Anti-HLA alloantibodies of the IgA isotype in re-transplant candidates part II: Correlation with graft survival.

We reported previously on the widespread occurrence of anti-HLA alloantibodies of the IgA isotype (anti-HLA IgA) in the sera of solid-organ re-transplantation (re-tx) candidates (Arnold et al., ). Specifically focussing on kidney re-tx patients, we now extended our earlier findings by examining the impact of the presence and donor specificity of anti-HLA IgA on graft survival. We observed frequent concurrence of anti-HLA IgA and anti-HLA IgG in 27% of our multicenter collective of 694 kidney re-tx patients. This subgroup displayed significantly reduced graft survival as evidenced by the median time to first dialysis after transplantation (TTD 77 months) compared to patients carrying either anti-HLA IgG or IgA (TTD 102 and 94 months, respectively). In addition, donor specificity of anti-HLA IgA had a significant negative impact on graft survival (TTD 74 months) in our study. Taken together, our data strongly indicate that presence of anti-HLA IgA, in particular in conjunction with anti-HLA-IgG, in sera of kidney re-tx patients is associated with negative transplantation outcome.

Effects of weak/non-complement-binding HLA antibodies on C1q-binding.

It is unknown under what conditions and to what extent weak/non-complement (C)-binding IgG subclasses (IgG2/IgG4) can block C1q-binding triggered by C-binding IgG subclasses (IgG1/IgG3). Therefore, we investigated in vitro C1q-binding induced by IgG subclass mixtures targeting the same HLA epitope. Various mixtures of HLA class II specific monoclonal antibodies of different IgG subclasses but identical V-region were incubated with HLA DRB1*07:01 beads and monitored for C1q-binding. The lowest concentration to achieve maximum C1q-binding was measured for IgG3, followed by IgG1, while IgG2 and IgG4 did not show appreciable C1q-binding. C1q-binding occurred only after a critical amount of IgG1/3 has bound and sharply increased thereafter. When both, C-binding and weak/non-C-binding IgG subclasses were mixed, C1q-binding was diminished proportionally to the fraction of IgG2/4. A 2- to 4-fold excess of IgG2/4 inhibited C1q-binding by 50%. Very high levels (10-fold excess) almost completely abrogated C1q-binding even in the presence of significant IgG1/3 levels that would usually lead to strong C1q-binding. In sensitized renal allograft recipients, IgG subclass constellations with ≥ 2-fold excess of IgG2/4 over IgG1/3 were present in 23/66 patients (34.8%) and overall revealed slightly decreased C1q signals. However, spiking of patient sera with IgG2 targeting a different epitope than the patient's IgG1/3 synergistically increased C1q-binding. In conclusion, if targeting the same epitope, an excess of IgG2/4 is repressing the extent of IgG1/3 triggered C1q-binding in vitro. Such IgG subclass constellations are present in about a third of sensitized patients and their net effect on C1q-binding is slightly inhibitory.

Donor Specificity but Not Broadness of Sensitization Is Associated With Antibody-Mediated Rejection and Graft Loss in Renal Allograft Recipients.

Panel-reactive antibodies are widely regarded as an important immunological risk factor for rejection and graft loss. The broadness of sensitization against HLA is most appropriately measured by the "calculated population-reactive antibodies" (cPRA) value. In this study, we investigated whether cPRA represent an immunological risk in times of sensitive and accurate determination of pretransplantation donor-specific HLA antibodies (DSA). Five hundred twenty-seven consecutive transplantations were divided into four groups: cPRA 0% (n = 250), cPRA 1-50% (n = 129), cPRA 51-100% (n = 43), and DSA (n = 105). Patients without DSA were considered as normal risk and received standard immunosuppression without T cell-depleting induction. Patients with DSA received an enhanced induction therapy and maintenance immunosuppression. Surveillance biopsies were performed at 3 and 6 months. Median follow-up was 5.7 years. Among the three cPRA groups, there were no differences regarding the 1-year incidence of ABMR (p = 0.16) and TCMR (p = 0.75). The 5-year allograft survival rates were similar and around 87% (p = 0.28). The estimated glomerular filtration rate at last follow-up was 50-53 mL/min (p = 0.45). On multivariable Cox proportional hazard analysis, the strongest independent predictor for ABMR and (death-censored) graft survival was pretransplantation DSA. cPRA were not predictive for ABMR, TCMR, or (death-censored) graft survival. We conclude that with current DSA assignment, the broadness of sensitization measured by cPRA does not imply an immunological risk.

Detection of newly antibody-defined epitopes on HLA class I alleles reacting with antibodies induced during pregnancy.

The determination of HLA mismatch acceptability at the epitope level can be best performed with epitopes that have been verified experimentally with informative antibodies. The website-based International Registry of HLA Epitopes (http://www.epregistry.com.br) has a list of 81 antibody-verified HLA-ABC epitopes but more epitopes need to be added. Pregnancy offers an attractive model to study antibody responses to mismatched HLA epitopes which can be readily determined from the HLA types of child and mother. This report describes a HLAMatchmaker-based analysis of 16 postpregnancy sera tested in single HLA-ABC allele binding assays. Most sera reacted with alleles carrying epitopes that have been antibody-verified, and this study focused on the reactivity of additional alleles that share other epitopes corresponding to eplets and other amino acid residue configurations. This analysis led in the identification of 16 newly antibody-defined epitopes, seven are equivalent to eplets and nine correspond to combinations of eplets in combination with other nearby residue configurations. These epitopes will be added to the repertoire of antibody-verified epitopes in the HLA Epitope Registry.

Predicted Indirectly Recognizable HLA Epitopes Presented by HLA-DRB1 Are Related to HLA Antibody Formation During Pregnancy.

Pregnancy can prime maternal immune responses against inherited paternal HLA of the fetus, leading to the production of child-specific HLA antibodies. We previously demonstrated that donor-specific HLA antibody formation after kidney transplantation is associated with donor-derived HLA epitopes presented by recipient HLA class II (predicted indirectly recognizable HLA epitopes presented by HLA class II [PIRCHE-II]). In the present study, we evaluated the role of PIRCHE-II in child-specific HLA antibody formation during pregnancy. A total of 229 mother-child pairs were HLA typed. For all mismatched HLA class I molecules of the child, we subsequently predicted the number of HLA epitopes that could be presented by maternal HLA class II molecules. Child-specific antigens were classified as either immunogenic or nonimmunogenic HLA based on the presence of specific antibodies and correlated to PIRCHE-II numbers. Immunogenic HLA contained higher PIRCHE-II numbers than nonimmunogenic HLA. Moreover, the probability of antibody production during pregnancy increased with the number of PIRCHE-II. In conclusion, our data suggest that the number of PIRCHE-II is related to the formation of child-specific HLA antibodies during pregnancy. Present confirmation of the role of PIRCHE-II in antibody formation outside the transplantation setting suggests the PIRCHE-II concept is universal.

Inter-individual differences in HLA expression can impact the CDC crossmatch.

How human leucocyte antigen (HLA) expression levels on human lymphocytes relate to clinically relevant in vitro cytotoxicity testing has not been defined. Here, cross-sectional (n = 14) and longitudinal (n = 6) semi-quantitative assessment of HLA expression on lymphocytes was performed. Complement-dependent cytotoxicity (CDC) and cellular allo-reactivity were assessed vis-à-vis target cells with defined levels of HLA expression. On CD4(+) and CD8(+) T-cells, and on B-cells, intra-individual HLA levels varied ≤1.5-fold, whereas inter-individual HLA expression varied 2.34-fold and 2.07-fold on CD4(+) and CD8(+) T-cells, respectively, and 2.90-fold on B-cells. Importantly, CDC crossmatch reactions induced by anti-HLA-A2 monoclonal antibody as well as patient sera solely containing HLA-A2 antibodies were significantly impacted by HLA-A2 expression levels on donor cells. Likewise, cytotoxicity of HLA-A2 reactive effector cells was induced proportionate to availability of HLA-A2. These data demonstrate that human HLA expression on lymphocytes from healthy blood donors is fairly stable intra-individually, yet varies significantly from person to person. Variability in HLA expression levels can impact functional cytotoxic reactions in vitro, including the widely used CDC crossmatch assay. Prospective studies are required to test the clinical relevance of this finding.

Algorithms for the determination of unacceptable HLA antigen mismatches in kidney transplant recipients.

One of the major tasks of human leukocyte antigen (HLA) laboratories is the pretransplant determination of unacceptable HLA antigen mismatches (UAM) in organ transplant recipients. HLA antigen specificities are determined against which the patient has circulating alloantibodies that are expected to harm the transplanted organ. Using the information on UAM, negative crossmatch (XM) prediction or 'virtual XM' is possible when a potential donor's complete HLA typing is available. Before the introduction of solid-phase antibody detection assays, UAM were determined using the complement-dependent cytotoxicity methodology. After the introduction of the single antigen bead technique, however, various UAM determination algorithms have emerged. In this report, six different laboratories worldwide present how they determine UAM in their collective of kidney transplant recipients in the pretransplant phase and proceed thereafter to transplantation.

Frequency and determinants of pregnancy-induced child-specific sensitization.

The aim of this study was to define the frequency and determinants of pregnancy-induced child-specific sensitization shortly after full-term delivery using sensitive single HLA-antigen beads (SAB) and high resolution HLA-typing of the mothers and their children (n = 301). A positive SAB result was defined by a background normalized ratio >1 or a mean fluorescence intensity (MFI) >300, >500 and >1000, respectively. The overall frequency of pregnancy-induced sensitization determined by SAB shortly after full-term delivery was between 45% (MFI > 1000 cut-off) and 76% (ratio cut-off). The rate of child-specific sensitization at the HLA-A/B/C/DRB1 loci was between 28% (MFI > 1000 cut-off) and 38% (ratio cut-off). The number of live birth was associated with a higher frequency of sensitization, which was driven by child-specific, but not third party HLA-antibodies. There was a clear hierarchy of sensitization among the investigated loci (B-locus: 31%; A-locus: 26%; DRB1-locus: 20%; C-locus: 15%; p < 0.0001). Some mismatched paternal HLA-antigens led to a significantly higher rate of sensitization than the average (e.g. HLA-A2, HLA-B49, HLA-B51, HLA-C*15). Furthermore, the mother's own HLA-phenotype--especially HLA-A/B homozygosity--was associated with a higher rate and broadness of sensitization. The number of mismatched HLA-A/B/C eplets strongly correlated with the rate of child-specific class I sensitization.

Telomeric rather than centromeric activating KIR genes protect from cytomegalovirus infection after kidney transplantation.

Cytomegalovirus (CMV) infection is a common complication after organ transplantation. Previous studies have demonstrated that activating killer-cell immunoglobulin-like receptors (KIR) may reduce the rate of CMV infection. KIR genes can be divided into haplotype A (containing a fixed set of inhibitory receptors) and haplotype B (carrying additional activating KIR genes). The KIR locus is divided into a centromeric and a telomeric portion, both of which may carry A or B haplotype motifs. We studied a cohort of 339 kidney transplant recipients to elucidate which KIR genes protect from CMV infection. CMV infection occurred in 139 patients (41%). Possession of telomeric (hazard ratio 0.64, 95% confidence interval 0.44-0.94, p = 0.02) but not centromeric (HR 0.86, 95% CI 0.60-1.23, p = 0.41) B motifs was associated with statistically significant protection from CMV infection. Due to linkage disequilibrium, we were not able to identify a single protective gene within the telomeric B complex (which may contain the KIR2DS1, KIR3DS1, KIR2DL5A and KIR2DS5 genes). The presence of known or putative ligands to activating KIR did not significantly modify the influence of telomeric B group genes. We confirm that B haplotypes protect from CMV infection after kidney transplantation and show that this arises from telomeric B haplotype genes.

Efficacy of induction therapy with ATG and intravenous immunoglobulins in patients with low-level donor-specific HLA-antibodies.

Low-level donor-specific HLA-antibodies (HLA-DSA) (i.e. detectable by single-antigen flow beads, but negative by complement-dependent cytotoxicity crossmatch) represent a risk factor for early allograft rejection. The short-term efficacy of an induction regimen consisting of polyclonal anti-T-lymphocyte globulin (ATG) and intravenous immunoglobulins (IvIg) in patients with low-level HLA-DSA is unknown. In this study, we compared 67 patients with low-level HLA-DSA not having received ATG/IvIg induction (historic control) with 37 patients, who received ATG/IvIg induction. The two groups were equal regarding retransplants, HLA-matches, number and class of HLA-DSA. The overall incidence of clinical/subclinical antibody-mediated rejection (AMR) was lower in the ATG/IvIg than in the historic control group (38% vs. 55%; p = 0.03). This was driven by a significantly lower rate of clinical AMR (11% vs. 46%; p = 0.0002). Clinical T-cell-mediated rejection (TCR) was significantly lower in the ATG/IvIg than in the historic control group (0% vs. 50%; p < 0.0001). Within the first year, allograft loss due to AMR occurred in 7.5% in the historic control and in 0% in the ATG/IvIg group. We conclude that in patients with low-level HLA-DSA, ATG/IvIg induction significantly reduces TCR and the severity of AMR, but the high rate of subclinical AMR suggests an insufficient control of the humoral immune response.

Regulatory allospecific NK cell function is differentially associated with HLA C allotypes.

Major histocompatibility complex I (MHC I) molecules 'silence' natural killer (NK) cell activity. Conversely, NK cell activity is triggered through cells lacking expression of autologous MHC I. Unexpectedly we found that a subset of NK cells is activated rather than silenced when interacting with cells expressing normal levels of autologous MHC I. Instead of inducing an inflammatory phenotype, however, activation led to the secretion of the regulatory cytokines TGF-beta and IL-10. Importantly, in vitro models of allogeneic interactions showed that targets co-expressing HLA C1 and C2 epitopes best supported, or even enhanced, this cell-contact-mediated regulatory NK cell function. Together, these data ascribe a novel pattern of reactivity to NK cells, with potential implications both in autologous and allogeneic systems.

The number of activating KIR genes inversely correlates with the rate of CMV infection/reactivation in kidney transplant recipients.

Viral infection is a common complication after kidney transplantation. The role of natural killer cells (NK cells) in this setting remains unknown. NK cells express activating and inhibitory killer cell immunoglobulin-like receptors (KIR). We analyzed whether activating KIR genes carried by kidney transplant-recipients influence the rate of viral infection during the first year after transplantation. In patients with a KIR A/A genotype (n = 40, KIR2DS4 only activating KIR) the rate of cytomegalovirus (CMV) infection and reactivation was 36%, as compared to 20% in transplant recipients with more than one activating KIR gene (KIR B/X genotype, n = 82, p = 0.04). Adjusting for other risk factors in Cox regression, the relative risk of B versus A genotype patients was 0.34 (95% CI 0.15-0.76, p = 0.009). The degree of protection increased with the number of activating KIR genes. Symptomatic CMV disease was only observed in four individuals, all carrying a KIR A/A genotype. As for viral infections other than CMV, and for bacterial infections, no KIR-linked protective effect could be detected. Also, graft function and the rate-rejection episodes were similar in KIR A/A and KIR B/X genotype individuals. This study supports a role for activating KIR in the control of CMV infection after kidney transplantation.

Pretransplant risk assessment in renal allograft recipients using virtual crossmatching.

Preformed donor-specific HLA-antibodies antibodies (DSA) are a major risk for early antibody-mediated rejection (AMR). This prospective study evaluated the accuracy of pretransplant risk assessment using virtual crossmatching (virtualXM) (i.e. comparing HLA-typing of the donor with the recipient's HLA-antibody specificities determined by flow-beads). Sixty-five consecutive patients were stratified according to virtualXM results: patients without DSA (n= 56) were considered low risk and received standard immunosuppression; patients with DSA (n= 9) were considered high risk and received additional induction with anti-T-lymphocyte-globulin (ATG) and intravenous immunoglobulins. Despite induction therapy 4 of 9 patients with DSA (44%) had clinical/subclinical AMR, whereas only 2 of 56 patients without DSA (4%) (p = 0.002). Notably, one of these two patients had early AMR likely induced by non-HLA-antibodies; the other had subclinical AMR at month 6 consistent with de novo DSA. The results of virtualXM and retrospectively obtained flow-cytometric crossmatches (FCXM) (n= 59) were concordant in 51 patients (86%), four patients (7%) were virtualXM-/FCXM+ and none had AMR, four patients (7%) were virtualXM+/FCXM- and one had AMR. VirtualXM can accurately define absence or presence of DSA and may become an invaluable tool for organ allocation and pretransplant risk assessment. However, further studies need to address whether all HLA-antibodies detected by flow-beads are clinically relevant.

Molecular dosimetry of DNA adducts in C3H mice treated with bisphenol A diglycidylether.

The formation of a glycidaldehyde-DNA adduct in skin of C3H mice treated with [14C]bisphenol A diglycidylether has been previously reported and it was assumed that the modification occurred on guanine residues. We were interested in elucidating the structure of this glycidaldehyde-DNA adduct by using a non-radioactive approach. Male C3H mice were treated with a single topical dose of 2 mg bisphenol A diglycidylether in acetone for 48, 96 or 192 h. An additional two mice were treated with 2 mg glycidaldehyde in acetone for 24 h. Epidermal DNA was isolated and enzymatically digested to nucleoside-3'-monophosphates. Aliquots of the DNA hydrolysates were separated on HPLC using a reverse-phase column with a potassium dihydrogen phosphate/methanol gradient. Fluorescence analysis of the eluent indicated the presence of a fluorescent DNA adduct, which was identified as hydroxymethylethenodeoxyadenosine-3'-monophosphate by comparison with a synthetic reference standard. Amounts of adducts were determined by fluorescence measurements using a calibration curve obtained with the authentic adduct standard. Irrespective of duration of exposure, all DNA hydrolysates of treated mice contained similar amounts of the deoxyadenosine adduct. The alkylation frequency was 0.1-0.8 and 166 adducts/10(6) normal nucleotides for the treatment with bisphenol A diglycidylether and glycidaldehyde respectively. The limit of detection using 500 micrograms DNA samples for analysis was approximately 0.03 adducts/10(6) normal nucleotides.