In vivo tumor localization using tumor-specific monkey xenoantibody, alloantibody, and murine monoclonal xenoantibody

Specific in vivo localization of antibodies reactive with human melanoma cell membrane tumor associated antigens (TAA) has been attempted using congenitally athymic nude mice bearing subcutaneous human melanoma tumor xenografts as the experimental model. IgG fractions were prepared from each of several immune and control sera. Antimelanoma antibody sources included human alloantibody obtained from melanoma patients immunized against allogeneic melanoma cells, a monkey antiserum raised by immunization against a single human melanoma cell line, and a murine monoclonal antimelanoma antibody-secreting hybridoma cell line. Localization of these radiolabeled antibodies and of control IgG preparations to tumor tissue was determined by whole body scintigraphy and by differential tissue counting. Compared with the different control IgG preparations, each of the antimelanoma IgG preparations exhibited significant specific accumulation within the melanoma tissue. However, variation existed in the ability of each antimelanoma IgG to tumor preparation to localize despite attempts to control model parameters such as tumor source, in vivo passage number and mass. This variation appears to reflect basic biologic differences between tumors in different animals and possibly differences in the antigen-binding capacities of each IgG preparation following radioiodination. This technique for tumor localization is very promising and has obvious potential for clinical application.     

Ontogeny of antipig xenoantibody and hyperacute rejection

BACKGROUNDS: Neonatal primates have been reported to receive pig hearts without hyperacute rejection (HAR). We examined the ontogeny of the anti-pig xenoantibody (XenoAb) and HAR in the neonatal and infant monkeys. METHODS: Twenty-six serum samples from 15 monkeys ages 14-192 days were subjected to hemagglutination titration against pig erythrocytes. Ten pig hearts were heterotopically transplanted into the monkeys. RESULTS: Six monkeys, ages 52-114 days, received pig hearts without HAR, and those ages 129-191 days hyperacutely rejected them. XenoAb titers were increased according to the age (Spearman's rank correlation value=0.909 (P<0.01)). XenoAb titers in 16 monkeys <4 months were significantly (P<0.01) lower than those in 10 monkeys >4 months. CONCLUSIONS: Anti-pig XenoAb titers increased with the age of the monkeys. XenoAb levels in monkeys >4 months are high enough to reject pig hearts hyperacutely.     

Serology of liver transplantation in the rat. I. Alloantibody responses and evidence for tolerance in a nonrejector combination

The antibody response against class I and class II RT1 antigens has been studied in PVG rats grafted with DA liver. In this nonrejector combination, liver grafts survive permanently in all normal recipients and in about 50% of recipients presensitized by a DA skin graft, with concurrent induction of transplant tolerance for other DA organs and skin. Using a two-stage radioimmunoassay, the anti-class I (RT1Aa) levels in sera of normal PVG recipients of DA liver grafts were found to be low (maximal titer 1:50 serum dilution or less); after peaking at 2 weeks posttransplantation, they diminished to background levels by 6 weeks. The anti-RT1Aa response showed a close parallel to cell-mediated rejection events in the liver graft recipients. In contrast, anti-class II (RT1Ba/Da) responses reached much higher titers (over 1:1000), which were maintained for several weeks before declining after 4 months. Similar observations were made in presensitized recipients. The induction of tolerance in the alloantibody response was indicated by the inability of DA skin grafts to restimulate anti-RT1 antibody in liver recipients. The observations support the picture of "split tolerance" indicated by previous cellular studies in this combination.     

人血清中非Gal异种抗体检测方法的探讨

目的  探讨人血清中抗非半乳糖（Gal）异种抗原抗体的最佳检测条件。方法  选用α-1, 3半乳糖基转移酶基因敲除（GTKO）五指山小型猪的外周血单核细胞（PBMC) 作为靶细胞,与健康人血清混合,在不同血清浓度（4.8%、16.7%、100%）和孵育时间（0.5、1.0、2.0、3.0、6.0 h）条件下,利用流式细胞仪检测GTKO猪的PBMC上结合IgM和IgG的水平。结果  在16.7%血清浓度下,孵育时间从0.5 h延长至3.0 h,能显著提高非Gal IgM结合PBMC的水平（P < 0.01）,而IgG水平的提高则差异无统计学意义（P > 0.05）。提高血清浓度也可提高非Gal IgM的结合水平,采用100%的血清浓度孵育3 h,IgM结合PBMC水平最高且有统计学意义（P < 0.01）。采用100%的血清孵育6 h,IgG结合水平升高有统计学意义（P < 0.05）。延长孵育时间和提高血清浓度不会影响PBMC的活力。结论  检测人血清中抗非Gal异种抗原抗体的最佳条件为每1×10⁵个猪PBMC,采用100%人血清浓度孵育3 h检测IgM水平,或加100%人血清浓度孵育6 h检测IgG水平。这一条件的优化有助于筛选非Gal表达抗原低表达的供体猪。     

Critical Role of NKT Cells in Posttransplant Alloantibody Production

We previously reported that posttransplant alloantibody production in CD8‐deficient hosts is IL‐4⁺CD4⁺ T cell–dependent and IgG1 isotype‐dominant. The current studies investigated the hypothesis that IL‐4‐producing natural killer T cells (NKT cells) contribute to maximal alloantibody production. To investigate this, alloantibody levels were examined in CD8‐deficient WT, CD1d KO and Jα18 KO transplant recipients. We found that the magnitude of IgG1 alloantibody production was critically dependent on the presence of type I NKT cells, which are activated by day 1 posttransplant. Unexpectedly, type I NKT cell contribution to enhanced IgG1 alloantibody levels was interferon‐γ‐dependent and IL‐4‐independent. Cognate interactions between type I NKT and B cells alone do not stimulate alloantibody production. Instead, NKT cells appear to enhance maturation of IL‐4⁺CD4⁺ T cells. To our knowledge, this is the first report to substantiate a critical role for type I NKT cells in enhancing in vivo antibody production in response to endogenous antigenic stimuli.     

Mechanisms of Alloantibody Production in Sensitized Renal Allograft Recipients

While clinical protocols have been developed to allow for successful kidney transplantation in patients with high levels of donor-specific alloantibody (DSA), significant limitations still exist including high rates of early humoral rejection and decreased long-term graft survival compared to conventional transplants. A better understanding of the mechanisms of alloantibody production at baseline and at various phases posttransplant would be an important step toward the development of improved therapeutic approaches. The goal of this review is to outline recent studies regarding antibody production in general and specific studies that illustrate what is known about alloantibody production in sensitized patients.     

Alloantibody to proteoglycan induced by osteochondral homotransplantation in rabbits

The role of possible alloantigenicity of matrix proteoglycan (PG) in the osteochondral homotransplantation of knee joints was studied in rabbits. The incidence of acute rejection could be reduced by removing marrow tissues from grafts, but 5 of 25 recipients produced IgG anti-PG, even though the rabbits immunized with pooled PG in Freund's complete adjuvant failed to produce antibody. The reactivity of antibody in grafted animals was restricted to a core protein fraction of 600k dalton and to a relatively small fraction of pooled PG. No reactivity with completely glycosylated PG was detected. Grafts of anti-PG positive recipients were stained weakly with toluidine blue and exhibited minor degenerative changes of cartilage cells. These findings suggest that either glycosylation or sulfation of PG is impaired in the grafts and that such defective PG is antigenic to the host.     

Transplant Glomerulopathy: Subclinical Incidence and Association with Alloantibody

Transplant glomerulopathy (TG) usually has been described as part of a constellation of late chronic histologic abnormalities associated with proteinuria and declining function. The current study used both protocol and clinically-indicated biopsies to investigate clinical and subclinical TG, their prognosis and possible association with alloantibody. We retrospectively studied 582 renal transplants with a negative pre-transplant T-cell complement dependent cytotoxicity crossmatch. TG was diagnosed in 55 patients, 27 (49%) based on protocol biopsy in well-functioning grafts. The cumulative incidence of TG increased over time to 20% at 5 years. The prognosis of subclinical TG was equally as poor as TG diagnosed with graft dysfunction, with progressive worsening of histopathologic changes and function. Although TG was associated with both acute and chronic histologic abnormalities, 14.5% of TG biopsies showed no interstitial fibrosis or tubular atrophy, while 58% (7/12) of biopsies with severe TG showed only minimal abnormalities. TG was associated with acute rejection, pretransplant hepatitis C antibody positivity and anti-HLA antibodies (especially anti-Class II), with the risk increasing if the antibodies were donor specific. We suggest that subclinical TG is an under-recognized cause of antibody-mediated, chronic renal allograft injury which may be mechanistically distinct from other causes of nephropathy.     

Class II Alloantibody and Mortality in Simultaneous Liver‐Kidney Transplantation

Hyperacute kidney rejection is unusual in crossmatch positive recipients of simultaneous liver–kidney transplants (SLKT). However, recent data suggest that these patients remain at risk for antibody‐mediated kidney rejection. To further investigate the risk associated with donor‐specific alloantibodies (DSA) in SLKT, we studied 86 consecutive SLKT patients with an available pre‐SLKT serum sample. Serum samples were analyzed in a blinded fashion for HLA DSA using single antigen beads (median florescence intensity ≥ 2,000 = positive). Post‐SLKT samples were analyzed when available (76%). Thirty patients had preformed DSA, and nine developed de novo DSA. Preformed class I DSA did not change the risk of rejection, patient or allograft survival. In contrast, preformed class II DSA was associated with a markedly increased risk of renal antibody mediated rejection (AMR) (p = 0.006), liver allograft rejection (p = 0.002), patient death (p = 0.02), liver allograft loss (p = 0.02) and renal allograft loss (p = 0.045). Multivariable modeling showed class II DSA (preformed or de novo) to be an independent predictor of patient death (HR = 2.2; p = 0.043) and liver allograft loss (HR = 2.2; p = 0.044). These data warrant reconsideration of the approach to DSA in SLKT.     

Alloantibody and transferable suppressor activity induced by cyclosporine and blood transfusions in the rat

The effect of cyclosporine on the alloantibody response to blood transfusion was investigated in inbred strains of rats by IHA and CELISA; recipient animals differed from the donors at the class I (RT1A) or both class I and class II (RT1B) antigens of the major histocompatibility complex. Alloantibody titers stimulated in high responder PVGu/c animals by blood transfusions were attenuated by cyclosporine; this effect was not demonstrated in low responder PVGc rats, as alloantibody titers decreased after further blood transfusions whether or not cyclosporine was given. Cyclosporine not only reduced the initial IgM response but suppressed the subsequent production of IgG. Splenocytes from rats receiving cyclosporine and blood transfusions from donors that differed from the recipients at the class I antigen were effective in suppressing the subsequent antibody response to blood transfusion. When blood transfusions from donors which differed from the recipients at both class I and class II antigenic loci were given after splenocyte transfer, a greater degree of immunosuppression was detected than if the transfusion donor differed only at the class I locus. These data suggest that the sensitization produced by blood transfusions and the persistence or decline of the alloantibody response depend upon the responder status of the recipient. Blood transfusions given with cyclosporine are capable of inducing suppressor activity that is transferable in spleen homogenates. Subsequent alloantibody responses are influenced by the class I and class II disparities of the donor and recipient animals. If these results can be extrapolated to clinical practice, cyclosporine should be given with pretransplant blood transfusions to prevent sensitization, and the transfusion donor should differ from the recipient at both class I and class II antigenic loci.     

Enhanced De Novo Alloantibody and Antibody‐Mediated Injury in Rhesus Macaques

Chronic allograft rejection is a major impediment to long‐term transplant success. Humoral immune responses to alloantigens are a growing clinical problem in transplantation, with mounting evidence associating alloantibodies with the development of chronic rejection. Nearly a third of transplant recipients develop de novo antibodies, for which no established therapies are effective at preventing or eliminating, highlighting the need for a nonhuman primate model of antibody‐mediated rejection. In this report, we demonstrate that depletion using anti‐CD3 immunotoxin (IT) combined with maintenance immunosuppression that included tacrolimus with or without alefacept reliably prolonged renal allograft survival in rhesus monkeys. In these animals, a preferential skewing toward CD4 repopulation and proliferation was observed, particularly with the addition of alefacept. Furthermore, alefacept‐treated animals demonstrated increased alloantibody production (100%) and morphologic features of antibody‐mediated injury. In vitro, alefacept was found to enhance CD4 effector memory T cell proliferation. In conclusion, alefacept administration after depletion and with tacrolimus promotes a CD4+memory T cell and alloantibody response, with morphologic changes reflecting antibody‐mediated allograft injury. Early and consistent de novo alloantibody production with associated histological changes makes this nonhuman primate model an attractive candidate for evaluating targeted therapeutics.     

Transplant Accommodation in Highly Sensitized Patients: A Potential Role for Bcl-xL and Alloantibody

Transplantation of renal allografts into recipients with circulating anti-HLA antibodies results in hyperacute rejection. In some cases, however, antibodies return without causing harm; this phenomenon has been termed 'accommodation'. We have investigated this process in human allotransplantation. We removed anti-HLA antibodies by immunoadsorption in seven highly sensitized dialysis patients who subsequently underwent renal transplantation. Immunohistochemistry of renal biopsies for IgG and antiapoptotic proteins was performed. We also developed a model of 'accommodation' using anti-HLA antibodies eluted from sensitized patients and incubated with human umbilical vein endothelial cells (HUVECs) at different concentrations. Their effect on HUVEC phenotype was then analysed. Anti-donor antibody returned in 4/7 patients, without evidence of hyperacute rejection. Three out of four of these 'accommodated' grafts showed specific endothelial up-regulation of Bcl-xL and 2/2 tested positive for endothelial IgG deposition. HUVECs incubated with subsaturating concentrations of anti-HLA antibody showed increased expression of Bcl-xL, were rendered refractory to endothelial cell activation and became resistant to complement-mediated lysis. In contrast, HUVECs incubated with saturating concentrations underwent activation and expressed low levels of Bcl-xL. In conclusion, endothelial Bcl-xL expression defines the accommodation process in human allografts and this phenotype may be initiated by exposure of endothelium to low concentrations of anti-donor HLA antibodies.     

Alloantibody Levels and Acute Humoral Rejection Early After Positive Crossmatch Kidney Transplantation

We examined the course of donor ‐ specific alloantibody (DSA) levels early after transplant and their relationship with acute humoral rejection (AHR) in two groups of positive crossmatch (+XM) kidney transplant recipients: High DSA group—41 recipients with a baseline T‐ or B‐cell flow crossmatch (TFXM, BFXM) channel shift ≥300 (molecules of equivalent soluble fluorochrome units (MESF) of approximately 19 300) who underwent pretransplant plasmapheresis (PP), and Low DSA group—29 recipients with a baseline channel shift <300 who did not undergo PP. The incidence of AHR was 39% (16/41) in the High DSA group and 31% (9/29) in the Low DSA group. Overall, mean DSA levels decreased by day 4 posttransplant and remained low in patients who did not develop AHR. By day 10, DSA levels increased in patients developing AHR with 92% (23/25) of patients with a BFXM >359 (MESF of approximately 34 000) developing AHR. The BFXM and the total DSA measured by single antigen beads correlated well across a wide spectrum suggesting that either could be used for monitoring. We conclude that AHR is associated with the development of High DSA levels posttransplant and protocols aimed at maintaining DSA at lower levels may decrease the incidence of AHR.