Effect of Treatment With Tabalumab, a B Cell-Activating Factor Inhibitor, on Highly Sensitized Patients With End-Stage Renal Disease Awaiting Transplantation.

B cell-activation factor (BAFF) is critical for B cell maturation. Inhibition of BAFF represents an appealing target for desensitization of sensitized end-stage renal disease (ESRD) patients. We conducted a Phase 2a, single-arm, open-label exploratory study investigating the effect of tabalumab (BAFF inhibitor) in patients with ESRD and calculated panel reactive antibodies (cPRAs) >50%. The treatment period duration was 24 weeks. Eighteen patients received tabalumab, at doses of 240-mg subcutaneous (SC) at Week 0 followed by 120-mg SC monthly for 5 additional months. Patients were followed for an additional 52 weeks. Immunopharmacologic effects were characterized through analysis of blood for HLA antibodies, BAFF concentrations, immunoglobulins, T and B cell subsets, as well as pre- and posttreatment tonsil and bone marrow biopsies. Significant reductions in cPRAs were observed at Weeks 16 (p = 0.043) and 36 (p = 0.004); however, absolute reductions were small (<5%). Expected pharmacologic changes in B cell subsets and immunoglobulin reductions were observed. Two tabalumab-related serious adverse events occurred (pneumonia, worsening of peripheral neuropathy), while the most common other adverse events were injection-site pain and hypotension. Three patients received matched deceased donor transplants during follow-up. Treatment with a BAFF inhibitor resulted in statistically significant, but not clinically meaningful reduction in the cPRA from baseline (NCT01200290, Clinicaltrials.gov).

Identification of a novel HLA-DRB1 allele, DRB1*09:20, by sequence-based typing in an unrelated bone marrow donor.

The novel allele HLA-DRB1*09:20 differs from HLA-DRB1*09:01:02 by one nucleotide substitution at codon 9 (AAG->CAG) and differs from HLA-DRB1*09:07 by one nucleotide substitution at codon 13 (TAT->TTT).

Effect of desensitization in solid organ transplant recipients depends on some cytokines genes polymorphism.

Desensitization (DS) is widely used to decrease PRA in solid organs transplant candidates (TC). Various numbers of cycles of DS are required to reduce or eliminate donor specific antibodies (DSA). The goal of this study was to investigate if there was a correlation between polymorphism (PM) of some cytokine genes and intensity of DS required to make the recipient/donor cross match compatible. Thirty-one TCs were included in the study. Antibody specificity, percent of reactive antibodies (PRA) and serum concentration of cytokines were analyzed using the LUMINEX platform. PCR-SSP method was used for IL-1alpha, IL-1beta, IL-1R, IL-1Ralpha, IL-4Ralpha, IL-12, IFNgamma, TGFbeta1, TNFalpha, IL-2, IL-4, IL-6 and IL-10 gene PM analysis. Significant relationship between PM of genes encoding IL-4Ralpha, IFNgamma and IL-12 (p70) and susceptibility to DS was demonstrated (p=0.04, p=0.01 and p=0.05 respectively). Correlation between elevated serum level of IL-12 (p70) and A/A or C/A genotype at -1188 position was found in resistant to DS TCs (p=0.015). These results indicate that analysis PM of genes encoding IL-4R, IFNgamma and IL-12 enables to define the DS strategy in TCs more accurately regarding the number of plasmapheresis (PP) cycles and dose of intravenous immunoglobulin (IVIG).

Quantitative analysis of chimerism using a short tandem repeat method on a fluorescent automated DNA sequencer.

Stem cell transplantation (SCT) is the treatment of choice for a number of malignant and nonmalignant diseases. Monitoring of SC engraftment or microchimerism (MC) is important for diagnosis of relapse, rejection or graft vs. host disease (GVHD). The goal of this study was to develop a sensitive and relatively simple method for MC lineage analysis using the Visible Genetics fluorescence automated sequencer. Sensitivity of the method was studied by polymerase chain reaction (PCR) amplification of informative short tandem repeats (STR) using donor/recipient DNA mixtures as the templates and DNA extracted from donor and recipient CD3+, CD19+ and CD15+ cells mixed at various ratios. Semi-quantitative analysis was performed using the Visible Genetics software and percent of donor specific signal was calculated. The sensitivity of this method varied from 0.8% to 6.2% for both DNA and cellular MC in CD3+, CD19+ and CD15+ subsets. Regression analysis revealed linearity (r = 0.94) between the number of donor cells in the mixture and intensity of MC fluorescent signal. These data indicate that the Visible Genetics polyacrylamide gel sequencer can be successfully used for MC analysis in SC recipients providing a relatively high level of sensitivity.

The number of amino acid residues mismatches correlates with flow cytometry crossmatching results in high PRA renal patients.

Highly sensitized renal transplant candidates present a group at high risk for acute and chronic rejection. The probability of finding compatible donors for these recipients is significantly lower in comparison to those who have low PRA values. As a consequence, these patients spend longer time on the waiting list and become tethered to dialysis. The results of final cross match (XM) are critical for making a decision about whether such a candidate receives an organ or not. The degree of donor and recipient HLA compatibility predicts the results of XM. The goal of this study was to expand a variety of acceptable HLA-AB mismatches (MM) for high PRA kidney recipients using the HLAMATCHMAKER algorithm. This strategy focuses on the fine structural features of HLA polymorphism comprising amino acid residues or triplets (AAT), which are located in alpha-helical coils of HLA molecules and are available to antibodies. We analyzed serum samples from thirty-nine highly alloimmunized recipients (PRA > or = 85%). The level of sensitization was detected using FlowPRA Class I Screening Test. This group of transplant candidates included thirteen recipients who demonstrated negative results of final T/B FCXM and twenty-six, who were FCXM positive. The application of the HLAMATCHMAKER algorithm based on the HLA class I donor and recipient typing allowed us to detect the total number of AATMM as well as the number of immunogenic AAT in both FCXM negative and FCXM positive groups of recipients. Significantly greater numbers of both total and highly immunogenic AATMM have been emerged in the group of FCXM positive patients. Furthermore, the results of this analysis have shown a high degree of probability of positive FCXM if the number of highly immunogenic AATMM was > or = 1 (chi(2) = 22.9 Yate's correction; p = 0.000001). We did not observe overlapping between antibody specificity and permissible HLA-AB MM detected using the HLAMATCHMAKER strategy. Thus, the number of highly immunogenic AATMM can serve as a reliable predictive value for final FCXM results in highly sensitized renal transplant candidates. The HLAMATCHMAKER algorithm appears to be the proper strategy to find donors for high PRA recipients.

Allele-specific in situ analysis of microchimerism by fluorescence resonance energy transfer (FRET) in nonhuman primate tissues.

Rhesus monkeys are relevant models for tolerance induction. Hematopoetic chimerism is believed to be one of these strategies. The purpose of this study was to detect donor class I A locus allele specific mRNA in Rhesus monkey kidney recipient. We report here for the first time the results of frequency resonance energy transfer (FRET) hybridization technology in frozen tissues. Frequency resonance energy transfer hybridization was performed by using two Mamu-A*05 allele specific oligonucleotides: a donor probe labeled with FITC and acceptor probe conjugated to Texas Red. The PCR-SSP microchimerism analysis method produced 0.05% and 0.5% of donor DNA for Mamu-DRB1*1002 and Mamu-DRBw301/3 alleles, respectively. The donor cells were detected in mesenteric and/or inguinal lymph nodes, spleen, and liver, where the signal was the strongest. The results of FRET hybridization demonstrated the identical staining pattern in the recipient frozen tissues to that determined by PCR-SSP. Following FRET hybridization, the sections underwent immunohistochemical analysis, which revealed that donor cells had CD8+ phenotype. We demonstrate here for the first time that FRET in situ hybridization technique can be utilized for microchimerism analysis in frozen tissues. We conclude that using two donor mRNA specific oligonucleotide probes, rather than one, produce higher specificity.

Phenotypic and functional analysis of T-cell recovery after anti-CD3 immunotoxin treatment for tolerance induction in rhesus macaques.

T-cell reduction utilizing specific antibody has been widely used in human transplantation, and is a cornerstone of several tolerance induction strategies in nonhuman primates. We have established a population of long-term tolerant rhesus macaques induced with an anti-CD3epsilon immunotoxin (IT). This treatment effects transient, specific and profound ablation of T cells in blood and lymphoid tissues. In most instances the IT was used in combination with the NF-kappaB inhibitor, 15-Deoxyspergualin. This 2-week long protocol produces a "window of opportunity" for tolerization in which the animal exhibits an enduring quiescent state of unresponsiveness to the allograft, all accomplished without maintenance immunosuppressive drugs. During this induction period, the treated immune system bears some resemblance to that of the neonate, in that T cell numbers are abnormally low and antigen presentation by dendritic cells is precluded by an arrest in their NF-kappaB dependant maturation. In addition, IL-4 production is prominent during and after the tolerance induction interval. For this study we focused on measuring the monkey's ability to repopulate T cells with particular emphasis on the memory T-cell phenotype. Three "memory" phenotypes were utilized; CD3(+)CD45RO(+), CD3(+)CRTH2(+), and CD3(+)CD4(+)CD8(+). All three phenotypes exhibited different patterns of recovery, all of which included transient bursts in their numbers during repopulation. We also estimated thymic activity after T-cell ablation with the use of a newly-described RTE or recent thymic émigré phenotype (a naïve CD8(+)CD103(+) T cell). This marker revealed production of RTE cells including supranormal levels at approximately 6 months post-transplant, implicating thymic function in the repopulation of T-cells. Finally, we measured antibody responses to a panel of antigens (vaccines, environmental antigen, and foreign proteins) that indicated there was no apparent loss of immunologic function during or after the tolerance induction period. Results of studies of T-cell receptor repertoire expression suggest preservation of the pretreatment repertoire, which is consistent with rapid recovery of immune competence to the test antigens. Taken together, these results suggest that while aggressive, this tolerance induction protocol does not appear to incur a prolonged immunologically-compromised state, if at all.

Six mamu-A locus alleles defined by a polymerase chain reaction sequence specific primer method.

Rhesus monkeys are relevant models for human diseases and transplantation. In each case, a complete understanding of these models requires knowledge of macaque MHC. Due to high polymorphism and multiple genes per haplotype, it has been difficult to develop a rapid typing method for rhesus monkey MHC class I. We developed a simple and rapid PCR-SSP strategy for rhesus monkey Mamu-A locus typing. Fifty-two rhesus monkeys were included in the study. Six rhesus monkey allel-specific primer pairs were designed based on published Mamu-A locus gene sequences. Allele-specific PCR products ranged in size from 346 to 788 bp; 5' and 3' Mamu-A locus allele specific primers were located in the second and third exons, respectively. Specific PCR product gel purification was followed by direct sequencing, without subcloning, in both directions. Our data showed variability in the number of Mamu-A alleles ranging from 1 to 4 per genotype. The highest frequencies were observed for Mamu-A*02, -A*04, and -A*03 alleles. Thus, we report here the first PCR-SSP typing method for Mamu-A*02, -03, -04, -05, -06, and -07 array of class I alleles. This technique appears to be a highly reproducible and discriminatory method for detecting this subset of class I A locus genes in rhesus monkeys.

Durable donor-specific T and B cell tolerance in rhesus macaques induced with peritransplantation anti-CD3 immunotoxin and deoxyspergualin: absence of chronic allograft nephropathy.

Tolerance induction can prevent acute kidney allograft rejection without chronic immunosuppression. It is uncertain whether specific tolerance can prevent chronic allograft nephropathy (CAN), which involves both nonimmune and immune injury. This report provides evidence that immunologically tolerant macaques, induced with immunotoxin and deoxyspergualin, developed neither acute rejection nor CAN. Long survivors, bearing MHC-mismatched grafts without chronic immunosuppression for 0.8 to 3.4 years, exhibited general immune competence with donor-specific T and B cell tolerance and no functional or histological evidence of CAN. Stringent criteria for tolerance were satisfied by specific prolongation of donor skin grafts with rapid rejection of third-party skin, followed by indefinite acceptance of a second donor kidney graft and establishment of microchimerism. Primate tolerance with documented absence of CAN may give impetus to the clinical application of tolerance.

Specificity of preformed alloantibodies causing B cell positive flow crossmatch in renal transplantation.

The specificity of alloantibodies (alloAb) and their clinical significance in association with T-/B+ flow cytometry crossmatch (FCXM) in kidney transplantation are not clearly defined. This study was undertaken to examine the HLA specificity and clinical relevance of Ab causing B+ FCXM in pre-transplant (final XM) recipients' serum samples. Final FCXM serum samples were analyzed from 457 renal transplant patients followed for 10 months post-transplantation. Two hundred and sixty patients had T-/B+ final FCXM. The control group included 197 recipients with T-/B- FCXM at time of transplantation. Class I/class II PRA and specificity of anti-HLA class I and class II Ab in final FCXM serum samples were analyzed by FlowPRA Class I Screening Test and FlowPRA Class II Screening Test. We found no correlation between graft outcome and pre-transplant T-/B- and T-/B+ FCXM status. Additionally, we observed no clinical relevance of B+ FCXM in retransplant patients. However, MCS > or =200 in B+ FCXM retransplant recipients was associated with anti-class II Ab to previous mismatches in regrafted patients (n = 46). This finding was confirmed by specificity analysis of anti-DR/DQ Ab in patients with high ( > or =15%) class II PRA. In 63% (12 of 19) of retransplants having T-/B+ FCXM, we defined the specificity of alloAb to first graft mismatched class II antigens. In contrast, anti-class II Ab was detected in only 5.7% (2 of 35) of single-graft recipients with different PRA values. Significantly greater MCS (240 +/- 61 vs. 163 +/- 48; p = 0.022) was observed in retransplant patients having short ( < or =5 m) previous graft survival time (PGST) than in those with long PGST ( > or =5 m). Only 2% of retransplant recipients with B + FCXM had non-HLA Ab. In contrast, the overwhelming majority of primary recipients had no detectable alloAbs. No significant difference in class I PRA was found between B- and B+ FCXM recipients. However, class II PRA was significantly higher in patients having B + FCXM (p = 0.028). Collectively, these data show that MCS intensity is not always a reliable criterion for anti-HLA Ab detection because of the presence of non-HLA Ab. These results can be explained by low titers of anti-class II Ab, at which concentration these Ab cannot produce a deleterious effect. FlowPRA and Flow screen beads appeared to be reliable and sensitive methods for detection and specificity analysis of anti-class II alloAb.

Peritransplant tolerance induction in macaques: early events reflecting the unique synergy between immunotoxin and deoxyspergualin.

BACKGROUND: Day of transplant T cell depletion with anti-CD3 immunotoxin or F(Ab)2 immunotoxin induces stable tolerance to renal allografts in rhesus monkeys given 15-deoxyspergualin (DSG), a NF-kappaB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-kappaB are involved in dendritic cell (DC) maturation, we asked if impaired DC maturation and Th2-type cytokine deviation might be related to the synergistic effect of DSG in this novel model. METHODS: Immunosuppression was initiated 4 hr before transplanting a major histocompatibility complex mismatched renal allograft. Some groups received a supplemental 5-day course of cyclosporine A or DSG or a 15-day course of DSG. Peripheral lymph nodes were sequentially examined for presence of mature DC. In vitro effects of DSG on PC-induced maturation of DC were also examined. RESULTS: Allografts survived without rejection in 87% of recipients given immunotoxin or F(Ab)2 immunotoxin with DSG x 15 days, in 50% with DSG x 5 days, and 0% with cyclosporine A. The longest DSG survivors are >1000 days with normal graft function and tolerance validated, including acceptance of challenge second donor kidneys without treatment. DSG-treated recipients were unique in developing polarized Th2-type plasma cytokines. In DSG recipients, mature DC were significantly reduced in day +5 lymph node biopsies, with complete repopulation by 30 days. In vitro studies verified an inhibitory effect of DSG on DC maturation. CONCLUSIONS: The study suggests DSG arrests DC maturation. The unusual synergy of immunotoxin and DSG apparently involves coincidental reduction in lymph node T cell mass and mature DC, a transient circumstance favoring development of stable tolerance.

DR non-B1 mismatches influence allogeneic MLR-induced TH1- or TH2-like cytokine responses in rhesus monkeys.

Human and nonhuman primates have multiple DR B1 and non-B1 alleles. However, the role of mismatched DR non-B1 alleles in primary alloimmune responses is not well understood. Macaques, which share close DNA homologies with human MHC genes and have a high number of beta-chain genes in the DR subregion, are preeminent preclinical models for immunologic studies of transplant tolerance and immunosuppression. In this study, we examined the effect of allogeneic MHC Class II DRB mismatches in Th1- and Th2-like cytokine responses elicited in one-way MLR cultures in rhesus macaques. An ELISPOT method was used to estimate cytokine secretion at the single cell level. Molecular typing for DRB1 and DR non-B1 alleles was performed by a moderate-high resolution PCR-SSP method using a panel of 55 primer pairs covering 74 DRB alleles and clusters. Of 35 unrelated combinations, 66% had multiple (> or = 2) allelic MM at DRB1 and DR non-B1 with no significant correlation between numbers of DRB1 and DR non-B1 mismatches. Pairs with 1 or 0 MM were assigned to a mono/null MM group to obtain sufficient numbers for statistical analysis. The pairs differing by multiple vs. mono/null DRB1 MM showed no significant difference in cytokine prevalence (P = 0.69). In contrast, high IFN-gamma/ IL4 SFC ratios were noted in pairs with multiple vs. mono/null DR non-B1 MM (p = 0.0009). IFN-gamma/IL-10 spot forming cell (SFC) ratios were consistent with IFN-gamma/IL-4 SFC ratios (r = 0.98). Multiple DR non-B1 mismatches showed a trend towards higher MLR proliferative responses, although the stimulation index did not reflect the dominant cytokine response. These observations suggest a bias towards Th1-like cytokine production under allostimulation with multiple DR non-B1 gene products. Further study of the primary structure of DR non-B1 determinants may be helpful in understanding the fine molecular mechanisms governing the regulation of cytokine profiles during allostimulation in primates.

Peritransplant tolerance induction with anti-CD3-immunotoxin: a matter of proinflammatory cytokine control.

BACKGROUND: Tolerance is gaining momentum as an approach to reduce lifelong immunosuppressive therapy while improving transplant longevity. Anti-CD3 immunotoxin (IT), FN18-CRM9, has potential to induce tolerance owing to its exceptional ability to deplete sessile lymph node T cells. However, if initiated at the time of transplantation, alpha-CD3-IT alone elicits a proinflammatory cytokine response, precluding establishment of tolerance. METHODS: Four groups of rhesus monkeys received kidney allografts and immunosuppression. Three groups received alpha-CD3-IT alone or alpha-CD3-IT supplemented with 15-deoxyspergualin (DSG) and/or methylprednisolone (MP). One group received alpha-CD3-monoclonal antibody with DSG and MP. Cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: Supplementing peritransplant alpha-CD3-IT treatment with a brief course of DSG and MP promoted rejection-free kidney allograft acceptance in 75% of macaques followed for up to 550 days. Among those given alpha-CD3-IT alone or with MP, none were long-term survivors. Tolerance developed after alpha-CD3-IT, DSG, and MP treatment, but not when the unconjugated a-CD3 monoclonal antibody was substituted for IT. Systemic production of proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha induced after peritransplant alpha-CD3-IT was prevented only in animals given DSG. Despite high levels of interleukin (IL)-12 in the first month after transplant, tolerant recipients exhibited IL-12 resistance, as evidenced by baseline plasma levels of IFN-gamma but elevated IL-4. DSG was shown to inhibit IL-12-driven IFN-gamma production by a mechanism associated with inhibition of nuclear factor kappa-B. CONCLUSIONS: In this model, peritransplant induction of tolerance is promoted by efficient elimination of sessile lymph node T cells and control of the proinflammatory IFN-gamma response by a mechanism that appears to involve resistance to IL-12.

Preclinical studies of allograft tolerance in rhesus monkeys: a novel anti-CD3-immunotoxin given peritransplant with donor bone marrow induces operational tolerance to kidney allografts.

A major challenge in clinical transplantation today is to design a practical and effective protocol for tolerance induction compatible with cadaver organ transplantation. A preclinical rhesus monkey kidney allograft model using immediate peritransplant anti-CD3 immunotoxin (anti-CD3-IT) and donor bone marrow (DBM) is shown here to induce operational tolerance with prolonged graft survival in the absence of chronic immunosuppressive drugs. Bone marrow harvested from the kidney donor was depleted of mature alloantigen-presenting cells and T cells by removing DR(bright) cells and CD3(bright) cells, respectively. In outbred, major histocompatibility complex-incompatible donor-recipient pairs with high pretransplant mixed lymphocyte response and cytotoxic T lymphocyte precursor activity, four of six allografts survived for periods of 120 days to >1.5 years. Graft acceptance after peritransplant treatment followed robust elimination of both peripheral blood T cells and lymph node T cells. In most recipients given anti-CD3-IT and DBM infusion, anti-donor immunoglobulin G responses were completely inhibited. Microchimerism was observed in all recipients studied, including those not given DBM, but levels of microchimerism did not correlate with graft survival. Anti-CD3-IT induction in combination with modified DBM protocols such as the depletion of mature T cells and DR(bright) antigen-presenting cells may offer new opportunities to improve clinical tolerance protocols beyond those attempted in the clinic to date. Overall, these results with anti-CD3-IT show promise for development of cadaver transplant tolerance induction.