Low risk of anti-human leukocyte antigen antibody sensitization after combined kidney and islet transplantation

Anti-human leukocyte antigen (HLA) antibody could lead to humoral rejection and a decrease in graft survival after kidney transplantation. A recent report has suggested that islet transplantation alone is associated with a high rate of sensitization. The withdrawal of the immunosuppressive therapy because of the progressive nonfunction of the islets could explain the high rate of sensitization. Because the specific risk of immunization of multiple islet infusions remains unknown, we studied the immunization rate in our cohort of multiple islet infusions transplant recipients. De novo anti-HLA antibodies were analyzed in 37 patients after islets alone (n=8), islet-after-kidney (n=13), and simultaneous islet-kidney (n=16) transplantation by solid phase assays over time. The rate of immunization was 10.8% that is comparable with the risk of immunization after kidney transplantation alone. Multiple islet infusions do not represent a specific risk for the development of anti-HLA antibodies after combined kidney-islets transplantation.     

The effect of rabbit antithymocyte globulin on human mesenchymal stem cells

Mesenchymal stem cells (MSCs) possess immunomodulatory properties which are of key interest for their application in autoimmunity and transplantation. In transplantation, administration of MSCs has shown promising results in preclinical models and has recently moved to clinical trials. Therefore, it is important to study the interactions between MSCs and immunosuppressive drugs currently used in transplantation. We aimed to analyze the effect of rabbit antithymocyte globulin (rATG) MSCs. MSCs were obtained from perirenal fat of kidney donors and exposed to ranging doses of rATG (Thymoglobulin^®, Genzyme; 0.5–100 μg/ml). Binding of rATG, effects on viability and susceptibility to be killed by cytotoxic lymphocytes as well as effects on their immunosuppressive potential of MSCs were tested. rATG binds dose‐dependently to MSCs. This binding was associated with slightly impaired viability after 48 and 72 h when compared with nonexposed MSCs. In contrast to nontreated MSCs, rATG preexposed MSCs were susceptible to be lysed by cytokine‐activated CD8⁺ cytotoxic cells and NKT cells. The capacity of MSCs to suppress the proliferation of anti‐CD3/CD28 activated CD4 and CD8 T cells were reduced by the presence of rATG in the culture. rATG reduces the viability and antiproliferative capacity of MSCs in a dose‐dependent manner and converts them into targets for CD8 T cells and NKT cell lysis.     

Rabbit anti-human leukocyte polyclonal antibody inhibits xenogeneic cell-mediated immune responses

We studied the interactions between human monocytes and porcine endothelial cells (PEC) as well as the effects of a new generation of rabbit anti-human leukocyte polyclonal antibody (newRALG) to inhibit xenogeneic cell-mediated immune responses. Human peripheral blood mononuclear cells (PBMC) were cocultured with the florescent dye PKH-26 labeled-PEC, which showed membrane uptake by monocytes detected by florescence activated cell scanning (FACS). Scavenger receptor (SR) ligand poly-(G) or the newRALG or Thymoglobulin was added into the cocultures followed by FACS. Lymphocyte proliferation upon exposure to PEC with or without newRALG or thymoglobulin was evaluated by a xenogeneic mixed-lymphocyte-endothelial cell reaction (xMLER). FACS analysis demonstrated that CD14⁺ monocytes became positive for PKH-26 following their interaction with PKH-26-labeled PEC. These PKH-26⁺ monocytes displayed up-regulated CD40 and CD80 expression during the PBMC-PEC interaction. Furthermore, SR blockade with poly-(G) prevented PEC membrane uptake by CD14⁺ monocytes. The newRALG from rabbits immunized with activated human monocytes and lymphocytes greatly reduced SR-mediated PEC membrane uptake. xMLER demonstrated strong lymphocyte proliferation in response to PEC. Lymphocyte proliferation was dramatically inhibited in dose-dependent manner by the newRALG. In summary, monocytes up-regulate costimulatory molecules during xenogeneic interactions, indicating that they may serve as a source of T-cell costimulation during xenogeneic reactions, enguling PEC membranes. This phenomenon was inhibited by poly (G), suggesting that PEC membrane uptake was via SR. The newRALG inhibited monocyte SR-mediated PEC membrane uptake and lymphocyte proliferation in response to PEC suggesting that this new polyclonal preparation may impair the initiation of xeno-specific immune responses.     

Polymeric IgA antibody response to rabbit antithymocyte globulin in renal transplant recipients

Treatment of transplant recipients with heterologous antithymocyte globulin (ATG) can induce the production of antibodies to the ATG itself. Such responses have, however, not been fully defined in terms of the kinetics, class, and quantities of antibodies produced. We have studied these parameters in 32 renal transplant recipients who had received rabbit ATG as treatment for acute rejection episodes. Antibodies to rabbit IgG were detected in the sera of all patients; employing an enzyme-linked immunosorbent assay (ELISA), the majority of patients were shown to produce specific antibodies of the IgG, IgA, and IgM class. Anti-ATG antibodies were first detected 6-48 days after the initial injection of ATG and usually attained peak values within 23 days. The IgM and IgA responses decreased within 1-2 months, whereas the IgG response remained elevated for 2-12 months. Gel filtration studies indicated that the IgA and IgM antibodies directed to the rabbit ATG were polymeric. Furthermore, the polymeric IgA bound secretory component, indicating the presence of J chain. In 6 patients, circulating immune complexes that contained rabbit IgG were detected. The clinical symptoms and laboratory findings did not correlate with the production or quantities of the different classes of antibodies. Possible explanations for the prominent IgA response to intravenous injections of ATG are discussed.     

Stability studies on horse antithymocyte globulin.

Two equine antithymocyte globulin preparations were studied with respect to their relative rosette inhibition activity after storage at 4 and 25 C. After storage at 4 C for 48 months no activity loss was observed; 25 C storage resulted in fragmentation and eventual loss of relative rosette inhibition activity. A third preparation was studied by monkey skin graft prolongation. During a 1-year period, there was no loss of immunosuppressive activity as measured by this method and the rosette inhibition assay.     

Tools for human leukocyte antigen antibody detection and their application to transplanting sensitized patients

In recent years there have been major advances in the technology for the detection and definition of human leukocyte antigen antibodies. In this overview we describe the evolution in laboratory technology, the techniques currently available and consider their application in antibody specificity definition and in understanding a patient's sensitization profile. We discuss the importance of antibody specificity definition in facilitating efficient national organ allocation and informing clinical discussion regarding the appropriate pathway for sensitized patients awaiting renal transplantation.     

Development of anti-human leukocyte antigen class 1 antibodies following allogeneic islet cell transplantation

Currently there is minimal concern that islet allograft failure could result from the development of anti-human leukocyte antigen (HLA) antibodies reactive to the allograft. We report here a case of islet allograft failure where the recipient developed immunoglobulin G anti-HLA class I antibodies reactive to HLA antigens present in two of the three islet cell donors. The patient had no detectable anti-HLA antibodies prior to the transplant but these antibodies were detected approximately 4 months posttransplant. Of concern, these antibodies developed despite induction with anti-IL2R antibodies (Zenapex) prior to intraportal islet cell infusion, low-dose tacrolimus (12-hour troughs 3 to 5 ng/mL) and rapammune (target troughs 12 to 15 ng/mL). The patient was not presensitized with blood products or a previous allograft. Her husband, however, shared antigens present in one of the islet donors and the recipient could have been presensitized to her husband during her two pregnancies. This case clearly demonstrates that islet allografts can lead to development of anti-HLA antibodies, which can cause islet allograft failure, as is the case with solid organ transplants, and hence emphasizes the need to monitor for such antibodies pre- and posttransplant. Additionally it appears that currently recommended immunosuppression may not be sufficient to inhibit a humoral response to both alloantigens and autoantigens.     

Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antigen antibody specificities

BACKGROUND: It is difficult to assign antibody specificity for highly sensitized patients using a cell panel with multiple antigens per reaction. We describe here a single antigen bead panel for accurate identification of human leukocyte antigen (HLA) antibody specificities by flow cytometry. METHODS: A total of 110 single recombinant HLAs, including 34 A locus alleles, 57 B locus alleles, and 19 C locus alleles, were produced by a mammalian expression system. These single antigens were coated onto eight different colored microbeads, which were mixed together in one tube for simultaneous detection of HLA antibodies against eight different antigens per flow cytometry test. RESULTS: Single HLA reacted specifically with the serologically defined monoclonal antibodies. The single antigen panel provided higher resolution than the regular cell panel for antibody detection by uncovering the masked specificities. Single antigens also provided higher sensitivity than the multiple antigens coated onto beads for HLA antibody detection as demonstrated by serum dilution studies. In 10 sera from patients who had rejected a kidney transplant, single antigen beads identified antibodies to 31 of 35 antigens that were mismatched in the donor. Most important, none of the reactions were against antigens present in the recipient. CONCLUSION: An accurate and sensitive HLA antibody detection method is described using flow cytometry beads coated with single HLAs produced by recombinant technology. The single antigen beads should be useful in predicting negative crossmatch in highly sensitized organ recipients and highly sensitized patients requiring platelets.     

Clinical importance of anti-human leukocyte antigen-specific antibody concentration in performing calculated panel reactive antibody and virtual crossmatches

BACKGROUND: Highly sensitized patients develop multi-human leukocyte antigen (HLA) specific antibodies. This study measures concentrations of anti-HLA antibodies in multispecific sera by converting fluorescence intensity into molecules of equivalent soluble fluorochrome (MESF) units. This was used to determine MESF units required for a positive T and B flow crossmatches (FLXM). METHODS: MESF values of negative controls and sera from patients devoid of HLA antibodies were measured by FLXM and flow panel reactive antibody (PRA) screening beads. Fluorescence intensity values of anti-HLA specific antibodies determined by FlowPRA single antigen beads of highly sensitized patients were converted into MESF units. In addition, endpoint titers, MESF units, and % PRA of 26 sera were established. RESULTS: MESF analysis accurately predicted the outcomes of 100% of T and B FLXM of sera with strong (MESF units>18,000) donor-specific antibody (DSA). The predictive values of T and B FLXM declined to 95% and 88% with weak DSA (6,000 MESF<10,000). Endpoint titers of sera from highly sensitized patients ranged from 1:512 to 1:8 with corresponding MESF values of 452,596 to 20,000 units. However, there was no statistical difference in PRA values among these sera (95%-100%). We successfully transplanted five patients who had weak donor-specific HLA antibodies (MESF units>2,000). The graft survival at 1 year was 100% and there was no evidence of DSA posttransplant. CONCLUSION: MESF analysis is both a time and cost efficient way of measuring antibody strength. The strength of the antibody present in the sera of transplant candidates is critical for crossmatch prediction.     

The clinical significance of human leukocyte antigen antibody development in kidney transplantation

Background

This retrospective study uses the LAT-M (One Lambda Inc., Calif) screen assay to reexamine the impacts (a), of pretransplant human leukocyte antigen (HLA) antibody on long-term graft survival; (b) posttransplant HLA antibody on long-term graft survival and (c) immunosuppressive regimen on posttransplant HLA antibody development.

Patients and methods

Pretransplant sera from 222 renal transplant recipients and posttransplant sera from 216 renal transplant recipients were studied for the impact of HLA antibody on long-term graft survival.

Results

Among the patients who did not display pretransplant HLA antibodies, 85% enjoyed 5-year and 59% 10-year graft survival, whereas the patients who tested positive were 83% and 83% (P = .5596). Among the patients who did not show posttransplant HLA antibodies, 99% enjoyed 5-, 91% 10-, and 65% 15-year graft survival, whereas for the 44 patients who tested positive they were 59%, 44%, and 30%, respectively (P < .0001). Patients prescribed cyclosporine + myfortic (odds ratio 0.17, P = .05) or FK + Cellcept (odds ratio 0.36, P = .04) showed the lowest posttransplant HLA antibody development.

Conclusion

Both regimens improve graft survival.     

Effect of antithymocyte globulin on islet of Langerhans transplantation.

Lewis rats were treated with streptozotocin to induce hyperglycemia and glycosuria (400-600 mg/dl). Transplantation of approximately 1,000 dissociated islets obtained from collagenase-treated pancreases from 4 donors will promptly correct induced diabetes. Functional survival of islet allografts is related to genetic disparity between donor and recipient strains. In the closely matched Fisher-to-Lewis combination, islets functioned for a mean of 4.2+/-1 days while in the AgB-incompatible Wistar/Furth-to-Lewis combination, islets functioned for a mean of only 2.1+/-0.5 days. Treatment of recipients with antithymocyte globulin (ATG) for 3 days extended islet survival to a mean of 11.8 +/- 1.9 days in the Wistar/Furth-to-Lewis combination and to as long as 184+/-87.5 days in the Fischer-to-Lewis combination. ATG may have a role in trials of clinical islet transplants.     

新一代多克隆抗体抑制异种细胞免疫反应的研究

目的 研究新一代兔抗人免疫细胞多克隆抗体(newRALG)对异种细胞免疫反应的抑制作用.方法 应用活化的淋巴细胞和单核细胞致敏新西兰兔后获得newRALG.将PKH-26标记的猪血管内皮细胞(PEC)和正常人单个核细胞(PBMC)建立混合培养体系,培养液中分别加入newRALG、正常兔IgG、Thymoglobulin和Scavenger受体(SR)阻断剂Poly G,培养后收集PBMC,然后分别加入异硫氰基荧光素(FITC)标记的鼠抗人CD14、CD40、CD80、CD86和HLA-DR单克隆抗体.通过流式细胞术检测单核细胞对PEC膜的吞噬作用;通过淋巴细胞与PEC的混合培养体系,加入newRALG,以观察淋巴细胞的增殖反应和newRALG对增殖反应的阻断作用.结果 PBMC与PKH-26标记的PEC共培养后,PBMC中的CD86+单核细胞表达PKH-26,进一步研究发现PKH-26阳性的CD86+单核细胞不但表达CD14、CD86和HLA-DR,同时上调共刺激分子CD40和CD80的表达水平.正常兔IgG(作为阴性对照)对单核细胞吞噬PEC膜无阻断作用,Thymoglobulin具有较低的阻断效果,newRALG与Poly G相似,均具有较高的阻断作用.正常未经刺激的人淋巴细胞不具备增殖能力,经灭活的PEC刺激后,人淋巴细胞具有高水平的免疫增殖反应.正常兔IgG不能阻断PEC对人淋巴细胞的免疫增殖反应;Thymoglobulin的抑制作用随浓度的降低而增强;高浓度的newRALG不能抑制人淋巴细胞的免疫增殖反应,但低浓度的newRALG则显示出强大地抑制人淋巴细胞免疫增殖反应的作用.结论 单核细胞在异种免疫反应过程中发挥重要作用;newRALG可有效抑制单核细胞的吞噬功能和淋巴细胞的免疫增殖反应,从而有效的抑制异种移植排斥反应.     

Interferon-alpha therapy and anti-human leukocyte antigen antibodies in hepatitis C virus-positive patient: case report

Interferon-alpha (IFN-alpha) is currently the only treatment for patients with chronic hepatitis C. Yet it can induce acute renal transplantation rejection possibly by stimulating humoral responses. We tested patient sera for detection of donor-specific anti-human leukocyte antigen (HLA) antibodies observing an increased panel-reactive antibodies value after IFN-alpha therapy. Then, we also investigated whether antiviral treatment with IFN-alpha was related to an increased and/or different production of class I and class II anti-HLA antibodies. Patient sera analysis performed by a cytofluorimetric method using flow PRA tests showed the appearance of new HLA-antibody specificities. This study underlined that INF-alpha therapy modifies a patient's immune profile; hence, it is recommended to confirm HLA-antibody specificities after treatment in order to protect recipients from enhanced rejection risk owing to a false-negative donor-specific cross-match.