cultures. Moreover, the stimulatory activity of these DR-positive T cells was suppressed by the anti-alpha chain, not by the beta chain-specific antibody. Since continuous proliferation of T cells requires IL-2 and since nonactivated T cells are not sensitive to IL-2 and are unable to absorb this growth factor, we conclude the following: (1) The alpha, not the beta chain of HLA-DR antigens seems to be the structure responsible for enabling resting T cells to respond to IL-2 and induce production of IL-2 in AMLR. (2) Once T cells have acquired responsiveness to IL-2 and the growth factor has been produced, there is no further requirement for HLA-DR antigens, but the availability of IL-2 determines the level and extent of proliferation of IL-2 sensitive T cells.

The central role of immune lymphoid cells in the manifestation of diverse im-munological reactions in delayed hypersensitivity, transplantation, and autoim-munity is well established, but the mechanism by which the sensitized cells exert their effect is still largely unknown. The introduction of tissue culture methods represents a major advance in this area, and cell-mediated immunity against histoin-compatible target cells can now be demonstrated by the capacity of specifically sensitized lymphoid cells to kill normal or neoplastic target cells in vitro (1-8). Cytotoxicity by sensitized cells fulfils requirements for immunological specificity (9), is preceded as a rule by aggregation of the lymphoid cells around the target cells, and is abolished if the two cell types are separated by impermeable diffusion membranes (8). However, not only specifically sensitized cells but normal allogeneic (though not syngeneic) cells were also found to kill H-2 incompatible target cells of mouse origin in vitro (10-12) if heterologous antibodies or phytohemagglutinin was present in the medium. Although this reaction may represent the induction of a primary immune response in vitro, this seemed less probable since lymphoid cells, which would be genetically unable to react immunologically against the targets, were still capable of mediating target cell death (11). The only variable that determined cytotoxicity in this case seemed to be the presence of foreign histocompatibility antigens on the lymphoid cells. Presumably, target cells were damaged by dose confrontation with cells carrying incompatible surface isoantigenic determinants. An analogous concept was also put forward by HellstrSm et al. (13) as an explanation of the phenomenon of reduced tumor growth in semisyngeneic F1 hybrid mice as compared to tumor growth in syngeneic hosts (14). The reaction was termed "allogeneic inhibition". In the present study, attempts have been made to test the validity of the above hypothesis concerning contact-induced cytotoxicity by incompatible lymphoid cells. Interest has been directed to the demonstration of cytotoxocity

We have monitored the humoral immune responses of 10 type I diabetic patients, xenotransplanted with fetal porcine islet-like cell clusters for up to 8 years after xenotransplantation. We investigated the immunoglobulin subclass distribution as well as specificity differences of xenoreactive antibodies. Hemagglutintion tests, using pig erythrocytes, showed that some patients maintained higher titers of xenoreactive IgM antibodies during the entire follow up period, compared with pretransplant levels. In microcytotoxicity tests all but one patient tested showed higher than pretransplant levels of cytotoxic antibodies against pig peripheral blood mononuclear cells (PBMC) 6-8 years after transplantation. Levels of Gal alpha 1,3Gal specific antibodies, were also high. Antibody dependent cellular cytotoxicity (ADCC) activity against a Gal alpha 1,3Gal expressing human B cell line was detected in four patients while ADCC reactivity against adult pig islet cells was detected in only two patients, 6-8 years after transplantation. Immune sera collected 30 days and 1 year after transplantation showed positive staining of adult pig islet cells in fluoromicroscopy whereas sera from later time points did not. Western blot experiments showed that some patients had IgG1 antibodies reactive against epitopes on pig cells other than Gal alpha 1,3Gal, while xenoreactive IgM and IgG2 antibodies mainly reacted with Gal alpha 1,3Gal-containing epitopes as shown by absorption experiments. These results show that patients continue to produce higher than pretransplant levels of IgM and IgG2 xenospecific antibodies against Gal alpha 1,3Gal for extended time periods following xenotransplantation. Some patients also produce xenoreactive IgG1 antibodies directed against non-Gal alpha 1,3Gal epitopes.

Immune complexes from the synovial fluid of rheumatoid arthritis (RA) patients, or artificial rheumatoid factors (RF)‐like (antibody–antibody) immune complexes, induce a remarkably intense, sustained and selective immunoglobulin (Ig)G1 response under certain experimental conditions in mice. Because the IgG1 antibody response is extraordinarily strong, the role of interleukin (IL)‐4, important for IgG1 synthesis, was investigated. Both C57BL/6 and NZB IL‐4‐deficient mice produced IgG1‐RF antibodies after injection with RF‐like immune complexes, although the antibody levels were slightly delayed compared to wild type mice. This shows that IL‐4 is not obligatory in RF‐like immune complex induced IgG1‐RF production. A discrepancy in the decline of serum IgG1–RF was noted between NZB and C57Bl/6 mice. Serum IgG1–RF declined 43 days postinjection (p.i.), in C57BL/6 mice whereas high serum levels of IgG1–RF were maintained more than 100 days in the NZB mice, indicating different regulatory mechanisms in these mice. To study if the affinity for mouse IgG increased with time in NZB mice and thus become more directed against self, the cross‐reactivity of the IgG1–RF antibodies with IgG from other species was investigated early and late after injection. It was, however, found that the cross‐reactivity with IgG of human, goat and rabbit origin did not change between the two time points.

Rheumatoid factor (RF)‐like (antibody‐antibody) immune complexes induce a selective and intensive immunoglobulin (Ig)G1‐RF response after a single injection in mice. However, the longevity of the response differs between mouse strains: serum IgG1‐RF antibody titres decline 40 days after injection in C57Bl/6 mice whereas levels are maintained for more than 100 days in NZB mice. In order to elucidate whether this difference was owing to a lower ability of NZB mice to clear the injected immune complexes, sections of kidney, spleen, liver and mesenteric lymph nodes were harvested at different time points after injection with RF‐like immune complexes. Immunohistochemical staining revealed that NZB mice have a delayed clearance of the injected immune complexes, because the immune complexes are retained for more than 40 days in their spleens and 100 days in their kidneys, compared to only 14 days in C57Bl/6 mice. Germinal centres were also present for a longer period in the spleens of the NZB mice, accompanying the presence of the immune complexes, and were abnormally large compared to C57Bl/6 mice. The clearance of immune complexes from the spleen coincided with the decline in serum levels of IgG1‐RF, indicating that prolonged retention of immune complexes is responsible for the sustained IgG1‐RF response.

Mercuric chloride (HgCl2) has been proposed to be a mitogen for human blood lymphocytes in vitro. In our previous study, we demonstrated that HgCl2 preferentially stimulates the CD4+ T cell subset to blast transformation and DNA synthesis and that the reaction is dependent on CD14+ accessory cells. In order to characterise the responding cells further and to elucidate the mechanism of T cell activation, the T cell receptor (TCR) Vβ chain expression of the blast–transformed cells was analysed by monoclonal antibodies and flow cytometry. The 22 TCR–Vβ–specific antibodies used were found to react with 55–80% of the naive CD4+ and CD8+ blood T cells from the different donors. Six to 18% of the lymphocytes, mainly CD4+ T cells, were blast transformed after addition of HgCl2. The distribution of the lymphoblasts carrying certain TCR Vβ chains were skewed, and 15–40% expressed the TCR Vβ2 chain. Furthermore, if cells were pretreated for 5 days with HgCl2, whereafter recombinant interleukin–2 in fresh medium was added, the TCR Vβ7+ T cell subset was also stimulated to blast transformation. The superantigen staphyloccal enterotoxin B, as a control, induced blast transformation in 10–26% of the lymphocytes, mainly CD4+ T cells, which were, as expected, positive for Vβ3, Vβ12, Vβ14 or Vβ17. We conclude that HgCl2 has characteristics of a superantigen, activating the human lymphocytes in a Vβ–chain–selective manner in vitro.

Blood lymphocytes from 20 patients with oral contact lesions to dental amalgam and 10 healthy individuals were analyzed for HgCl2-induced proliferation in vitro, using both a modified assay and a conventional assay. The release of interferon-gamma (IFN-gamma) was measured in cell supernatants. Six patients displayed positive reactions in patch tests to mercuric compounds. No significant differences were recorded in HgCl2-induced proliferation in cells from patients and controls, since only few in the whole material responded to submitogenic concentrations. IFN-gamma was detectable in cell supernatants from some patients but also from controls and is not predictive of mercury allergy. Neither the phenotypes of peripheral lymphocyte subsets, the frequency of circulating cells expressing the interleukin-2 (IL-2) receptor, spontaneous lymphocyte proliferation nor concentrations of serum interleukin-6 differed between patient and control samples. In contrast to what has been claimed before, we did not find any evidence for specific in vitro lymphocyte reactivity in patients with oral contact lesions.

HLA-specific humoral immunity, as a result of recipient allosensitization, induces hyperacute rejection of allogeneic kidney grafts. Cross-match tests are performed to avoid this complication. However, present techniques do not allow determination of HLA specificity of donor-reactive antibodies in the acute necro-donor situation. New methods are described and discussed in this communication as well as the alloantibody specificities which are of clinical importance. Alloantibodies not only mediate hyperacute rejection, but may also participate in the acute rejection of organ grafts. Clinical associations between early immunological complications, such as acute rejection, in heart, liver and kidney allografted patients and pre-transplantation humoral alloimmunity emphasize the need for proper determination of donor-specific humoral immunity prior to transplantation. Acute rejection episodes are associated with an increased risk of subsequent chronic rejection. Cytomegalovirus (CMV) infection is also an important risk factor for chronic organ graft rejection as well as for chronic graft-versus-host (GvH) disease in bone marrow transplanted patients. CMV infection triggers autoantibody formation against CD13 in transplanted patients which, in turn, has been shown to be associated with the development of chronic GvH disease. Recently, alloactivation of immune reactivity in vitro was found to induce reactivation of latent CMV infection. We present here a hypothesis to explain the series of clinically associated events: acute rejection--CMV infection--chronic rejection.

Rheumatoid factors (RF) are autoantibodies with specificity for the constant regions of IgG molecules. They are found in several immunopathological diseases. The mechanism(s) by which these autoantibodies are produced is largely unknown. We have previously shown that a single injection of RF-like immune complexes (ICs) into mice selectively induced an intense IgG1-antibody production with RF activity. This response was sustained for several months and did not resemble a conventional immune response to an antigen or other immune complexes. In the present study, we sought to elucidate the mechanism for the IgG1 RF response to RF-like ICs. Therefore, the roles of CD4+T cells, complement and Fcγ receptors were analysed. In order to characterize the role of CD4+T cells, RF-like induced IgG1-RF production was analysed in NZB mice treated with a monoclonal antibody (mAb) against the CD4 molecule, which resulted in complete abrogation of IgG1 RF production. To evaluate the importance of FcγRs, the effect of RF-like ICs was tested in mice deficient for RFγRI/III. A significant decrease in the numbers of IgG1 antibody secreting cells, as well as in serum IgG1 RF levels, was found in the deficient mice, as compared with their normal outbred littermates. The role of complement in RF-like ICs mediated IgG1 RF was tested in complement depleted NZB mice, using Cobra venom factor. The IgG1 RF response in complement depleted and intact mice was comparable. Thus, our results demonstrate that RF-like immune complexes selectively induce an FcγR-dependent, complement independent antibody response in mice.

The triggering or immunogenic stimulus for human autoimmune diseases is unknown. It is not even known whether the stimulus is endogenous, i.e. truly “self” or exogenous, “non‐self”. Many autoimmune diseases are human lymphocyte antigen (HLA)‐associated and demonstrate linkage to the HLA chromosome in family investigations. For most of these diseases, evidence is strong that the association is directly dependent on specific HLA class I or II molecules rather than on other genes located in the HLA region. Since HLA polymorphic HLA molecules have so far only been shown to have two distinct functions, both of which are immunological, a HLA association supports the notion that a particular disease is autoimmune. Furthermore, an association to a specific HLA allele implies that the immunogenic stimulus for autoimmunity would be one specific HLA‐binding peptide and that, at least initially, autoimmunity is dependent on the reactivity of one or a limited number of potentially autoaggressive T cell clones. These findings are encouraging and form the basis for future preventive measures. One current theory is that autoimmune disease is precipitated by an environmental agent, such as a viral infection. Several different mechanisms to explain how a viral infection could induce autoimmune disease in humans are described and one specific example is presented for a virus‐induced autoimmune disease in humans. The question of whether ITP could also be dependent on such a mechanism is briefly discussed.

The synovial fluid (SF) of rheumatoid arthritis (RA) patients contains a mixture of inflammatory mediators. In order to determine whether certain cytokine patterns locally in the joint are specifically related to the chronic inflammation in RA, the concentrations of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, transforming growth factor-beta (TGF-beta), tumour necrosis factor-alpha (TNF-alpha) and IgG2b-inducing factor (IgG2bIF) were measured in SF from 22 patients with RA and 22 patients with other types of arthritic lesions. High levels of IL-10, latent and active TGF-beta and the presence of IgG2bIF are significantly correlated with RA when corrected for age. As these factors have the capacity to promote antibody production, they might contribute to the maintenance of local antibody production in RA synovial tissues. All RA-SF samples contained detectable levels of IL-10 and all except one contained IL-1beta, while concentrations in several non-RA-SF samples were below detection limits. IL-6 and TGF-beta were present in all SF samples from both RA and non-RA patients. The presence of IgG2bIF was strongly correlated with high levels of IL-10 and IL-1beta in SF. However, no distinct cytokine profile specific for the chronic inflammation characteristic of RA was found.

In vitro studies of human NK cell-mediated cytotoxicity and ADCC against porcine target cells were performed. Stimulation of human PBMC responder cells with either allogeneic or xenogeneic porcine cells led to a marked increase in NK cell reactivity, Maximum reactivity was reached following 3-6 days of in vitro culture. The sensitivity of target cells ranked as follows: K562 > porcine PHA-induced lymphoblasts > resting porcine PBMC. Limiting dilution analysis showed that allo-and xeno-stimulation in vitro led to differentiation of similar frequencies of effector NK cells. Split culture experiments showed that single NK effector cells were cytotoxic against both K562 and porcine lymphoblasts, demonstrating that individual NK cells lack species specificity. NK effector cell generation stimulated by xenogeneic cells was cyclosporin A (CsA) sensitive and dependent on the presence of autologous responder T lymphocytes, a dependence that was completely reconstituted by the sole addition of human IL-2. Xenostimulation of enriched CD3+ cells also led to a preferential appearance of CD16+ or CD56+ lymphoblasts. Natural xenoreactive human anti-porcine antibodies are mainly of IgM and IgG2 subclasses, but antibodies in xenoimmunised patients reactive against porcine lymphocytes and fetal porcine islet cells were also of IgG1 and IgG3 subclasses. The same subclass distribution was found among antibodies specific for gal alpha 1,3 gal epitopes as shown by tests performed with alpha 1,3 galactosyltransferase-transfected Raji cells (human Burkitt lymphoma cells). Natural antibodies did not mediate ADCC, whereas gal alpha 1,3 gal-specific antibodies in sera from xenoimmunised patients did. Fetal porcine islet cells were sensitive to human NK cell-mediated cytotoxicity and to ADCC mediated by xenoimmune sera.

Patients with rheumatoid arthritis (RA) produce a variety of autoantibodies, not only demonstrable in the circulation, but also locally in the inflamed joint. We investigated the local production of several autoantibodies in the synovial fluid (SF) of 24 patients with RA and of 26 patients with other arthritic lesions. RA patients had higher titres of immunoglobulin M (IgM) and immunoglobulin G (IgG) rheumatoid factors (RFs) and of collagen type II antibodies in SF, whereas there were no demonstrable differences between groups with regard to antibodies against double-stranded (ds) DNA, C1q or the hapten 2,4,6-trinitrobenzene sulfonic acid (TNP). No differences were observed for total synovial levels of IgM or IgG. There was no autoantibody pattern that was typical of RA patients, except for the local presence of RF, primarily in seropositive RA patients. Our findings therefore support the notion that RF and collagen type II antibodies are induced by immunogenic material present in the local inflamed environment. In the accompanying paper we studied various synovial fluid cytokines in the same patient groups. Here we correlated the level of these cytokines with autoantibody titres in SF, but no specific cytokine associated with the production of RF was found. Hence, we conclude that several different inflammatory mediators might contribute to the chronic inflammation and autoantibody production in the joint of RA patients. An inverse correlation was established between concentrations of tumour necrosis factor-alpha (TNF-alpha) and levels of total IgG.

Rheumatoid arthritis synovial fluid (RA-SF) contains a factor that induces IgG2b antibody production in LPS-stimulated murine B cells and therefore is called IgG2b inducing factor (IgG2bIF). When LPS, together with crude RA-SF or semi-purified IgG2bIF, was added to highly purified LPS-stimulated B cells, the number of IgG2b-producing cells was substantially enhanced. This shows that IgG2bIF acts directly on activated B cells, presumably by binding to a receptor expressed on LPS-activated B cells. In vivo LPS-activated B blasts were not able to respond to RA-SF unless LPS was present in vitro, showing that LPS is needed to maintain cell viability and responsiveness to the IgG2bIF. To elucidate the mechanism for the IgG2bIF effect on highly purified B cells, the IgG2b response of LPS-stimulated, Bruton's tyrosine kinase-defective, xidB blasts was studied. Purified B blasts from the btk-defective CBA/N mouse strain were sensitive to IgG2bIF. These findings show that IgG2bIF acts directly on B cells and activates cells through a btk-independent pathway.

The immunological rejection of HLA-identical kidney transplants indicates that non-HLA antigens may also be targets for transplant rejection. Interest in the possible role of endothelial specific antigens has grown steadily over the years. Most of the studies published, regarding the association of such antibodies with rejection, have demonstrated the reactivity of endothelial antibodies also with monocytes and keratinocytes, but not with lymphocytes. Such antibodies escape detection in conventional crossmatch tests.In this paper, we present a case report of a 10-year-old girl, whose two consecutive kidney allografts, (one living and one cadaveric donor) were hyperacutely rejected in spite of the fact that she had neither been alloimmunized, nor had any HLA-specific antibodies. Endothelial cell specific antibodies were detected in vivo and in vitro after transplantation only 11 days apart, which were considered to be responsible for rejection. The third cadaveric kidney was lost within 1 week post-transplant. Immunopathological investigation of the three rejected grafts revealed deposition of IgM in the endothelium of arteries and in some glomeruli. No deposition of IgG antibodies was found. Antibodies from this patient did not react with lymphocytes, monocytes or keratinocytes. Patient serum had IgM antibodies that were specifically reactive with cultured endothelial cells, demonstrated by binding in vitro and by complement-dependent cytotoxicity of IL-P stimulated endothelial cells. No HLA antibodies were found following the first two transplantations, but were demonstrated 1 week after the third transplantation, at the time of an acute irreversible rejection. Western blots of proteins solubilized from endothelial cell membranes, indicated that the antibodies reacted with a 97-110 kD protein. Endothelial cell antigen preparations were made from several different umbilical cord veins. Some primary cell cultures, but not all, reacted with the patient's serum. Therefore, we suggest that the target determinant might be polymorphic.These findings imply that the non-HLA endothelial cell specific molecules may function as target(s) for hyperacute antibody-mediated destruction of kidney allografts.