Minor H antigen matches and mismatches are equally distributed among recipients with or without complications after HLA identical sibling renal transplantation.

Studies of the effect of minor H antigen mismatching on the outcome of renal transplantation are scarce and concern mainly single center studies. The International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 16 laboratories of the IHIW, the role of 15 autosomal, 10 Y-chromosome encoded minor H antigens and 3 CD31 polymorphisms, was investigated in relation to the incidence of renal graft rejection and graft loss in 444 human leukocyte antigens (HLA)-identical sibling renal transplantations. Recipient and donor DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, C19Orf48, LB-ECGF-1, CTSH, LRH-1, LB-ADIR and HY. The correlation between minor H antigen mismatch and the primary outcome graft rejection or graft loss was statistically analyzed. The incidence of rejection was very low and no correlation was observed between one or more minor H antigen mismatch(es) and a rejection episode (n = 36), of which only eight resulted in graft loss. In summary, in our study cohort of 444 renal transplants, mismatching for neither autosomal nor HY minor H antigens correlate with rejection episodes or with graft loss.

Progressive Fibrosis Is Driven by Genetic Predisposition, Allo-immunity, and Inflammation in Pediatric Liver Transplant Recipients.

AIM: To determine predisposing factors of idiopathic allograft fibrosis among pediatric liver transplant recipients. BACKGROUND: Protocol biopsies (PB) from stable liver transplant (LT) recipient children frequently exhibit idiopathic fibrosis. The relation between allograft inflammation, humoral immune response and fibrosis is uncertain. Also the role of HLA-DRB1 genotype has not been evaluated, though it's associated with fibrosis in autoimmune hepatitis. PATIENTS AND METHODS: This observational study, included 89 stable LT recipient transplanted between 2004-2012 with mean follow-up of 4.3years, 281 serial PBs (3.1 biopsy/child) and human leukocyte antigen (HLA) antibody data. PBs were taken 1-2, 2-3, 3-5, 5-7, and 7-10years post-LT, and evaluated for inflammation and fibrosis using liver allograft fibrosis score (LAFSc). The evolution of fibrosis, inflammation and related predisposing factors were analysed. FINDINGS: HLA-DRB1*03/04 allele and Class II DSA were significantly associated with portal fibrosis (p=0.03; p=0.03, respectively). Portal inflammation was predisposed by Class II DSA (p=0.02) and non-HLA antibody presence (p=0.01). Non-portal fibrosis wasn't predisposed by inflammation. Lobular inflammation was associated with non-HLA antibodies. INTERPRETATION: We conclusively demonstrated that allograft inflammation results in fibrosis and is associated with post-LT Class II DSA and non-HLA antibodies. The HLA-DRB1*03/04 allele caused genetic predisposition for fibrosis. FUNDING: None.

Optimal flagging combinations for best performance of five blood cell analyzers.

INTRODUCTION: Corelab automation needs increasingly more efficient hematology analyzers and algorithms to adequately detect abnormal samples. The aim of this study is to assess the effect of combining flags or to adjust their trigger level to identify positive samples for further detection within a smear. METHODS: Five hundred and seventeen EDTA samples from patients followed for hematological malignancies were randomly analyzed on Sysmex XE2100 and XN2000, Abbott Cell-Dyn Sapphire, Beckman Coulter DXH800 and Siemens ADVIA 2120. A blood smear as well as a buffy coat was further performed for each of them. RESULTS: Our results shows that depending on the flags, the combinations of them and the thresholds we use, analyzers can provide extremely variable results in their performances for detecting abnormal cells. ADVIA and XN2000 show remarkable performance for blasts detection. DXH800 is the most sensitive for the detection of abnormal lymphocytes, while XN outperforms the market for immature granulocytes and nucleated red blood cell. CONCLUSION: Flagging performances have been shown to be inconsistent among the different manufacturers. This article should help laboratory professionals in their quest for the best flagging schemes and give them a baseline in the selection of the most appropriate analyzer.

A new and simple method to study the role of soluble factors in antibody-mediated cell modulation.

This paper describes an in vitro method to study how interactions between cell suspensions incubated in two culture compartments can occur by diffusion of small molecules through a dialysis membrane. This consists of in vitro cultures of a human PBMC suspension, divided into two parts separated by a dialysis membrane. In one of the cultures, a mitogenic monoclonal antibody (mAb) is added. The porosity of the membrane permits cytokines secreted by activated cells to pass while blocking the antibodies. As a model, PBMC from the same blood donor were divided into two parts and one portion was incubated with an anti-CD3 monoclonal antibody (OKT3 Ortho-Cilag), known to induce IL-2 secretion whereas the other was enclosed in a dialysis bag without antibody. After a 4 day incubation, the cells incubated outside the bag proliferate and secrete cytokines which pass through the membrane and induce cell proliferation inside the bag. This method could complement the currently used methods for the analysis of antibody-mediated cell activation.

Tolerance to discordant xenografts. I. Sharing of human natural antibody determinants on miniature swine bone marrow cells and endothelial cells.

Xenotransplantation could potentially overcome the organ shortage that currently limits the field of transplantation. Because of their breeding characteristics as well as their size and physiologic similarities to humans, we have chosen miniature swine as possible xenograft donors, and are currently attempting to develop a means of using mixed xenogeneic chimerism as an approach to tolerance induction in swine-to-primate species combinations. One major barrier to organ grafting from pig to man is the presence in human serum of preformed natural antibodies (NAb) reacting with antigens expressed on porcine endothelial cells and causing hyperacute rejection. Previous experiments performed in our laboratory have shown that both humoral and cellular tolerance can be induced in a concordant xenogeneic species combination (rat-->mouse) using donor bone marrow infusion following conditioning with a nonmyeloablative regimen. Induction of chimerism in these animals was associated with a marked reduction in the level of IgM natural antibodies that recognize rat bone marrow cells. A similar approach could also lead to humoral and cellular tolerance induction in the swine-->human species combination, permitting transplantation of vascularized organs from the swine donor. To determine the potential of bone marrow transplantation to induce a state of "natural antibody tolerance," it was essential to determine whether or not all human NAb target antigens expressed on swine EC are also expressed on cells derived from swine bone marrow. We have addressed this question by evaluating the ability of various swine bone marrow-derived cells to absorb human IgM and IgG NAb that bind to swine EC. Our results demonstrate that swine bone marrow cells and their progeny can absorb almost all IgM NAb that bind to swine EC, as detected by flow cytometric and ELISA assays. Specificity of absorption was demonstrated, as total serum IgM levels declined only minimally after absorption on swine BMC and to an extent comparable to that observed following absorption with human cells, which did not deplete swine EC-binding NAb. Human IgG binding to swine EC was also completely absorbed by swine BMC. These results suggest that a state of "NAb tolerance" could be induced by successful swine marrow engraftment in man. Furthermore, swine PBL, platelets, and EC were able to absorb most IgM NAb that bound to swine BMC, suggesting that absorption using antigen from any of these tissues might facilitate marrow engraftment, and hence tolerance induction, in this species combination.

In vivo depletion of xenoreactive natural antibodies with an anti-mu monoclonal antibody.

Hyperacute rejection of vascularized discordant xenografts, such as pig-to-primate kidney or heart xenotransplants, is thought to be mediated by xenoreactive natural antibodies (XNA) of the IgM isotype and the activation of the classic pathway of complement. Using the guinea pig-to-rat discordant xenograft model, we have developed a potential therapeutic protocol leading to long-term depletion of circulating IgM in adult animals. This protocol consists of the injection into adult LOU/C rats of an antirat IgM MAb (MARM-7) after splenectomy, plasma exchange, and the administration of an anti-B cell immunosuppressant, mycophenylate mofetil (RS61443). Splectomized plasma exchanged adult rats receiving RS61443 showed strongly decreased IgG and IgM serum concentrations for a relatively short period during which these isotypes remained nevertheless detectable by a sensitive ELISA technique. In contrast to IgM, IgG in serum returned, shortly after the end of this treatment, to normal concentrations. Splenectomy alone was able to significantly decrease, for a long period (more than 70 days), IgM but not IgG serum concentrations in these rats. During this treatment, IgM XNA concentration mirrored total IgM. The injection of MARM-7 MAb to adult LOU/C rats was able to deplete circulating IgM and IgM XNA for a period of several weeks during which IgM was undetectable by a sensitive ELISA technique. Depletion time was dose-dependent--the higher the dose of injected MARM-7, the longer the period for which IgM and IgM XNA remained undetectable. Moreover depletion of circulating IgM was correlated with the detection in the serum of these rats of noncomplexed, free MARM-7. Finally, MARM-7 administration was significantly more efficacious in rats that had decreased levels of circulating IgM after splenectomy, plasma exchange, and administration of RS61443. These experiments suggest that the anti-mu approach may allow depletion of IgM XNA for a sufficiently long period to test the hypothesis of "accommodation" in other xenograft models such as the pig-to-primate xenograft or even in ABO-incompatible allografts.

Effect of graft preservation and IgM depletion on guinea pig to rat cardiac xenograft survival.

BACKGROUND: Heterotopic guinea pig (GP) cardiac xenografts (XG) are hyperacutely rejected within minutes when transplanted into rats. METHODS: In this GP to rat cardiac XG model, we studied the effect on graft survival of a short cold preservation time (1 hr at 4 degrees C) in the presence or absence of rat anti-GP IgM preformed antibodies. The complete depletion of circulating IgM was obtained by two intraperitoneal injections of anti-rat IgM monoclonal antibody (MARM-4) on preoperative days -3 and -1. RESULTS: When the GP cardiac XG was cold preserved for 1 hr before transplantation, the mean graft survival time (MST) was 13.5+/-2.8 min, whereas without previous cold preservation, the MST was significantly prolonged to 51.5+/-12.3 min (P<0.001). Interestingly, the complete depletion of preformed circulating IgM before grafting significantly prolonged the MST of a cold-preserved XG to 37.1+/-11.3 min in comparison with a nondepleted recipient of a cold-preserved XG (P<0.02), but did not prolong the graft survival of a XG that was not cold preserved (42.5+/-14.1 min). To assess the effect of cold preservation and/or ischemia reperfusion, we intravenously injected a superoxide-dismutase mimetic (EUK-134) just before transplantation of a cold-preserved XG. This antioxidant regimen improved the MST from 13.5+/-2.8 min to 35.3+/-7.3 min (P<0.001). These results clearly suggested that either preservation lesions or preformed IgM are capable of accelerating the loss of the cardiac graft function, but also that the presence of preformed IgM seems to be especially deleterious when the cardiac XG has previously been ischemically injured. Analyzing the histological data, we also observed that the prompt cessation of cardiac function seen in cold-preserved grafts was uniformly associated with massive interstitial hemorrhage, thereby suggesting a particular susceptibility of the GP cardiac XG to cold preservation. To assess the effect of preservation on the GP cardiac function in a nonimmunological model, we performed syngeneic GP cardiac grafts and found that 1 hr of cold preservation provoked massive interstitial hemorrhage capable of promptly inducing the cessation of the heartbeat. CONCLUSIONS: Overall, this study demonstrated that both ischemic lesions and immunological processes might induce the cessation of cardiac graft function in the GP to rat model and this cessation of graft function is probably often misinterpreted as a XG rejection only.

Administration of exogenous interleukin-2 abrogates spontaneous rat liver allograft acceptance but does not affect long-term established graft survival.

BACKGROUND: Spontaneous tolerance to the orthotopic liver allograft uniformly occurs in the DA (RT1a) to PVG (RT1c) rat combination despite a fully allogeneic barrier. METHODS: To assess whether spontaneous acceptance might be the consequence of a T cell help deficit at the time of the first exposure of alloantigens to the host, we studied the effect of exogenous interleukin (IL)-2 injections at the time of liver transplantation and during long-term follow-up. RESULTS: Although spontaneous acceptance of the liver allograft constantly ensued in the DA to PVG combination, a daily injection of recombinant IL-2 (3 x 10(5) U) uniformly provoked acute cellular rejection of the liver allograft and consequently the death of animals by postoperative day 5-6. Simultaneous to the graft loss, hepatic enzymes (alanine aminotransferase) increased more than 50-fold in IL-2-treated recipients, whereas similar IL-2 treatment did not produce any hepatic dysfunction in syngeneic animals. By immunohistology, the expression of the alpha chain of the IL-2 receptor, usually undetectable in untreated animals, was evident on CD4 and CD8 lymphocytes infiltrating the liver graft. In contrast, a similar IL-2 regimen and even higher IL-2 doses (x 10(6) U) did not abrogate the liver allograft survival during long-term follow-up. CONCLUSIONS: Our results demonstrate that spontaneous rat liver allograft acceptance may be abolished by exogenous IL-2 injections, which suggests that an "inherent T cell help deficit" might be implicated in the spontaneous acceptance mechanisms of DA to PVG liver allografts.

Specific depletion of preformed IgM natural antibodies by administration of anti-mu monoclonal antibody suppresses hyperacute rejection of pig to baboon renal xenografts.

BACKGROUND: The elimination of circulating anti-porcine preformed antibodies is crucial for avoiding hyperacute vascular rejection (HAVR) of primarily vascularized xenograft in discordant pig to baboon model. Previously described methods used for eliminating natural antibodies, however, constantly removed both anti-porcine IgM and IgG antibodies, as well as often complement proteins. To study specifically the role of preformed anti-porcine IgM antibodies, a specific anti-IgM monoclonal antibody (mAb) has been designed and evaluated in vivo. METHODS: Iterative injections of anti-IgM mAb (LO-BM2) at high dose (20 mg/kg) depleted to undetectable level the circulating IgM and therefore anti-porcine IgM antibodies but did not change the concentration of anti-pig IgG antibodies. The serum concentration of IgM and IgG antibodies was assessed by ELISA and the level of anti-pig natural IgM and IgG antibodies by flow cytometry (FC). Anti-rat sensitization was assessed by specific ELISA as well as the serum concentration of LO-BM2. RESULTS: Iterative injections of LO-BM2 allowed to specifically eliminate the anti-porcine IgM antibodies to undetectable levels at ELISA. Despite a normal serum level of anti-porcine IgG and complement proteins, HAVR was avoided. Without immunosuppression, the specific elimination of preformed anti-porcine IgM prolonged the survival of a renal xenograft in baboon up to 6 days, whereas without IgM antibody elimination, the renal xenografts were hyperacutely rejected within hours. The lost of activity of LO-BM2 after 10 days was concomitant to an IgM and IgG antibody rebound, which caused an acute vascular rejection of the xenograft. CONCLUSION: Specific elimination of natural anti-porcine IgM antibodies allows to avoid HAVR of a pig to baboon renal xenograft, whereas anti-porcine IgG antibodies and complement proteins were present in the serum. This result confirms previous in vitro reports and demonstrates for the first time in vivo that preformed IgM antibodies alone are responsible for HAVR, while preformed anti-porcine IgG antibodies are unable alone to cause HAVR. Anti-IgM therapy appears as an important tool to transiently but completely eliminates xeno-IgM antibodies in vivo.

Effects on human and nonhuman primate immune response of a new rat anti-CD2 monoclonal antibody.

BACKGROUND: Nonhuman primate models are highly clinically relevant in transplantation. The development of immunosuppressive tools or a tolerogenic regimen for primate models therefore represents an important goal of transplantation immunological research. Hence, we have developed a rat monoclonal antibody (mAb) that recognizes the CD2 molecule (LO-CD2b) on both human and nonhuman primate cells. METHODS: The LO-CD2b mAb has been characterized by flow cytometry, E-rosetting inhibition, and Western blotting. In vitro inhibition of immune responses by LO-CD2b was assessed after both mitogenic and allogeneic stimulation in mixed lymphocyte reactions (MLR). Several LO-CD2b dose and time responses were tested. In vivo, peripheral and lymph node T-cell depletion was examined both by flow cytometry and immunohistology in 10 baboons that received intravenous injection of LO-CD2b at different doses and time courses. Xenosensitization (anti-rat) was assessed by ELISA. Renal allograft survival was followed in two baboons treated with iterative LO-CD2b injections. RESULTS: In vitro, LO-CD2b binds a lymphocyte antigenic determinant of 52 kDa that is recognized by other well-characterized anti-CD2 mAbs (T11, Leu5b). LO-CD2b recognized natural killer CD2+ cells. Administration of 200 ng/ml LO-CD2b almost completely inhibited human and baboon mitogenic stimulation. Allogeneic baboon and human MLR were completely inhibited by the addition of LO-CD2b (at 312 ng/ml) on the day of the initiation of culture; when added after 1 or 2 days, LO-CD2b still provided a significant MLR inhibition (>50%). Incubation of LO-CD2b with baboon peripheral blood mononuclear cells produced very low cytokine levels (interferon-y, tumor necrosis factor-alpha, interleukin 2). In secondary MLR, baboon peripheral blood mononuclear cells previously incubated with LO-CD2b were unable to respond to a second allogeneic stimulation but were able to react to mitogens. In vivo, within the first hour after LO-CD2b injection (at 0.15, 0.5, and 2 mg/kg), an 85-90% peripheral depletion of CD2+ cells was observed. A partial T-cell depletion in inguinal lymph nodes was seen after 1 week. The mechanism of peripheral T-cell depletion could have been antibody-dependent cell cytotoxicity or opsonization but was complement independent. Iterative LO-CD2b injections (12 days at 0.35 mg/kg) slightly prolonged the renal allograft survival in two baboons. CONCLUSION: LO-CD2b is a nonactivating rat anti-CD2 mAb able to strongly inhibit both mitogenic and allogeneic responses in human and nonhuman primates. In vivo, LO-CD2b provides a rapid peripheral T-cell depletion, which is reversible within days after the cessation of injections. This rat mAb represents a very important tool for in vivo experimental investigation in nonhuman primates because it similarly reacts against human T cells in vitro.

Monoclonal antibodies in prophylactic immunosuppression after liver transplantation. A randomized controlled trial comparing OKT3 and anti-IL-2 receptor monoclonal antibody LO-Tact-1.

A prospective trial was conducted to assess the efficacy of induction immunosuppression with antilymphocyte monoclonal antibodies in 129 primary liver transplant patients who were randomly divided into three groups according to immunosuppression during the first 10 days post-OLT: triple drug therapy only (TDIS: cyclosporine, steroids, azathioprine) (group I: n = 42); TDIS with a 10-day course of OKT3 (group II: n = 44); and LO-Tact-1 (anti-IL-2 receptor mAb) (group III: n = 43). Biopsy-proved acute rejection (AR) was treated using the same biopsy-guided protocol in the 3 groups. One-year patient survival rates were 67%, 84%, and 93% in groups I, II, and III, respectively (I vs. II, NS; I vs. III, P = 0.001; II vs. III, P = 0.044). Incidences of AR were studied in the subgroup of 100 patients who were exposed to the risk of developing rejection, with an overall rate of 89% during the first 3 months post-OLT, similar in the 3 groups. However, incidences of steroid-resistant rejection diagnosed during the 10 first days post-OLT were 54%, 24%, and 34% in groups I, II, and III and 46%, 26%, and 11%, respectively, during the 10-90 days interval. Sixteen patients with CMV had received OKT3, whereas the 5 remaining CMV cases had not (P = 0.019). In summary: (1) mAbs did not modify crude incidence of AR; (2) in the early period (< 10 days), TDIS immunoprophylaxis combined with OKT3 was more efficient than TDIS alone; (3) when compared with groups I and II, LO-Tact-1 apparently better prevented steroid-resistant rejection during the 10-90 days post-OLT; (4) OKT3 significantly increased incidence of CMV infection. In conclusion, TDIS with LO-Tact-1 seemed to achieve the better risk-benefit ratio in induction immunosuppression after OLT.

Suppression of delayed xenograft rejection by specific depletion of elicited antibodies of the IgM isotype.

BACKGROUND: Hamster hearts transplanted into untreated rats undergo delayed xenograft rejection (DXR). This acute inflammatory response is associated with the deposition of anti-graft antibodies of the immunoglobulin (Ig)M isotype in the vasculature. We have previously shown that these antibodies are generated in a T cell-independent manner. In this study, we tested whether the generation of anti-graft IgM antibodies is involved in the pathogenesis of DXR. In addition, we tested whether the suppression of this antibody response would overcome DXR. METHODS: Hamster hearts were transplanted into rats treated with an anti-mu monoclonal antibodies (mAb) to deplete circulating IgM or with an isotype-matched control mAb recognizing the dinitrophenyl epitope. T cell immunosuppression was achieved with cyclosporin A (CsA). RESULTS: Depletion of circulating IgM by anti-mu mAb inhibited DXR, whereas the control mAb had no effect on DXR. In anti-mu-treated rats, xenografts were rejected 5-7 days after transplantation through a T cell-dependent mechanism associated with the generation of antibodies of the IgG isotype. Combination of anti-mu with CsA suppressed the anti-graft IgM and IgG response and resulted in long-term xenograft survival (> 50 days). Xenograft long term survival occurred despite the return of anti-graft IgM antibodies to the circulation, a phenomenon referred to as accommodation. CONCLUSION: This study demonstrates that the pathogenesis of DXR can be initiated by anti-graft antibodies of the IgM isotype, which are generated in a T-cell independent manner. In addition, we show that under T cell immunosuppression, specific depletion of this IgM response by anti-mu mAb administration results in xenograft long-term survival and accommodation.

Differential inhibition of B-cell development and xenoreactive natural antibody production by administration of anti-mu or anti-delta monoclonal antibodies in adult rats.

BACKGROUND: Given the role of xenoreactive natural antibodies (XNA) in the pathogenesis of xenograft rejection, we tested whether the administration of anti-mu or anti-delta monoclonal antibodies (mAbs) in adult rats would suppress the generation of XNA. METHODS: Adult LOU/C (Igkappa-1a) rats were treated with anti-mu or anti-delta mAbs after nonlethal total body irradiation and bone marrow transplantation from congenic LOU/C (Igkappa-1b) rats. The differentiation of donor bone marrow (BM)-driven Igkappa-1b+ B cells and XNA production were analyzed. RESULTS: Both anti-mu and anti-delta mAbs arrested B-cell differentiation in the BM. In anti-mu-treated rats, there was a total depletion of donor-driven, peripheral Igkappa-1b+ B cells, secreting cells, and circulating XNA of the Igkappa-1b allotype. In anti-delta-treated rats, a significant number of Igkappa-1b+ B cells, which did not express membrane IgD, "escaped" deletion and partially repopulated peripheral lymphoid organs. This B-cell population was active in the production of XNA, as revealed by the high serum levels of XNA in these animals. CONCLUSIONS: Anti-mu administration resulted in arrest of B-cell differentiation and in down-regulation of IgM and IgG XNA production in adult rats. These data suggest that the use of anti-mu mAbs may be a useful approach to suppress the production of XNA and prevent xenograft rejection. Furthermore, we suggest that the B-cell population responsible for the production of XNA in adult rats belongs to a B-cell lineage expressing low levels of membrane IgD and "escaping" deletion in the BM upon anti-delta treatment.

Differential effects of injections of anti-mu and anti-delta monoclonal antibodies on B-cell populations in adult mice: regulation of xenoreactive natural antibody-producing cells.

BACKGROUND: The depletion of differential B cell and xenoreactive natural antibodies (XNA) by anti-delta and anti-mu injections was analyzed in adult mice. Sequential treatment with anti-delta and then anti-mu induces a complete depletion of B cells and XNA and represents a potential approach to induce xenograft tolerance. METHODS: Adult mice were injected with anti-mu, anti-delta, anti-delta then anti-mu, or control isotype monoclonal antibodies from day 0 to day 14. The different B-cell populations were analyzed by FACS and immunohistology. Ig production was tested by ELISA. XNA were analyzed by FACS. RESULTS: Anti-mu injections induced a depletion of IgMhigh, immature B cells, marginal zone B cells, and B1 cells and an increase of IgG-XNA production. Anti-delta injections induced mature conventional IgDhigh B-cell depletion and increased IgM-XNA production. Interestingly, sequential injections of anti-delta then anti-mu induced a depletion of immature B cells, mature B cells (MZ, B2, and B1), and XNA. CONCLUSIONS: These results demonstrate that mature B-cell depletion in adult mice can be obtained by mAb injections and depends on the surface immunoglobulin cross-linking threshold. Indeed, anti-mu mAb depleted IgMhigh B cells (MZ and B1) and anti-delta, IgDhigh B cells (B2). The differential B-cell suppression shows that conventional B cells are responsible in the IgG-XNA production and MZ and B1 cells in the IgM-XNA production. Sequential repeated injections of anti-delta then anti-mu mAb depleted all B-cell populations and suppressed the whole XNA production.

In vivo IgM depletion by anti-mu monoclonal antibody therapy. The role of IgM in hyperacute vascular rejection of discordant xenografts.

Xenoreactive natural antibodies (XNA) and complement activation are thought to be the 2 main factors responsible for the hyperacute vascular rejection (HVR) of discordant xenografts. The aim of this work was to study the role of IgM XNA in the HVR of guinea pig to rat cardiac xenografts, a discordant model. Adult LOU/C rats were depleted of circulating IgM and therefore of IgM XNA using an anti-mu mAb (mouse anti-rat IgM mAb 7 [MARM-7]). Rats were injected with 10 mg and 5 mg of MARM-7 at days -3 and -1, respectively, and guinea pig cardiac xenografts were performed on day 0. Control animals were injected on the same days with 10 mg and 5 mg of anti-alpha mAb (MARA-1) or equivalent volumes of PBS. Xenografts were performed on day 0. Guinea pig cardiac xenograft survival time was significantly prolonged in IgM-depleted animals (62 min, P < 0.01) compared with controls using PBS (18 min) or MARA-1 mAb (12 min). This prolongation was not due to a decrease in the complement activity in IgM-depleted rats, since no significant variation of the C1q, C4, C3, and C5 complement hemolytic activity was observed between control and treated animals before HVR. Prolongation of the xenograft survival time in the MARM-7-treated group was correlated with an undetectable serum level of IgM and IgM XNA and a lack of IgM XNA deposits on the rejected xenograft vascular endothelium. Contrarily, both IgM-depleted and control animals showed C3 deposits on the rejected xenograft vascular endothelium and myocardium, as well as diffuse deposits of IgG2a XNA. Although HVR was not abrogated by the depletion of IgM XNA, our data indicate that IgM is implicated in the HVR and that the anti-mu approach is a potential therapeutic treatment for discordant xenografts. Finally, we suggest that other factors such as IgM-independent activation of complement might be one of the mechanisms responsible for the pathogenesis of HVR in the guinea pig to rat xenograft model.

Prophylactic immunosuppression with anti-interleukin-2 receptor monoclonal antibody LO-Tact-1 versus OKT3 in liver allografting. A two-year follow-up study.

A prospective trial was conducted in 129 recipients of primary liver transplantation, to compare induction immunosuppression using triple drug therapy (cyclosporine, steroids, and azathioprine; group 1, n = 42), versus triple drug therapy with a 10-day course of OKT3 (group 2, n = 44) or of the anti-interleukin-2 receptor monoclonal antibody LO-Tact-1 (group 3, n = 43). Two-year actual patient survival rates were 64%, 79%, and 93% in groups 1, 2, and 3, respectively (1 vs. 2, NS; I vs. III, P = 0.003; 2 vs. 3, NS). Up to 2 years after transplantation, 18%, 44%, and 53% of the grafts in groups 1, 2, and 3, respectively, had not experienced steroid-resistant acute rejection (1 vs. 2, P = 0.002; 1 vs. 3, P = 0.007; 2 vs. 3, NS). The overall incidence of chronic rejection was 4%. OKT3 therapy, but not LO-Tact-1, significantly increased the incidence of cytomegalovirus infections (P = 0.019). In conclusion, immunoprophylaxis with LO-Tact-1 seemed to provide a liver graft acceptance rate at least as satisfactory as that with OKT3, without an increase in the incidence of infections.

The experimental (in vitro) and clinical (in vivo) immunosuppressive effects of a rat IgG2b anti-human CD2 mAb, LO-CD2a/BTI-322.

BACKGROUND: CD2 is a cell surface glycoprotein expressed on most human T cells and natural killer (NK) cells, working as a cell adhesion and costimulatory molecule. The aim of this paper is to analyze the mechanism of action of a rat IgG2b anti-human CD2 monoclonal antibody (mAb) (LO-CD2a/BTI-322 mAb), which is a potent immunosuppressive agent and inducer of cell death. In vivo, this mAb is able to prevent or treat kidney allograft rejection. METHODS: The mechanisms by which the LO-CD2a/BTI-322 mAb is able to induce inhibition of cell activation and cell death were analyzed by mixed lymphocyte reactions and by flow cytometry. After in vivo treatment, levels of circulating mAb were measured by ELISA as well as anti-rat immunization and cytokine release. RESULTS: We show that the inhibition of cell activation induced by LO-CD2a/BTI-322 mAb after allogeneic or OKT3 stimulation is due to an Fcgamma receptor-dependent CD2 down-modulation and to T-cell depletion through an antibody-dependent cell-mediated cytotoxicity mechanism mediated by NK cells or activated monocytes. Peripheral T- and NK-cell depletion was observed after in vivo treatment with LO-CD2a/BTI322. Cytokine release (TNFalpha) was correlated with some side effects, but only after the first injection, and the effects were never severe or life threatening. CONCLUSION: The correlation between the in vitro and in vivo data suggests that T-cell depletion, especially of activated cells, and inhibition of cell activation after CD2 down-modulation are the main mechanisms of action of the LO-CD2a/BTI-322 mAb.

Association of natural killer cell depletion with induction of mixed chimerism and allograft tolerance in non-human primates.

BACKGROUND: Nonmyeloablative T cell depletion followed by donor bone marrow infusion has proved to be an effective approach to induction of mixed chimerism and tolerance of organ allografts in non-human primates. To help define the mechanisms involved we have compared T cell depletion with ATG versus anti-CD2 monoclonal antibody with respect to establishment of mixed chimerism and induction of tolerance. METHOD: Both nonmyeloablative regimens included low dose total body irradiation (1.5 Gy x 2), thymic irradiation (7 Gy), splenectomy and kidney plus donor bone marrow transplantation, followed by a 4-week posttransplant course of cyclosporine. In addition, the ATG group (13 recipients) received antithymocyte globulin, although the LOCD2b group (10 recipients) were treated with an anti-CD2 monoclonal antibody (LOCD2b). RESULTS: In the ATG group, 11 of 13 monkeys developed multilineage chimerism and 9 survived for more than 100 days without kidney allograft rejection. In contrast, 0/10 monkeys in the LOCD2b group developed chimerism, 5 died of infection and 5 suffered progressive rejection; only 1 recipient survived beyond 100 days. Sequential monitoring of peripheral blood mononuclear cells revealed greater T cell (CD3+) depletion in the LOCD2b-treated animals compared to those receiving ATG. However, NK cells (CD16+CD8+) were significantly more depleted in the ATG group and NK function remained abrogated longer after ATG than LOCD2b treatment (3 weeks vs. <5 days). CONCLUSION: Despite excellent T cell depletion by LoCD2b, ATG was more effective in inducing chimerism and tolerance. This difference correlated with anti-NK activity of the two reagents. These data suggest that NK cells may also resist engraftment of allogeneic bone marrow cells in this model.

Apoptosis of human naive NK cells mediated by a rat IgG2b anti CD2 mAb through a fractricidal ADCC reaction.

LO-CD2a/BTI-322, a rat anti human CD2 mAb, shows in vitro and in vivo immunosuppressive properties and induces T-cell depletion resulting partially from an antibody dependent cellular cytotoxicity (ADCC) mediated by NK cells. The aim of this paper is to study the in vitro effect of LO-CD2a/BTI-322 on NK cells, the majority of them also expressing the CD2 molecule. The addition of the mAb to purified naive NK cells induces apoptosis of CD2+ cells. The apoptosis is rapid, Fas ligand independent and completely inhibited by the calcium chelator EGTA, suggesting a fractricidal ADCC reaction and implying that NK cells are not resistant to lysis when used as target cells. At the end of the reaction, the CD2 - remaining cells are still capable of natural cytotoxicity against K562 cells, but at a lower rate than untreated cells.

Human recombinant interleukin-4 (HurIL-4) improves SCID mouse reconstitution with human peripheral blood lymphocytes.

The application of human-scid mouse chimera (hu-scid) technology has some limitations, because of the variable amounts and functional heterogeneity of human cells recovered from engrafted mice. Attempts to optimize the construction of hu-scid chimeras with human peripheral blood mononuclear cells (Hu-PBMC) by using in vivo anti-asialo GM1 antibody treatment in mice in order to eliminate natural killer cells have been published by other authors with interesting results. In this study, Hu-PBMC were incubated in vitro with human recombinant interleukin-4 (HurIL-4) or human recombinant interleukin-2 (HurIL-2) for two hours and were intraperitoneally transferred into scid mice. Total human IgM and IgG levels in hu-scid sera, human cell markers in the thymus and in the spleen of grafted mice were considered as parameters of successful engraftment. HurIL-4 significantly enhanced human immunoglobulin production while HurIL-2 did not show any obvious effect. Human cell markers (CD2 and CD19) were significantly higher in the group of HurIL-4 treated Hu-PBMC than in the other groups, despite the high variability in human cell proliferation in the recipients. Thus, HurIL-4 can be used as an adjuvant growth factor which improves successful engraftment of human peripheral blood lymphocytes in scid mice.