Non-cytotoxic Antibodies Research Articles

Occurrence of Fatal and Nonfatal Adverse Outcomes after Heart Transplantation in Patients with Pretransplant Noncytotoxic HLA Antibodies.

HLA antibodies (HLA ab) in transplant candidates have been associated with poor outcome. However, clinical relevance of noncytotoxic antibodies after heart transplant (HT) is controversial. By using a Luminex-based HLA screening, we retested pretransplant sera from HT recipients testing negative for cytotoxic HLA ab and for prospective crossmatch. Out of the 173 consecutive patients assayed (52 ± 13y; 16% females; 47% ischemic etiology), 32 (18%) showed pretransplant HLA ab, and 12 (7%) tested positive against both class I and class II HLA. Recipients with any HLA ab had poorer survival than those without (65 ± 9 versus 82 ± 3%; P = 0.02), accounting for a doubled independent mortality risk (P = 0.04). In addition, HLA-ab detection was associated with increased prevalence of early graft failure (35 versus 15%; P = 0.05) and late cellular rejection (29 versus 11%; P = 0.03). Of the subgroup of 37 patients suspected for antibody mediated rejection (AMR), the 9 with pretransplant HLA ab were more likely to display pathological AMR grade 2 (P = 0.04). By an inexpensive, luminex-based, HLA-screening assay, we were able to detect non-cytotoxic HLA ab predicting fatal and nonfatal adverse outcomes after heart transplant. Allocation strategies and desensitization protocols need to be developed and prospectively tested in these patients.

298 Pre-Transplant Non-Cytotoxic Anti-HLA Antibodies Predict Multiple Adverse Events after Heart Transplantation

298 Pre-Transplant Non-Cytotoxic Anti-HLA Antibodies Predict Multiple Adverse Events after Heart Transplantation

59-P: Cytotoxic and Non-Cytotoxic Anti-HLA Antibodies Identified in Lung Transplant Recipients

59-P: Cytotoxic and Non-Cytotoxic Anti-HLA Antibodies Identified in Lung Transplant Recipients

Intact HLA Not β2m-free Heavy Chain-Specific HLA Class I Antibodies Are Predictive of Graft Failure

We investigated the effects of intact and beta2m-free heavy chain (HC)-specific human leukocyte antigen (HLA) class I antibodies on long-term graft survival. HLA class I mixed antigen beads were used to detect intact and beta2m-free HC-specific antibodies, whereas elution buffer-treated beads were used to detect antibodies against beta2m-free HC. Donor-specific antibodies (DSAs) were identified using single-antigen beads. Complement-dependent cytotoxicity assays were performed to determine the cytotoxicity of DSA. Three hundred seventy-nine of 994 of patients (38%) had antibodies against intact HLA and beta2m-free HC. There was no survival rate difference between antibody-positive and -negative groups. When the 379 antibody-positive patients were further tested with beta2m-free HC-coated beads, 179 of them with antibodies only against intact form of antigens had a 4-year graft survival rate of 76%, which is significantly lower than that of 200 patients with antibodies against beta2m-free HC of HLA antigens (88%, P=0.0056). Patients with intact antigen specific DSAs had a significantly lower graft survival rate as compared with those with no DSAs (70% vs. 89%, P=0.0073). More patients with strong donor-specific cytotoxic antibodies lost allografts than those with weak-cytotoxic or noncytotoxic antibodies. However, cytotoxic activity of DSA was not correlated to antibody level. We concluded that intact antigen-specific antibodies, especially DSAs, are predictive of graft failure. DSAs were not always cytotoxic. Strong cytotoxic activity of DSA was associated with a higher rate of graft loss but not correlated to the antibody level. Antibodies against beta2m-free HC negatively interfere with the predictive value of intact antigen-specific antibodies.

Progesterone and the immunology of pregnancy

The foetal–placental unit is a semi-allograft and the immunological recognition of pregnancy, together with the subsequent response of the maternal immune system, is necessary for a successful pregnancy. This recognition of pregnancy results in an upregulation of progesterone receptors on activated lymphocytes amongst placental cells and decidual CD56+ cells. In the presence of sufficient progesterone, these cells synthesise progesterone induced blocking factor (PIBF), a mediator that exerts substantial anti-abortive activities. PIBF affects B cells and induces an increased production of asymmetric, non-cytotoxic antibodies. It also alters the profile of cytokine secretion by activated lymphocytes resulting in an increase in the production of non-inflammatory, non-cytotoxic interleukins (IL) (e.g. IL-3, IL-4 and IL-10) and a reduction in the production of inflammatory, cytotoxic cytokines (e.g. interferon (IFN)-δ, tumour necrosis factor (TNF)-α and IL-2). PIBF also inhibits the cytotoxity of natural killer (NK) cells by blocking their degranulation and perforin release, as well as inhibiting IFN-δ, TNF-α and IL-2-mediated transformation of NK cells into detrimental lymphokine activated killer (LAK) cells.

Laboratory Diagnostics of HLA Antibodies

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Clinical relevance of antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test.

Recent literary data suggest that antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test may cause not only chronic, but also acute immunological complications after kidney transplantation. The aim of this study was to investigate the significance of non-cytotoxic antibodies to HLA antigens for the development of immunological complications and a worse graft prognosis after first kidney transplantation. Sera before and early after transplantation from 120 first kidney recipients were analyzed by flow cytometry (FCXM), ELISA and the standard complement-dependent cytotoxicity (CDC) test. Pre-transplant FCXM negativity was related to a lower incidence of rejection episodes in the first post-transplant year ( P<0.01). A significant association between acute rejection and the presence of antibodies to HLA class II antigens before and after transplantation was also found ( P<0.05). Our study supports the findings of other centers of the detrimental role to the kidney graft played by anti-HLA antibodies undetectable by the classical CDC test.

The human bone marrow as an immunoregulatory organ.

It was 45 years ago that in a virtual revolution in thinking in immunology there developed the acceptance and the subsequent expansion of two new dogmas: (1) that to eliminate toxins and pathogens as the major mode of defense, individual immune cells were, in their ontogeny of differentiation, internally programmed to react singly and then clonally against the virtually limitless individual stimuli of the outside world (1–3); (2) that before this programming was manifested the immune system would fail to recognize any antigenic stimulus as foreign, thereby not differentiating non-self from self-recognition. This allowed for non-self-antigens, if introduced in this early stage, to be immunologically tolerated on subsequent testing. In the chronology of the evolution of these two dogmas, there were the earlier descriptions of specific immunological paralysis and unresponsiveness to certain defined polysaccharides and haptens demonstrated in adult mice and guinea pigs respectively by Felton (4) and Chase (5). It remained for Medawar and his colleagues (6) in allotransplantation experiments to clearly define “acquired specific immunological tolerance” as an ontogenic concept involving the lack of maturation or differentiation were tolerance to be evoked. As early as 1953, Billingham, Brent, and Medawar demonstrated that allogeneic donor bone marrow-derived cells could confer such a specifically acquired tolerant state to the immune system of the murine recipient before self versus non-self recognition occurred in immune ontogeny, the test being subsequent acceptance of skin allografts from the same donors. The developmental processes of positive versus negative selection only a decade later began to be demonstrated to predominantly involve the thymus gland in maturational events of cells of the recipient immune system in studies first performed in neonatal rodents (7). By experimental manipulation of the immune system predominantly involving immunoablation by whole body x-irradiation, these findings of specific immunological tolerance as a result of the infusion of bone marrow-derived cells could be extended to adult animals as the system regenerated from stem cells in the bone marrow of either that of the donor (8), the recipient (9), or both (10). Nonetheless, except in the setting of clinical bone marrow transplantation using major (lethal) immunoablation followed by the salvage and replacement of the recipient immunohematopoietic system with donor cell lineages and only in the context of little or no donor-recipient MHC disparity, with few exceptions (11–15), to date, specific immunological tolerance in organ transplantation in humans has not been possible.

Role of antibodies in rejection

Recent evidence suggests that antibody plays a role in the pathogenesis of both acute rejection and accelerated coronary artery disease following human cardiac transplantation. There is strong evidence that posttransplant monitoring identifies patients at risk of developing transplant-associated coronary artery disease. That antibody is either essential for or influences the nature of the vascular lesion is supported by experimental models of cardiac graft vasculopathy. One of the major concerns in this area is that the specificities of antibodies (formed in patients) remain poorly defined. Major histocompatibility and non–major histocompatibility target antigens have been described. Traditionally, cytotoxic antibodies have been detected, but newer methods of detecting noncytotoxic antibodies and use of endothelial cells as targets should expand and clarify the range of antibodies being produced. The mechanism whereby antibody causes vascular lesions is not understood but almost certainly involves activation of donor endothelial cells. Indirect presentation of graft antigens (including non-HLA or minor antigens) and epitope spreading drive chronic rejection; such an expansion of the immune response probably includes antibody production against many different HLA and non-HLA antigens.

Rapamycin inhibits production of cytotoxic but not noncytotoxic antibodies and preferentially activates T helper 2 cells that mediate long-term survival of heart allografts in rats.

Rapamycin (RAPA) induces unresponsiveness toward heart allografts by at least two mechanisms: selective production of noncytotoxic IgG2c-blocking Ab and preferential activation of Th2 cells. RAPA (0.8 mg/kg/day) delivered via a 14-day osmotic pump to Wistar Furth (WF; RT-1u) recipients prolongs Buffalo (BUF; RT-1b) heart allograft survival from a mean survival time (MST) of 6.5 +/- 0.5 days to 75.0 +/- 18.9 days (n = 18; p < 0.001), with 6 of 18 grafts beating for more than 100 days. Recipient sera or their IgG but not IgM fraction, obtained after postgrafting day 40, passively transfer the unresponsive state to sublethally irradiated secondary recipients in a dose-dependent and immunologically-specific fashion. Sera obtained after untreated WF hosts rejected BUF hearts contained IgG moieties of all subclasses that bound to class I MHC BUF epitopes. In contrast, the unresponsive sera contained predominantly non-C'-fixing IgG2c and only marginal amounts of activated (C') fixing IgG1, IgG2a, and IgG2b Ab. The transcription of IL-2, IL-4, and IL-10 mRNAs was assessed using a PCR method. There were similar increases in the levels of IL-2, IL-4, and IL-10 mRNA in heart allografts from both untreated and RAPA-treated recipients on day 5 postgrafting. In contrast, on days 60 and 300 postgrafting heart allografts from RAPA-treated unresponsive recipients showed increased levels of IL-10 and IL-4 but not of IL-2 mRNA, suggesting preferential activation of Th2 cells. Thus, RAPA treatment selectively inhibits the synthesis of C'-binding of IgG subclasses, spares the non C-binding blocking IgG2c Ab, and preferentially activates Th2 cells.

Preoperative detection of IGG non-cytotoxic antibodies identifies primary renal transplant recipients at risk for early acute rejection and graft loss

Preoperative detection of IGG non-cytotoxic antibodies identifies primary renal transplant recipients at risk for early acute rejection and graft loss

Cytotoxic flow-cytometric crossmatches (flow-tox): A comparison with conventional cytotoxicity crossmatch techniques

Detection and avoidance of donor-reactive antibodies in the sera of potential organ transplant recipients is key to a successful transplant outcome. Techniques of antibody detection that use flow cytometry are more sensitive than those that rely upon a visual determination of cytotoxicity. However, as conventionally performed, flow-cytometric crossmatches do not distinguish between cytotoxic (complement fixing) and noncytotoxic antibodies because both types of antibodies can bind to a cell and be detected by laser-activated fluorochrome photon emission. In 1989 we described two techniques for detecting cytotoxic antibodies using flow-cytometric techniques [1]. In 1990, we expanded the application of these new techniques that we called flow cytotoxicity assays or “Flow-Tox” [2]. Flow-Tox crossmatches demonstrate an increase in both sensitivity and specificity over conventional cytotoxicity crossmatches.

Non-cytotoxic blocking antibodies and suppressor cells induced by donor-specific transfusions in healthy volunteers and potential kidney transplant recipients

Donor-specific transfusion (DST)-induced immunosuppression plays a significant role in clinical and experimental transplantation. To clarify the mechanism of suppression on alloreactivity the suppressor cell induction and the non-cytotoxic blocking antibody production and importance was studied in 15 healthy volunteers and 3 kidney transplant recipients (KTR) after DST on mixed lymphocyte culture (MLC). Significant decrease of anti-donor MLC response was found in all of KTR and in 12 cases of the 15 transfused volunteers. Of 18 cases, 15 had blocking factors in their post-DST serum which strongly (52-91%) inhibited the MLC response. The IgG fractions isolated from the "blocking sera" were responsible for this inhibition. Eighty-one percent of non-cytotoxic blocking antibodies affected the responder cells in MLC and reacted with third party responder cells as well. Both buffy coat and platelet transfusions evoked production of the non-specific blocking antibodies. Our data strongly suggest that DST induces not only the differentiation of suppressor cells and production of anti-idiotypic antibodies, but also the appearance of non-specific, non-cytotoxic antibodies, which may participate in the cell-mediated immune suppression of the alloimmune reactivity.

Complement killing of human neuroblastoma cells: A cytotoxic monoclonal antibody and its F(ab)' 2-cobra venom factor conjugate are equally cytotoxic

Only a few monoclonal antibodies mediate complement lysis of tumor cells, but for several antibodies it has been demonstrated that a complement-activating function can be introduced by covalent coupling of cobra venom factor (CVF), a non-toxic glycoprotein which is a structural and functional homologue of human complement component C3. In this study we compared the efficacy of complement killing of human neuroblastoma cells by the complement-activating monoclonal antibody 3F8 directed against the G D2 ganglioside antigen with that of its F(ab') 2-CVF conjugate. At equal numbers bound per cell the 3F8 antibody and the 3F8 F(ab') 2-CVF conjugate were found to be equally cytotoxic in the presence of complement from several species including human. Maximal killing reached up to 98%. The kinetics of killing and the bivalent metal requirement confirmed that the cytotoxic activity of the 3F8 antibody is mediated via the classical pathway and that of the 3F8 F(ab') 2-CVF conjugate via the alternative pathway. To achieve a comparable degree of killing, an approximately eight-fold higher concentration of the 3F8 F(ab') 2-CVF conjugate was required which appears to be a consequence of the approximately eight-fold lower binding activity of the 3F8 F(ab') 2-CVF conjugate compared to the intact 3F8 antibody. Our data suggest that the coupling of CVF to non-cytotoxic antibodies allows the generation of conjugates with a cytotoxic activity similar to that of inherently cytotoxic antibodies.

Pregnancy-maintaining antibodies: Workshop report (Giessen, 1988)

To analyse the nature of antibodies which are purported to be essential for the maintenance of normal human pregnancy, six centers participated in a workshop of “blind” tests on 19 allosera. Fc-receptor dependent assays detected antibodies with specificity only for HLA. In addition to cytotoxic antibodies, the Fc-receptor dependent immune phagocytosis inhibition test revealed two non-cytotoxic alloantibodies with HLA specificity. These antibodies had high titers and may, therefore, be essentially non-cytotoxic. Murine monoclonal antibodies to HLA-A, B, C or DR (W6/32 and 2MC3) were used to evaluate the methods. These antibodies inhibited immune rosette formation as well as immune phagocytosis. Diluted to concentrations below the threshold of complement-dependent cytotoxicity, the monoclonal antibodies still inhibited the mixed lymphocyte reaction and the immune phagocytosis. A human monoclonal immunoglobulin M with specificity for monomorphic non-HLA lymphocyte antigens inhibited the mixed lymphocyte reaction. The immune rosette inhibition test exhibited several false positive reactions, e.g. three out of four with a serum that did not contain alloantibodies to blood cells. Non-cytotoxic antibodies were therefore rare in the selected sera of the workshop and they exhibited HLA specificity only. No participant was able to identify pregnancy-maintaining non-HLA-antibodies.

Renal transplantation: HLA-linked, non-cytotoxic antibodies develop after blood transfusion.

1. A previously untransfused dialysis patient was given blood from a single donor on three occasions and sera were obtained from the recipient before and after transfusion. 2. The sera were tested against B-lymphocytes from the blood donor and his family in the erythrocyte-antibody rosette inhibition assay to determine whether any Fc-receptor-blocking activity which developed was HLA linked. 3. Antibody activity was noted after the second transfusion and was directed toward B-lymphocytes from the blood donor and his family members sharing the haplotype HLA A11, B12 with him. 4. These antibodies were removed by the absorption of active sera with lymphocytes from the blood donor but not from (a) his sibling who shared no HLA antigens with him or (b) the transfusion recipient. Absorption of the active sera with platelets from the blood donor did not remove activity. 5. These results indicate that Fc-receptor-blocking antibodies developing after blood transfusion are directed to HLA-linked antigens on donor lymphocytes.

Non-cytotoxic antibodies in chronic lymphocytic leukaemia.

Non-cytotoxic Fc receptor blocking antibodies against autologous B lymphocytes were sought in sera from patients with chronic lymphocytic leukaemia (CLL), using a rosette inhibition assay. They were found in 11 of 52 (21%) of patients with CLL, but were not associated with previous blood transfusion or pregnancy, suggesting that they were unlikely to have resulted from allogeneic stimulation. Fc receptor blockade was more commonly detected in sera from patients with stage B rather than stage A CLL (Binet classification), though this did not achieve significance beyond the 90% level, and it was noted in 62.5% of those with lymphocyte doubling times of less than one year, compared with 36.3% of those whose lymphocyte doubling time was more than one year. The results indicate that autologous Fc receptor blocking antibody activity occurs in sera from patients with CLL, is more likely to be generated by the tumour itself than by allogeneic stimulation, and is associated with increased tumour load. Such antibodies may permit tolerance of tumour by the host.

The significance of antilymphocyte antibodies in patients with acquired immune deficiency syndrome (AIDS) and their sexual partners.

Antilymphocyte antibodies have been demonstrated in autoimmune diseases, acute viral infections, and acquired immune deficiency syndrome (AIDS) by using either the conventional microlymphocytotoxicity or the double fluorescence technique. In the present study, we used both methods to detect the antilymphocyte antibodies and to characterize further their immunologic significance in patients with AIDS and their sexual partners. The results using the conventional microlymphocytotoxicity method demonstrated that 8 of 10 patients with AIDS and 6 of 10 partners had significant levels of antilymphocyte antibodies which were reactive with B and T cells at cold and warm temperatures. A significant loss in antibody activity following absorption with B, T, and Daudi cells and Staphylococcus aureus, but not platelets or red cells, indicated that these antibodies are not directed to HLA class I antigens but, rather, to antigens that are common to both groups of lymphocytes. There is a close association between antilymphocyte antibodies and lymphopenia in patients but not in partners. Antibodies against lymphocyte subclasses [helper (T4) and suppressor (T8)] were detected by the double fluorescence staining technique, which employs C6-deficient serum as a nonlytic source of complement, and demonstrated the binding of antibodies to target cells, in contrast to lysing of the target cells as in the microlymphocytotoxicity method. The results of this assay showed that antibodies were directed to both populations, and there was no correlation or association between the absolute numbers of peripheral T4 and T8 cells and the percentage of antibody binding. Taken together, there appear to be at least two kinds of antilymphocyte antibodies: lymphocytotoxic antibodies detected by the conventional microlymphocytotoxicity assay and noncytotoxic antibodies detected by the double fluorescence staining technique. The former may be responsible in part for the lymphopenia. The latter may alter lymphocyte function. The patients and partners who had antilymphocyte antibodies also had anti-HTLV-III antibodies, although there was not any close correlation between titers. These findings support the possibility that both types of antibodies occur as part of a generalized immune response, possibly stimulated by the same viral agent.

EFFECT OF CYCLOSPORIN, PREVIOUS THIRD-PARTY TRANSFUSION, AND PREGNANCY ON ANTIBODY DEVELOPMENT AFTER DONOR-SPECIFIC TRANSFUSION BEFORE RENAL TRANSPLANTATION

Non-cytotoxic and cytotoxic antibodies were sought after donor-specific transfusion (DST) in 12 potential renal transplant recipients given concomitant cyclosporin therapy and 13 given DST alone. Non-cytotoxic antibodies, which have been shown to develop after third-party transfusion and to be associated with successful transplantation, developed after DST whether or not cyclosporin was given. Donor and panel reactive lymphocytotoxic antibodies developed relatively infrequently after DST with or without cyclosporin. Donor-specific sensitisation occurred only in patients who were multiparous or had over 10 third-party transfusions. Non-cytotoxic Fc-receptor-blocking antibodies may play a part in the improved survival of one-haplotype-mismatched transplants pretreated with DST.

Immune phagocytosis inhibition by commercial immunoglobulins.

Sixteen commercially available immunoglobulins (Ig) and 5 anti-Rho (D) hyperimmune globulins were investigated for immune phagocytosis inhibition (IPI) factors as well as for T, B lymphocytotoxic and monocytotoxic antibodies. All Ig contained IPI factors with lowest inhibitory IgG concentrations ranging from 0.08 to 50 mg/ml. Pepsin-digested Ig was noninhibitory. IPI factors in anti-D preparations were uniformly high (inhibitory IgG concentrations 0.6-2.5 mg/ml). Cytotoxic antibodies against T, B lymphocytes and monocytes were found in 2,2 and 7 products, respectively. Since we have recently shown that IPI is caused by antibodies against major histocompatibility complex antigens, most likely HLA, the hypothesis is put forward that IPI factors in Ig are HLA-related, cytotoxic as well as noncytotoxic antibodies which act via Fc receptor blockade of human monocytes.