Donor-targeted serotherapy as a rescue therapy for steroid-resistant acute GVHD after HLA-mismatched kidney transplantation.

Acute graft-versus-host disease (GVHD) is a rare but frequently lethal complication after solid organ transplantation. GVHD occurs in unduly immunocompromised hosts but requires the escalation of immunosuppression, which does not discriminate between host and donor cells. In contrast, donor-targeted therapy would ideally mitigate graft-versus-host reactivity while sparing recipient immune functions. We report two children with end-stage renal disease and severe primary immune deficiency (Schimke syndrome) who developed severe steroid-resistant acute GVHD along with full and sustained donor T cell chimerism after isolated kidney transplantation. Facing a therapeutic dead end, we used a novel strategy based on the adoptive transfer of anti-HLA donor-specific antibodies (DSAs) through the transfusion of highly selected plasma. After approval by the appropriate regulatory authority, an urgent nationwide search was launched among more than 3800 registered blood donors with known anti-HLA sensitization. Adoptively transferred DSAs bound to and selectively depleted circulating donor T cells. The administration of DSA-rich plasma was well tolerated and notably did not induce antibody-mediated rejection of the renal allografts. Acute GVHD symptoms promptly resolved in one child. This report provides a proof of concept for a highly targeted novel therapeutic approach for solid organ transplantation-associated GVHD.

Epitope load identifies kidney transplant recipients at risk of allosensitization following minimization of immunosuppression.

Human leukocyte antigen (HLA) mismatching and minimization of immunosuppression are two major risk factors for the development of de novo donor-specific antibodies, which are associated with reduced kidney graft survival. Antibodies do not recognize whole HLA antigens but rather individual epitopes, which are short sequences of amino acids in accessible positions. However, compatibility is still assessed by the simple count of mismatched HLA antigens. We hypothesized that the number of mismatched epitopes, or ("epitope load") would identify patients at the highest risk of developing donor specific antibodies following minimization of immunosuppression. We determined epitope load in 89 clinical trial participants who converted from cyclosporine to everolimus 3 months after kidney transplantation. Twenty-nine participants (32.6%) developed de novo donor specific antibodies. Compared to the number of HLA mismatches, epitope load was more strongly associated with the development of donor specific antibodies. Participants with an epitope load greater than 27 had a 12-fold relative risk of developing donor-specific antibodies compared to those with an epitope load below that threshold. Using that threshold, epitope load would have missed only one participant who subsequently developed donor specific antibodies, compared to 8 missed cases based on a 6-antigen mismatch. DQ7 was the most frequent antigenic target of donor specific antibodies in our population, and some DQ7 epitopes appeared to be more frequently involved than others. Assessing epitope load before minimizing immunosuppression may be a more efficient tool to identify patients at the highest risk of allosensitization.

HLA-DRB3/4/5 mismatches are associated with increased risk of acute GVHD in 10/10 matched unrelated donor hematopoietic cell transplantation.

Matching for HLA-A, -B, -C, and -DRB1 loci (8/8 match) is currently the gold standard for unrelated donor hematopoietic cell transplantation (HCT). In Europe, patients are also matched at the HLA-DQB1 loci (10/10 match). However, there is increasing evidence that matching at HLA-DRB3/4/5 loci may help to lower transplant-related morbidity and mortality. We therefore investigated the impact of HLA-DRB3/4/5 mismatches on outcomes in 1975 patients who received a first 10/10 matched unrelated donor (MUD) HCT in France from 2000 to 2012 for a hematological malignancy. High-resolution typing was performed at HLA-A, -B, -C, -DRB1, -DQB1, -DPB1, and -DRB3/4/5 loci for all donor/recipient pairs. Compared with DRB3/4/5-matched pairs, patients who received a MUD HCT from a DRB3/4/5 mismatched donor had a significantly increased risk of grade II-IV acute graft-versus-host disease (aGVHD) (Adjusted Hazard Ratio (HR) 1.43 (1.07 to 1.90)) associated with lower graft-versus-host disease-free and relapse-free survival (GRFS) (Adjusted HR 1.20 (1.02 to 1.42)). Conversely, we observed no differences in terms of chronic GVHD, nonrelapse mortality, relapse and overall survival. However, we believe that patients stand to benefit from DRB3/4/5 loci being considered for unrelated donor selection to improve GRFS and then quality of life after unrelated HCT.

Impact of donor-specific anti-HLA antibodies on graft failure and survival after reduced intensity conditioning-unrelated cord blood transplantation: a Eurocord, Société Francophone d'Histocompatibilité et d'Immunogénétique (SFHI) and Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC) analysis.

Graft failure is a major complication after unrelated cord blood transplantation. Presence of HLA-antibodies before cord blood transplantation may impact graft failure. To analyze the effect of anti-HLA antibodies on unrelated cord blood transplantation outcomes, we analyzed 294 unrelated cord blood transplant recipients after reduced intensity conditioning regimen. The majority of the patients (82%) were transplanted for malignancies, 60% with double-unrelated cord blood transplant, 63% were HLA mismatched. Retrospectively, pre-unrelated cord blood transplant serum was tested for HLA-Ab using Luminex™ platform. Results were interpreted as mean fluorescence intensity (MFI) against donor-specific mismatch. Among 62 recipients (23%) who had anti-HLA antibodies before unrelated cord blood transplant, 14 patients had donor specific anti-HLA antibodies (DSA) (7 were donor-specific anti-HLA antibodies for single unrelated cord blood transplant and 7 for double unrelated cord blood transplant). Donor specific anti-HLA antibodies threshold ranged from 1620-17629 of mean fluorescence intensity (MFI). Cumulative incidence of Day-60 neutrophil engraftment was 76%: 44% for recipients with donor specific anti-HLA antibodies and 81% in those without donor specific anti-HLA antibodies (P=0.006). The cumulative incidence of 1-year transplant related mortality was 46% in patients with donor specific anti-HLA antibodies and 32% in those without antibodies (P=0.06). The presence of donor specific anti-HLA antibodies was associated with a trend for decreased survival rate (42% vs. 29%; P=0.07). Donor specific anti-HLA antibody in recipients of unrelated cord blood transplant is associated with graft failure and decreased survival. Patient's screening for donor specific anti-HLA antibodies before unrelated cord blood transplantation is recommended before choosing an HLA mismatched cord blood unit. Whenever possible it is important to avoid selecting a unit for which the patient has donor specific anti-HLA antibodies.

HLA graph, a free and ready-to-use bioinformatics tool to explore anti-HLA eplets reactivity pattern.

INTRODUCTION: HLA antigens are highly polymorphic, and their immunogenicity is dependent on the configurations of polymorphic amino acids. Monitoring anti-HLA immunization is essential in organ transplantation, as antibodies directed against HLA molecules are a major cause of rejection. Anti-HLA antibodies are not specific for HLA antigens, but recognize B-cell epitopes present on HLA molecules. METHODS: To better understand antibody reactivity patterns, we calculated the Spearman correlation of the mean fluorescence intensity (MFI) of anti-HLA antibodies identified by a single-antigen assay performed using a Luminex® immunobeads assay on a large number of samples. Then, we built a computer tool analyzing antibody reactivity patterns with an accessibility by a web browser linked to the International Epitope Registry. We also extended our model to Onelambda® and Lifecodes® single-antigen class I and class II assays. RESULTS AND DISCUSSION: The resulting MFI correlations reflect HLA antibody cross-reactivity and eplets similarity. We built HLA Graph, a computer tool that analyzes the eplets involved in antibody reactivity profiles. HLA Graph is usable with Onelambda® and Lifecodes® single-antigen class I and class II assays. The interpretation of reactivity against alleles not tested by the antibody assays and against the alpha and beta chains of HLA-DQ and HLA-DP loci were also developed. CONCLUSION: HLA Graph is a free and ready-to-use bioinformatics tool that can be used by all laboratories performing anti-HLA antibody identification by immunobead assay.

Monoclonal secondary reagents do not outperform polyclonal secondary reagents for detection of anti-HLA IgG using single antigen flow beads assays.

Luminex single antigen flow beads (SAFB) assays are used to monitor anti-HLA antibodies, which are associated with allograft loss. The primary antibody binding level is evaluated by the mean fluorescence intensity (MFI) provided by the tracing secondary antibody. The aim of this study was to compare the MFI obtained with two secondary antibodies and to evaluate their sensitivity to detect newly developed de novo anti-HLA donor-specific antibodies (dnDSA). A total of 23 and 46 different sera from 20 HLA-sensitized kidney transplant recipients were tested with class I and II SAFB, respectively, as well as sera from 17 patients with dnDSA. The conventional secondary antibody (IgHPolyFab) was the phycoerythrin (PE)-conjugated goat anti-human IgG constant heavy chain (HC)-binding polyclonal-F(ab')2. The alternate secondary antibody was a PE-conjugated anti-human IgG Fc-specific monoclonal IgG (FcMonoIgG). MFI of negative control sera were drastically lower with the FcMonoIgG than with IgHPolyFab, requesting a unique positivity threshold to be defined for each secondary antibody. MFI obtained with positive control sera were also lower with FcMonoIgG. In kidney transplant recipient's sera, the number of antigenic specificities detected by each secondary antibody was comparable, for dnDSA as well as for nonDSA. In conclusion, decision thresholds must be refined when changing the secondary tracing reagent in SAFB assays. FcMonoIgG did not demonstrate any superiority over IgHPolyFab for monitoring HLA antibodies.

Donor KIR3DL1/3DS1 gene and recipient Bw4 KIR ligand as prognostic markers for outcome in unrelated hematopoietic stem cell transplantation.

Given their antileukemic activity, natural killer (NK) cells can alter the outcome of hematopoietic stem cell transplantation (HSCT). The physiologic functions of NK cells are regulated by the interaction of killer immunoglobulin-like receptors (KIR) with specific HLA class I ligands. In the literature, different models based on HLA class I and/or KIR donor (D)/recipient (R) gene disparities are considered as predictors of NK cell alloreactivity. In this retrospective and multicentric French study, we analyzed the clinical impact of the different NK-alloreactivity models in 264 patients who underwent T repleted unrelated HSCT. First, we did not observe that the "KIR ligand-ligand" model had a significant clinical impact on unrelated HSCT outcome, whereas the "missing KIR ligand" model had a significant but limited effect on unrelated HSCT, because only the absence of C1 ligand in patients with myelogenous diseases was associated with a decreased overall survival (OS) (hazard ratio=2.17, P=.005). The "KIR receptor-receptor" and the "KIR receptor-ligand" models seemed the most capable of predicting NK alloreactivity because they had a significant impact on acute graft-versus-host disease (aGVHD) occurrence, OS, and relapse incidence in D/R unrelated pairs. In particular, KIR3DL1 gene mismatches in the GVH direction (D(+)R(-)) and the D KIR3DL1(+)/3DS1(+) and R Bw4(-) combination were respectively correlated with the lowest OS in HLA identical pairs (HR=1.99, P =.02) and the highest incidence of relapse in HLA nonidentical D/R unrelated pairs (HR=4.72, P =.03). Overall, our results suggest a detrimental effect of KIR3DL1(+)/3DS1(+) donor NK cells transplanted into HLA-Bw4(-) patients in the absence of an educational process via KIR3DL1/HLA-Bw4 interactions.

Improvement in HLA-typing by new sequence-specific oligonucleotides kits for HLA-A, -B, and -DRB1 loci.

Polymerase chain reaction sequence-specific oligonucleotide is commonly used for HLA-typing. We replaced our LabType SSO HD (HD) kits with LabType SSO XR (XR) kits (One Lambda, Inc., Canoga Park, California) for HLA-A, -B, and -DRB1 following acquisition of a LABScan3D analyzer. The XR kits have more bead regions than the HD kits, allowing for an extended number of probes and exon coverage. They are claimed to improve typing resolution and to diminish the number of allele ambiguities, including common and well-documented (CWD) and null alleles to be resolved. We retrospectively selected patients who had their first HLA-typing performed with the HD kits and their second determination with the XR kits between 2015 and 2016. Forty-two patients were selected for HLA-A typing comparison, and 48 for HLA-B and 41 for HLA-DRB1. XR kits significantly decreased the number of allele ambiguities for HLA-A and -B. On the other hand, the improvement was limited for the HLA-DRB1 locus. The XR kits did not resolve all the CWD HLA allele ambiguities, which may be important for organ and/or hematopoietic stem cell transplantations. The XR kits eliminated 88%, 62%, and 27% of null allele ambiguities for HLA-A, -B, and -DRB1 loci, respectively. In conclusion, the XR kits allow for a significant improvement of HLA-typing resolution for HLA-A and -B loci in comparison with HD kits. In contrast, the number of oligonucleotides in the XR HLA-DRB1 kit should be extended to include exon 3 at the very least. It could also be interesting to include oligonucleotides allowing HLA-DRB3, 4, and 5 typing.

Very low residual concentrations of rituximab long after infusion still induce positive B-cell complement-dependent cytotoxicity-crossmatch.

Rituximab may induce positive B-cell complement-dependent cytotoxicity crossmatch (CDC-XM) in the absence of donor-specific antibodies, as we report in these two cases. We retrospectively assessed the in vitro concentration-effect relationship of rituximab in sera. B-cell CDC-XM results were positive only in the presence of rituximab, even with low concentrations (inferior to 1 µg/mL). Moreover, rituximab neutralization with increasing concentration of an anti-rituximab-idiotype monoclonal antibody progressively reduced B-cell lysis. In conclusion, measurement of rituximab content may be useful to identify sera at risk of misinterpretation in immunized patients.

HLA Association with hematopoietic stem cell transplantation outcome: the number of mismatches at HLA-A, -B, -C, -DRB1, or -DQB1 is strongly associated with overall survival.

HLA matching between the donor and recipient improves the success of unrelated hematopoietic stem cell transplantation (HSCT). Because many patients in need of an unrelated transplant have only donors with mismatch, information is needed to evaluate the limits of HLA mismatching. We examined the association of survival, acute graft-versus-host disease (aGVHD) and relapse with HLA-A, -B, -C, -DRB, -DQB1, and -DPB1 mismatching in 334 patients coming from 12 French transplant centers and who received a non-T cell-depleted bone marrow graft from an unrelated donor. All patients were prepared with the use of myeloablative conditioning regimens. Our analyses demonstrate negative effects of HLA mismatching for either HLA-A, -B, -C, -DRB1, or -DQB1 loci on survival. Multivariate Cox analyses showed that a single mismatch was associated with a significant decrement in survival (P=.046, hazard ratio [HR]=1.41, confidence interval [CI] 95% 1.1-1.98). The presence of multiple mismatches was worse for survival (P=.003, HR=1.91, CI 95% 1.26-2.91) and severe aGVHD (grade III-IV) (P=.002, HR=2.51, CI95% 1.41-4.46). The cumulative incidences of aGVHD and relapse in those HLA-A, -B, -C, -DRB1, and -DQB1 identical pairs with 2, 1, or 0 DPB1 incompatibilities were 63%, 50%, and 51%, and 12%, 27%, and 20%, respectively, but these differences were not statistically significant. Similar differences of aGVHD and relapse, but not statistically significant, were observed in those HLA-A, -B, -C, -DRB1, and -DQB1 identical pairs with DPB1 disparities classified into permissive or nonpermissive mismatches according to Zino's classification based on a hierarchy of the immunogenicity of the HLA-DP molecules. "Missing killer cell immunoglobulin-like receptor (KIR) ligand" evaluated on the presence of HLA-C1, -C2, and Bw4 groups in the recipients was not associated with aGVHD, survival, and relapse in this cohort of non-T cell-depleted HSCT.