组织相容性Ⅰ类相关链A位点基因多态性在预判肾移植物排斥中的意义

目的 研究组织相容性Ⅰ类相关链A位点(MICA)基因多态性和抗MICA特异性抗体在肾移植排斥反应发生中的意义.方法 采用免疫磁珠液相芯片技术对40例肾移植患者在移植术前和移植术后1个月、3个月、6个月、1年和2年动态检测抗MICA抗体的特异性和阳性分值的变化,同时采用SSOP方法分析16对肾移植供受者的MICA基因分型...     

Anchored anti-HLA class I monoclonal antibody fails to induce inhibition of PHA-activated lymphocytes proliferation.

It is known that anti-HLA Class I antibodies inhibit the proliferative response of PHA-activated T-lymphocytes. We found that plastic- or sepharose-linked anti-HLA Class I monoclonal antibody 01.65 does not inhibit either [3H]Thymidine incorporation or recruitment in the cell cycle, nor does it reduce the expression of c-myc mRNA and the membrane expression of Interleukin-2 Receptor and Transferrin Receptor. Furthermore, particulate Protein Kinase C is not affected by anchored anti-HLA Class I monoclonal antibody 01.65. We suggest that anti-HLA Class I monoclonal antibody may act through crosslinking or internalization of HLA Class I antigens.     

Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure

The detection of donor-specific anti-HLA antibodies by standard procedures such as complement-dependent cytotoxicity assay (CDC) or flow cytometric (FACS) analysis is limited by its low sensitivity and the quality of the donor cells. Therefore, an ELISA-based technique was employed using solid phase-immobilized monoclonal antibodies to capture HLA class I or class II molecules of the donor, respectively. In this HLA class I and class II antibody monitoring system (AMS) the donor-specific anti-HLA antibodies from the sera of recipients bind to the HLA molecules of the donor which have been immobilized by monoclonal antibodies (mAb) recognizing non-polymorphic epitopes. Upon binding of donor-specific anti-HLA antibodies they are recognized by secondary enzyme-conjugated anti-human immunoglobulin (Ig) antibodies. A newly established modification of the standard protocol allows the differentiation between bound antibodies of the IgG and IgM isotype. Furthermore, this assay was adapted for investigating small amounts of solid tissue of donors from whom no other cells (e.g. from blood) were available. We here provide an overview of the classical crossmatch methods with their advantages and limits. In addition, the design of the novel AMS-ELISA is described in terms of quality and sensitivity of the approach using exemplary cases of different application. The selected cases show that the AMS-ELISA represents a valuable tool for the post-transplantation monitoring of donor-specific anti-HLA antibodies during reaction crisis, after transfusion reactions and in particular cases of tissue transplantations lacking single cells.     

Antibody-induced association between discrete regions of HLA class I and II antigens

The anti-HLA-DR + DP monoclonal antibody (MoAb) CR11-462 was unexpectedly found to cross-inhibit the binding to B lymphoid cells of the anti-HLA Class I MoAb CR10-215 and CR11-115. The latter two antibodies recognized the same or spatially close antigenic determinant. The cross-blocking of anti-HLA Class I MoAb CR10-215 and CR11-115 by MoAb CR11-462 reflects neither its contamination by anti-HLA Class I antibodies nor its cross-reactivity with HLA Class I antigens. On the other hand, the cross-blocking appears to reflect redistribution of HLA Class II antigens by the MoAb CR11-462, since the MoAb CR10-215 and CR11-115 are not susceptible to blocking when lymphoid cells are treated with 0.025% glutaraldehyde or are coated with Fab' fragments of the MoAb CR11-462. Furthermore, immunoprecipitates from B lymphoid cells preincubated with the MoAb CR11-462 before solubilization contain HLA Class I antigens. Therefore, these results have shown for the first time an antibody-induced association between discrete regions of HLA Class I and Class II antigens on the membrane of B lymphoid cells.     

HLA-typing analysis following allogeneic bone grafting for sinus lifting

According to the Brazilian Association of Organ Transplants, in 2015, 19,408 bone transplants were performed in Brazil, over 90% by Dental Surgeons. The surgical technique itself has a respectable number of reports regarding its clinical efficacy, as measured by long-term survival of dental implants in grafted areas. Uncertainty remains, however, as to whether fresh frozen grafts from human bone donors remain immunologically innocuous in the body of the host. Six male with no previous medical history of note, including systemic diseases, surgery or blood transfusion were selected. These patients underwent reconstructive procedures (sinus lifting) using fresh frozen human bone from a tissue bank. All patients had venous blood samples collected prior to surgery and 6 months after the procedure. Anti-HLA analysis for the detection of HLA (human leukocyte antigen) antibodies was performed using methods such as the LABScreen PRA Class I and Class II, LABScreen Single Antigen Class I and Class II, Luminex Platform. Reactive individuals to the screening tests (LABScreen PRA) were further investigated to determine the specificity of the antibodies detected (LABScreen Single Antigen) with a cutoff value of median fluorescence intensity ≥500. As a result, it was observed that two patients (33%) were positive in screening tests, one presenting with anti-HLA Class I and II sensitization and the other with anti-HLA class II. The specificity analysis showed that the patients sensitized to HLA class II presented 4 specificities, 3 of which immunologically relevant. In the second individual, 23 specificities were identified, 6 of which immunologically important for HLA class I and 4 specificities for HLA class II, 3 of these were immunologically important. All specificities detected had average fluorescence. These findings are suggestive that sinus-lifting procedures with allogeneic bone can induce immunological sensitization.     

Lack of a role of monocytes in the inhibition by monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens of PHA-P-induced peripheral blood mononuclear cell proliferation

This study aimed at characterizing the mechanism(s) underlying the regulatory role of distinct determinants of HLA Class I antigens in PHA-P-induced T cell proliferation and the involvement of monocytes in this phenomenon. The anti-HLA-A2,A28 monoclonal antibodies (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to the gene products of the I antigens HLA-B locus, and the MoAb CR10-215 and W6/32 to distinct monomorphic determinants of HLA Class I antigens were found to inhibit PHA-P-induced peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent fashion. The inhibition is specific and reflects neither inhibition of PHA-P binding to cells nor a toxic effect of the anti-HLA Class I MoAb. The latter differed in the concentration required to induce inhibition, in the influence of the concentration of PHA-P used as mitogen, in the differential effect on the donors used as a source of PBMC, and/or in the requirement of the Fc portion to induce inhibition. At variance with the information in the literature, the inhibitory effect of anti-HLA Class I MoAb on PHA-P-induced PBMC proliferation neither reflected their interaction with accessory cells nor was mediated by suppressor factors released by monocytes stimulated with PHA-P in the presence of anti-HLA Class I MoAb. Therefore, the regulatory role of HLA Class I antigens in T cell proliferation is not likely to be mediated by monocytes and/or factors released from them, but may reflect an involvement of these molecules in T cell activation pathways.     

Quantitation of HLA proteins incorporated by human immunodeficiency virus type 1 and assessment of neutralizing activity of anti-HLA antibodies

Human anti-human leukocyte antigen (HLA) antibodies were assessed for neutralizing activity against human immunodeficiency virus type 1 (HIV-1) carrying HLA alleles with matching specificity. Multiparous women carrying anti-HLA antibodies were identified. Plasma samples from those women were confirmed as having antibodies that specifically bound to HLA proteins expressed on the peripheral blood mononuclear cells (PBMCs) of their husbands. A primary HIV-1 isolate was cultured in the husband's PBMCs so that the virus carried matching HLA alleles. To determine the HIV-1-neutralizing activity of anti-HLA antibodies, the infectivity of the virus for GHOST cells (which express green fluorescent protein after HIV infection) was investigated in the presence of a plasma sample positive for the respective anti-HLA antibody. A neutralization assay was also performed using purified immunoglobulin G (IgG) from two plasma samples, and two plasma samples were investigated in the presence of complement. The prerequisite for anti-HLA antibody-mediated neutralization is incorporation of HLA proteins by HIV-1. Therefore, the extent of incorporation of HLA proteins by the primary HIV-1 isolate was estimated. The ratios of HLA class I protein to HIV-1 capsid (p24) protein cultured in the PBMCs of two healthy individuals were 0.017 and 0.054. These ratios suggested that the HIV-1 strain used in the assay incorporated more HLA proteins than gp160 trimers. Anti-HLA antibody-positive plasma was found to contain antibodies that specifically reacted to HIV-1 carrying cognate HLA alleles. However, incubation of HIV-1 with anti-HLA antibody- positive plasma or purified IgG did not show a reduction in viral infectivity. HIV-1-neutralizing activity was also not detected in the presence of complement. This study shows that HIV-1 primary isolates cultured in PBMCs contain significant amounts of HLA proteins. However, the binding of antibodies to those HLA proteins does not mediate a reduction in viral infectivity.     

Regulatory role of a monomorphic determinant of HLA Class I antigens in T cell proliferation

The role of HLA Class I antigens in T cell proliferation was investigated by using the anti-HLA Class I monoclonal antibodies (MoAb) CR10-215, CR10-325, and CR11-115. MoAb CR10-215 and CR11-115 recognize the same (or spatially close) monomorphic determinant, which is distinct and spatially distant from that reacting with MoAb CR10-325. Addition of MoAb CR10-215 and CR11-115 to cultures of peripheral blood mononuclear cells stimulated with MoAb OKT3, MoAb Pan T2, PHA, or PPD inhibited cell proliferation. The blocking is specific in that the anti-HLA Class I MoAb CR10-325 and the Pan T MoAb Pan T1 had no effect on the proliferation. The inhibitory activity of MoAb CR10-215 and CR11-115 does not reflect i) toxic effects, ii) induction of suppressor cells and factors, iii) blocking of the binding of mitogens to lymphocytes, iv) inhibition of the production of interleukin 1 (IL 1) and interleukin 2 (IL 2), or v) function of IL 2 receptor. Anti-HLA Class I MoAb were able to inhibit the proliferation of purified, Tac-, T cells. The inhibited cells did not express Tac antigen, as assayed by direct immunofluorescence, with MoAb anti-Tac, but released a normal amount of IL 2 in culture medium. These results indicate that monomorphic determinants of the HLA Class I complex are involved in the regulation of T cell proliferation. The effect appears to occur at the level of IL 2 receptor expression.     

The Clinical Relevance of Pre-Formed Anti-HLA and Anti-MICA Antibodies after Cord Blood Transplantation in Children

Preformed anti-HLA antibodies (AHA) are known to be associated with delayed engraftment and reduced overall survival after adult hematopoietic stem cell transplantation. However, limited data is available in pediatric patients. In this study, we explored the role of AHA on clinical outcomes in 70 pediatric patients who received a single unit of HLA mismatch cord blood for hematologic malignancies, immunodeficiencies or metabolic diseases. The presence of AHA was detected in 44% (31/70) of the patients. Preformed class I AHA was associated with an increased occurrence of grade 1–4 acute graft-versus host disease (p<0.05). The presence of anti- major-histocompatibility-complex class I–related chain A antigens (MICA) antibodies was significantly associated with a reduced platelet recovery after transplantation (p<0.05). AHA of class II with the strength of antibody titer measured as the mean fluorescence intensity above 2000 was associated with reduced event-free survival (p<0.05). A reduction of high titer of AHA and anti-MICA antibodies might have to be considered before cord blood transplantation in pediatric patients for better outcomes.     

Pregnancy in patients with chronic renal disease.

Pregnancy is not invariably contra-indicated in patients with pre-existing renal disease. Clinical data now exist that permit the clinician to distinguish such patients who are likely to experience difficulty during pregnancy from those in whom pregnancy can be undertaken with high expectation of success. Patients suffering from systemic lupus erythematosus, active or inactive, with or without lupus nephritis, should avoid pregnancy. Patients with other forms of chronic renal disease in whom the serum creatinine concentration prior to pregnancy is less than 1.5 mg/dL are not exposed to increased maternal or fetal risk. On the other hand, patients with serum creatinine values exceeding 1.6 mg/dL experience a high incidence of maternal and fetal complications and should avoid pregnancy. The life expectancy of recipients of a renal transplant is uncertain, and these patients should receive counselling as to the advisability of undertaking pregnancy. The maternal risk in such patients is not inordinately high, but the fetal risk is considerable.     

Regulatory role of monomorphic and polymorphic determinants of HLA class I antigens in the proliferation of T cells stimulated by autologous and allogeneic B and T lymphoid cells

Monoclonal antibodies (mAb's) to monomorphic and polymorphic determinants of HLA Class I antigens were shown to inhibit proliferation of T cells stimulated with autologous and allogeneic B and T lymphocytes. Inhibition of proliferative responses was lower when T cells were used as stimulators than when B cells were used. The inhibitory activity was similar for mAb's to monomorphic and polymorphic determinants of HLA Class I antigens, suggesting that the density of antigen-antibody complexes on the cell membrane does not play a major role in the phenomenon. The anti-HLA Class I mAb's exerted their inhibitory effect at the level of both the responding and the stimulating cells. Addition of exogenous interleukin 2 to the mixed cultures did not affect the mAb-mediated inhibition.     

Impact of ABO Incompatibility on the Development of Acute Antibody-Mediated Rejection in Kidney Transplant Recipients Presensitized to HLA

Whether the coexistence of anti-A/B antibody and donor specific anti-HLA antibody (HLA-DSA) has a synergistic impact on the development of acute antibody-mediated rejection (AAMR) in kidney transplant recipients (KTRs) is unclear. This study includes 92 KTRs who received a kidney from an ABO-incompatible (ABOi) donor or were presensitized to donor HLA (HLAs) and 292 controls (CONT). HLAs was defined as a crossmatch positivity or the presence of HLA-DSA. We compared the incidence of AAMR among ABOi (n = 58), ABOi+HLAs (n = 12), HLAs (n = 22), and CONT (n = 292) groups and evaluated the risk factors and antibody type (anti-A/B vs. HLA-DSA) responsible for AAMR. AAMR developed less frequently in ABOi and CONT than in the ABOi+HLAs or HLAs (P < 0.05 for all); however, there was no difference between the ABOi+HLAs and HLAs groups. AAMR developed more frequently with strong HLA-DSA at baseline; however, high baseline anti-A/B titer did not affect AAMR development. Strong baseline HLA-DSA was an independent predictor for AAMR, however the baseline anti-A/B titer was not. All four AAMR episodes in ABOi+HLAs were positive to HLA-DSA but not to anti-A/B. In conclusion, ABO incompatibility does not increase the risk for AAMR in HLAs KTRs.     

Polymorphisms of MICA recognized by human alloantibodies

MICA antigens are polymorphic glycoproteins expressed on the surface of human endothelial cells and other cells. Antibodies against MICA have been found in transplant recipients and were found to be associated with decreased survival of kidney allografts. In the present work, we investigated the polymorphisms that are recognized by antibodies against MICA. Soluble MICA recombinant proteins representing 11 common alleles, two hybrid alleles, and two single amino acid mutated alleles were produced. Patterns of reactivity were determined with MICA bound to Luminex beads. In some studies, sera containing antibodies against MICA were absorbed by cell lines transfected with MICA*001, MICA*002, MICA*008, and MICA*009 or with untransfected cells, followed by testing of antibody reactivity against MICA proteins bound to beads. The monoclonal antibodies and sera used in this study were found to recognize up to 14 distinct MICA epitopes as demonstrated by their differential absorption/reactivity patterns. Among these, nine epitopes correlated with a single unique amino acid: one shared two signature amino acids, one shared three signature amino acids in close proximity, and three epitopes involved multiple amino acids in a nonlinear sequence. Two groups of public epitopes (MICA-G1 and MICA-G2) were characterized. MICA shared epitopes were determined by reactivity loss in single MICA antigen bead assays by absorption with MICA transfectants. Since these epitopes may be targets for antibody binding and possibly antibody-mediated allograft rejection, epitope identification may help understand the development of MICA antibodies and to identify suitable donors for sensitized transplant recipients.     

HLA compatibility and survival of 51 chromium-labeled allogeneic thrombocytes

In 37 patients with thrombocytopenia (mostly with ITP) the survival time of 51Cr-labeled allogeneic platelets was investigated. The HLA antigens were typed in donors and recipients and the presence of HLA cytotoxins and specific thrombocyte antibodies in sera of patients were examined. In 7 cases the identity of 2 HLA antigens, in 15 cases that of 1 HLA antigen and in 15 cases the HLA incompatibility between donor and patient were found. The survival of platelets did not depend on the degree of HLA compatibility, unless the HLA cytotoxins in sera of patients appeared. The HLA, as well the specific platelet antibodies brought about the shortened platelet survival to 1 day and less. The importance of these observations for platelet kinetics is discussed.     

Renal transplant patients treated with total lymphoid irradiation show specific unresponsiveness to donor antigens the mixed leukocyte reaction (MLR)

A group of 25 cadaveric renal transplant recipients received total lymphoid irradiation (TLI) before transplantation, rabbit anti-thymocyte globulin on alternate days for 10 days after transplantation, and low dose prednisone (5 to 10 mg/day) as the sole maintenance immunosuppressive therapy. Allograft function and the mixed leukocyte reaction (MLR) were monitored serially. After 18 to 30 mo, nine patients were selected on the basis of a return of the MLR such that the mean stimulation index to a panel of normal stimulator cells was greater than or equal to 5, a stable serum creatinine level which was less than or equal to 2 mg/dl, and a history of no more than one rejection episode. The MLR of these patients' post-transplant peripheral blood mononuclear leukocytes (PBML) against cryopreserved donor cells was compared with that against cryopreserved normal third-party cells. In control experiments, the MLR of cryopreserved pre-TLI recipient PBML or fresh normal PBML were tested against the same panel of donor and third-party stimulator cells. Seven of the nine recipients showed a pattern of specific unresponsiveness to the donor cells more than 18 mo after transplantation. Preliminary attempts to identify antigen specific suppressor cells were unsuccessful. The pattern of unresponsiveness may indicate a state of specific immune tolerance to the allogeneic graft.     

Anti-idiotypic antibodies to HLA-DR4 and DR2

The aim of our study was to determine whether antibodies recognizing epitopes of HLA-DR antigens (idiotypic antibodies or Ab1) induce the production of anti-idiotypic antibodies (Ab2). We tested the capacity of the F(ab')2 fragment obtained from two sera, one with no anti-HLA antibodies (serum ES) and one depleted by absorption of anti-HLA lymphocytotoxins (serum FH), to block the anti-DR antibodies reacting with the HLA-DR antigens of the immunizing donor. The F(ab')2 fragment obtained from serum ES inhibited the anti-DR2 activity of an earlier post-delivery bleeding obtained from the same woman. The anti-idiotypic antibodies contained by this serum also inhibited the anti-DR2 activity of a reference anti-DR2 antiserum 8W907 and of an anti-MT1 antiserum 8W1231. Similarly, the F(ab')2 fragment obtained from serum FH, after absorption of her anti-DR4 antibody, inhibited the anti-DR4 activity of autologous and homologous antisera. These data suggest that sera of parous women contain anti-idiotypic antibodies directed against regulatory idiotypes of anti-DR antibodies.     

General insufficiency of the classical CDC-based crossmatch to detect donor-specific anti-HLA antibodies leading to invalid results under recipients' medical treatment or underlying diseases

Antibodies directed against HLA antigens of a given donor represent the most prominent cause for hyper-acute and acute rejections. In order to select recipients without donor-specific antibodies the complement-dependent cytotoxicity (CDC-) crossmatch as the standard procedure was established. As a functional assay it strongly depends on the availability of isolated donor lymphocytes and in particular on their vitality. However, due to several diseases or pharmacological treatment of a given recipient unexpected "false-positive" results of the CDC-crossmatch may arise. We here present three groups of patients which demonstrate the limits of the conventional crossmatch. 1) Kidney recipients before living donations exhibited positive CDC-reactions due to their conditioning using the therapeutical anti-CD20 mAb Rituximab (n=7), routinely used to deplete B-cells, or the anti-CD25 mAb basiliximab (n=2) to inhibit the proliferation of activated T-cells. 2) Recipients suffering from various leukaemias (n=5) exhibited "positive" CDC-crossmatches using PBL of the donors, although formerly these patients had never shown anti-HLA antibodies. Instead of donor-specific allo-antibodies, cytostatic agents such as 6-mercaptopurine led to an unspecific cell death. 3) Patients projected for post mortem or living kidney donations (n=44) exhibited "positive" CDC-crossmatch results which were not in accordance with their former antibody status and, partially, with high degrees of HLA-matching. These implausible results were due to underlying auto-immune diseases, mainly of the systemic immune complex type III such as lupus erythematosus, mainly leading to false-positive B-cell crossmatches by immune complexes binding to Fcγ-receptors. In all these 58 cases the alternatively performed ELISA-based "Antibody Monitoring System" (AMS-) crossmatch assay was not artifically affected, suggesting that this assay may be comprehensively established at least for the cases described.     

Superiority of B locus matching over other HLA matching in renal graft survival.

Graft survival after 348 consecutive first cadaver-donor renal transplants was significantly improved by HLA matching when recipients who had received pretransplant blood transfusions were matched with their kidney donor for two HLA-B locus antigens. No other type of HLA matching significantly improved graft survival in transfused recipients nor did any type of HLA matching in non-transfused recipients. Matching for one HLA-DR antigen had no benefit in transfused recipients. Only two patients received kidneys matched for both DR antigens and only two of those in whom DR matching had been performed had not been transfused. These results indicate that pretransplant blood transfusion and selection of graft recipients predominantly on the basis of HLA-B matching has significantly reduced the renal graft rejection rate in Newcastle upon Tyne over two years. Thus, HLA-B antigen matching should be adopted as the main criterion for kidney sharing between transplant centres.     

Adverse effect of donor-specific anti-human leukocyte antigen (HLA) antibodies directed at HLA-DP/-DQ on engraftment in cord blood transplantation

Background aimsWhile donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) in the recipient before transplantation are associated with graft failure in cord-blood transplantation (CBT), effects of DSAs other than against HLA-A, -B or -DRB1 on transplantation outcomes remained poorly understood.MethodsWe retrospectively analyzed 567 single-unit CBT recipients to evaluate impact of DSAs against HLA-DP and -DQ on CBT outcomes.ResultsAmong 143 recipients (25.2%) who had anti-HLA antibodies, nine harbored DSAs against HLA-DP or -DQ. DSAs against HLA-DP or -DQ were associated with a significantly lower neutrophil engraftment rate (55.6% versus 91.8%, P = 0.032) and with a marginally lower platelet engraftment rate (46.7% versus 75.3%, P = 0.128) at day 100 after transplantation, compared with patients without anti-HLA antibodies. Time to neutrophil and platelet engraftment in patients with DSAs for HLA-DP or -DQ was significantly longer than that in patients without anti-HLA antibodies (median, 25 versus 21 days, P = 0.002 in neutrophil; median 61 versus 46 days, P = 0.014 in platelet). Cumulative incidence of bacterial infection at day 100 was significantly greater (88.9% versus 57.1%, P = 0.024), and re-transplant-free survival was marginally lower (55.6% versus 76.8%, P = 0.132) in patients with DSAs against HLA-DP or -DQ, compared with those without anti-HLA antibodies. These findings suggest that DSAs against HLA-DP or -DQ lead to unfavorable engraftment, which may increase risk of bacterial infection, and reduce survival soon after CBT.ConclusionsOur results suggest the importance of evaluating DSAs against HLA-DP and -DQ in recipients before selecting CB units.