Gwendaline Guidicelli

Interpretation of Positive Flow Cytometric Crossmatch in the Era of the Single-antigen Bead Assay

Background. Prognosis of renal transplants with positive flow cytometric crossmatch (FCXM) remains controversial. Methods. We analyzed the outcome of these kidney transplant recipients by human leukocyte antigen (HLA) donor-specific antibodies (HLA-DSA) using single-antigen bead (SAB) assays in major histocompatibility complex classes I and II. We compared them with controls with a negative FCXM. Results. Forty-five patients consecutively transplanted with a positive FCXM had significantly more acute rejection episodes than the control patients (33.3% vs. 8.9%, P=0.002). Risk of acute rejection was increased with day 0 (D0) positive T-cell FCXM (odds ratio [OR]=9.04, P=0.002), D0 positive B-cell FCXM (OR=7.43, P=0.02), and D0 HLA-DSA identified by SAB assay (OR=6.5, P=0.03). The 21 patients with D0 positive FCXM and D0 HLA-DSA had more acute rejection (62%, P=0.0001) and a lower estimated glomerular filtration rate 1-year posttransplantation (P=0.0001), when compared with controls. Mainly anti-Cw and anti-DP HLA-DSA were found in patients displaying acute rejection. The remaining FCXM-positive patients displayed short-term outcomes similar to controls. The presence of HLA-DSA detected only by the SAB assay in the context of a negative FCXM crossmatch was not associated with increased risk of acute rejection. Conclusion. Identification of HLA-DSA in D0 sera by the two sensitive techniques FCXM and SAB assay indicates which patients are at highest risk of subsequent acute allograft rejection and chronic allograft dysfunction.

Non-Complement–Binding De Novo Donor-Specific Anti-HLA Antibodies and Kidney Allograft Survival

C1q-binding ability may indicate the clinical relevance of de novo donor-specific anti-HLA antibodies (DSA). This study investigated the incidence and risk factors for the appearance of C1q-binding de novo DSA and their long-term impact. Using Luminex Single Antigen Flow Bead assays, 346 pretransplant nonsensitized kidney recipients were screened at 2 and 5 years after transplantation for de novo DSA, which was followed when positive by a C1q Luminex assay. At 2 and 5 years, 12 (3.5%) and eight (2.5%) patients, respectively, had C1q-binding de novo DSA. De novo DSA mean fluorescence intensity >6237 and >10,000 at 2 and 5 years, respectively, predicted C1q binding. HLA mismatches and cyclosporine A were independently associated with increased risk of C1q-binding de novo DSA. When de novo DSA were analyzed at 2 years, the 5-year death-censored graft survival was similar between patients with C1q-nonbinding de novo DSA and those without de novo DSA, but was lower for patients with C1q-binding de novo DSA (P=0.003). When de novo DSA were analyzed at 2 and 5 years, the 10-year death-censored graft survival was lower for patients with C1q-nonbinding de novo DSA detected at both 2 and 5 years (P<0.001) and for patients with C1q-binding de novo DSA (P=0.002) than for patients without de novo DSA. These results were partially confirmed in two validation cohorts. In conclusion, C1q-binding de novo DSA are associated with graft loss occurring quickly after their appearance. However, the long-term persistence of C1q-nonbinding de novo DSA could lead to lower graft survival.

Deciphering complement interference in anti-human leukocyte antigen antibody detection with flow beads assays.

Background Anti–human leukocyte antigen (HLA) antibody detection in solid-phase flow beads assays can be quenched by complement activation, but the precise mechanism of this interference is not fully elucidated yet. Methods Using the Luminex flow beads screening assay for detection of anti-HLA antibodies, we analyzed the binding of high concentrations of the pan class I anti-HLA monoclonal antibody W6/32 in neat normal, ethylenediaminetetraacetic acid–treated normal and complement factors C1q, C4/C3, C2, C3, factor B or C5-depleted human sera, using anti-mouse immunoglobulin G as the detection antibody. Complement activation and binding to beads were revealed using anti-human C1q, C4d, and C3d antibodies. To translate our findings to the human setting, we used the class I and class II HLA single-antigen flow beads assays and sera from four patients with high titers of antibodies. Results Detection of W6/32 did not suffer any interference with C1q and C4/C3–depleted sera. A partial quenching was observed with C2, C3, and factor B-depleted sera, but was more pronounced with the factor B-depleted serum. W6/32 was undetectable in presence of C5-depleted serum. The binding of activation products derived from C3 principally, and also from C4, impaired immunoglobulin G and C1q detection. Accordingly, C4d detection was hindered by deposition of activated C3. Similar findings were obtained with patients’ sera. Conclusion Binding of C4 and C3 activation products is the main responsible for complement interference in flow beads assays. A complete quenching requires complement activation through C3 cleavage and its amplification by the alternative pathway.

Denatured class I human leukocyte antigen antibodies in sensitized kidney recipients: prevalence, relevance, and impact on organ allocation.

Background Single antigen flow beads assays may overestimate sensitization because of the detection of supposedly irrelevant antibodies recognizing denatured class I human leukocyte antigens (HLAs). Methods Sera of 323 HLA-sensitized kidney transplant candidates positive with a class I HLA single antigen flow beads assay were retested after acid treatment of the beads. Denatured HLA antibodies were identified according to ratio between the measured fluorescence intensity for treated and nontreated beads. T-lymphocyte flow cytometry crossmatches were performed to characterize the ability of these antibodies to recognize HLA on normal cells as a surrogate of their potential clinical relevance. Their impact on organ allocation was evaluated through a calculated panel reactive antibody. The utility of single antigen flow beads largely devoid of denatured HLA (iBeads) was also evaluated. Results Denatured HLA antibodies were detected in 39% of the patients. They provided much less positive flow cytometry crossmatches than anti-native HLA antibodies (16% vs. 83%, P<0.0001). Removing the HLA-A and HLA-B antigens targeted by denatured HLA antibodies from unacceptable antigens lowered the calculated panel reactive antibody for 90 patients, sometimes dramatically. The iBeads assay demonstrated nearly the same ability to predict crossmatch results than the acid treatment assay. Conclusion Denatured class I HLA antibodies are common, but the antigens they target should not be considered as unacceptable in most cases, because they negatively impact access to a transplant while predominantly providing negative sensitive crossmatches. The iBeads assay seems to be a valuable alternative to better define unacceptable antigens.

The complement interference phenomenon as a cause for sharp fluctuations of serum anti-HLA antibody strength in kidney transplant patients.

The single antigen flow bead (SAFB) assay greatly improves the identification of antigenic specificity of anti-HLA alloantibodies. However, it may underestimate or miss high titer antibodies due to the prozone phenomenon caused by a competition between the fluorescent anti-IgG conjugate and serum complement, for the alloantibody. We explored this effect in our cohort of transplant candidates and transplanted recipients. Among a total of 292 and 269 patients with at least three different sera tested with class I and/or II SAFB assays respectively, we identified 9 patients (6 in class I and 3 in class II) who displayed a profound drop (≥ 75%) followed by a subsequent rise (≥ 100%), in strong (mean fluorescence intensity >8000) antibody levels, across an 18-month period. We postulated that such abrupt fluctuations were not explainable by naturally occurring transient desensitization. Sera were analysed with the SAFB assay using EDTA-treated serum and direct complement C1q staining, and with complement-dependent cytotoxicity and flow cytometry crossmatches (CDCXM and FCXM respectively). The prozone phenomenon was involved in all cases. Because it relies on complement activation, the CDCXM was not sensitive to this phenomenon, but the FCMXM was not either, although it resembles in its principle to the SAFB assay. Four additional anti-human conjugates targeting the IgG Fc fragment or the light chains did not circumvent the SAFB drawback. Therefore, a quick decrease in antibody strength must alert against a potential risk for recipients at the time of the transplant, using virtual crossmatch strategies. A prospective pre-transplant crossmatch still remains an ultimate safeguard.

Kidney Intragraft Donor-Specific Antibodies as Determinant of Antibody-Mediated Lesions and Poor Graft Outcome

Allograft pathology, antibody–tissue interaction as demonstrated by C4d deposition and serological evidence of donor‐specific antibodies (DSA) are the cardinal diagnostic features of antibody‐mediated lesions (AML) in kidney transplantation. However, discrepancy between histological and serological findings is common, and more reliable diagnostic tools are called for. Here, we asked whether the in situ detection of DSA could serve as marker for AML. To that end, we applied the anti‐HLA single antigen flow bead assay to eluates from 51 needle core graft biopsies performed for cause. Intragraft antibody profiles were correlated to serum DSA (sDSA), histological data and transplant outcome. The prevalence and the mean number of intragraft DSA (gDSA) were lower than that of sDSA (15/51 gDSA+ vs. 37/51 sDSA+ patients; 1.64 gDSA vs. 2.24 sDSA per patient). DSA were detected in all anti‐HLA antibody‐positive biopsies (15/15). The presence of gDSA was significantly associated with (1) microcirculation lesions taken individually (g, cg) and analyzed in functional clusters (ptc + g + cg > 0, cg + mm > 0), (2) C4d positivity and (3) a worse short‐term transplant outcome (p = 0.05). These associations were not found for patients presenting only sDSA. Taken together, these results indicate that gDSA is a severity marker of antibody‐mediated pathogenic process.

Anti‐Cw Donor‐specific Alloantibodies Can Lead to Positive Flow Cytometry Crossmatch and Irreversible Acute Antibody‐Mediated Rejection

Figure 1: Identification of class I DSAs in serum (A) and graft eluate (B) using the single antigen bead assay on a Luminex R

The Immunosuppressor Mycophenolic Acid Kills Activated Lymphocytes by Inducing a Nonclassical Actin-Dependent Necrotic Signal

Mycophenolate mofetil (MMF) is an immunosuppressive agent used in transplantation. Over the last decade, MMF has also emerged as an alternative therapeutic regimen for autoimmune diseases, mainly for patients refractory to other therapies. The active compound of MMF, mycophenolic acid (MPA), depletes the intracellular pool of guanosine tri-phosphate through inosine monophosphate dehydrogenase blockade. The molecular mechanism involved in the elimination of T and B lymphocytes upon inhibition of inosine monophosphate dehydrogenase remains elusive. In this study, we showed that in contrast to the immunosuppressors azathioprine, cyclosporin A, and tacrolimus, MPA killed lymphocytes through the activation of a caspase-independent necrotic signal. Furthermore, the MPA-mediated necrotic signal relied on the transmission of a novel intracellular signal involving Rho-GTPase Cdc42 activity and actin polymerization. In addition to its medical interest, this study sheds light on a novel and atypical molecular mechanism leading to necrotic cell death.

Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation

Anti‐HLA donor‐specific antibodies are deleterious for organ transplant survival. Class I HLA donor‐specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti‐denatured HLA antibodies (anti‐dHLAs). Anti‐dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti‐dHLAs to be discriminated from anti‐nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid‐treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti‐dHLAs. However, some anti‐dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti‐nHLAs conserved significant reactivity toward acid‐treated LSAB. After depleting serum anti‐nHLA reactivity with HLA‐typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid‐treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti‐nHLAs and anti‐dHLAs, or anti‐nHLAs recognizing acid‐resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti‐HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti‐HLA antibodies in human serum.

Lung intragraft donor-specific antibodies as a risk factor for graft loss

BACKGROUND The effect of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) on graft survival is recognized in lung transplantation, but not all serum DSAs appear to be harmful. We wondered whether in situ DSA detection from graft biopsy specimens could help in identifying lung transplant recipients (LTRs) at higher risk for graft loss. METHODS Class I and II HLA antibody single-antigen flow bead assays were performed in 53 LTRs to identify immunoglobulin G DSA in biopsy specimen eluates and in sera and to evaluate C1q binding ability of DSA in sera. Intragraft DSAs (gDSAs) were correlated with serum DSAs (sDSAs), clinical and histologic data, and graft survival. RESULTS Twenty-eight (52.8%) LTRs had sDSAs, 12 (22.6%) had C1q-positive sDSAs, and 11 (20.8%) had gDSAs. Fifty sDSAs were found, among which 15 (30%) were C1q-positive and 14 (28%) were found in biopsy specimen eluates. One DSA was detected in the biopsy specimen only. Serum mean fluorescence intensity and biopsy fragment size were higher for sDSAs detected in biopsy specimens (p = 0.003 and p = 0.02, respectively). One-year post-biopsy graft survival was lower for LTRs with gDSAs (p = 0.008 by log-rank test). Presence of gDSA at the time of biopsy constituted a risk factor for graft loss in univariate (odds ratio, 6.67; 95% confidence interval [CI] 1.51-29.47; p = 0.008; hazard risk, 3.44; 95% CI, 1.47-8.01, p = 0.005) and multivariate (odds ratio, 5.85; 95% CI, 1.23-27.68; p = 0.03; hazard risk, 4.51; 95% CI, 1.83-11.13; p = 0001) analyses using logistic regression and a Cox proportional hazard model, respectively. CONCLUSIONS In lung transplantation, gDSA appears to be a valuable biomarker to identify pathogenic DSA and LTRs with a higher risk for graft loss.