Anne Devys

Discrimination between the main activating and inhibitory killer cell immunoglobulin-like receptor positive natural killer cell subsets using newly characterized monoclonal antibodies

Natural killer (NK) cells are key components of the innate anti‐viral and anti‐tumour immune responses. NK cell function is regulated by the interaction of killer cell immunoglobulin‐like receptors (KIR) with human leucocyte antigen (HLA) class I molecules. In this study, we report on the generation of KIR‐specific antibodies allowing for discrimination between activating and inhibitory KIR. For this purpose, BALB/c mice were immunized with human KIR2DS2 recombinant protein. The precise specificity of KIR2DS2‐specific clones was determined on KIR‐transfected BW cells and KIR‐genotyped NK cells. When used in combination with EB6 (KIR2DL1/2DS1) or GL183 (KIR2DL2/2DL3/2DS2), two KIR‐specific monoclonal antibodies (mAbs), 8C11 (specific for KIR2DL1/2DL2/2DL3/2DS2) and 1F12 (specific for KIR2DL3/2DS2), discriminated activating KIR2DS1 (8C11− EB6+) from inhibitory KIR2DL1 (8C11+ GL183−) and KIR2DL2 (1F12− GL183+), while excluding the main HLA‐Cw‐specific KIR. Using these mAbs, KIR2DS1 was shown to be expressed on the surface of NK cells from all individuals genotyped as KIR2DS1+ (n = 23). Moreover, KIR2DS1 and KIR2DL1 were independently expressed on NK cells. We also determined the amino acid position recognized by the 8C11 and 1F12 mAbs, which revealed that some KIR2DL1 allele‐encoded proteins are not recognized by 8C11. Because most available anti‐KIR mAbs recognize both inhibitory and activating forms of KIR, these newly characterized antibodies should help assess the expression of activating and inhibitory KIR and their functional relevance to NK biology.

Assessment of CD8 involvement in T cell clone avidity by direct measurement of HLA-A2/Mage3 complex density using a high-affinity TCR like monoclonal antibody

Peptide affinity for MHC molecules determines the number of MHC/peptide complexes stabilized at the cell surface in in vitro tests or in vaccination protocols. We isolated a high affinity monoclonal antibody specific for the HLA‐A2/Mage3 complex that enables an equilibrium binding assay to be performed on T2 cell line loaded with a range of Mage3 peptides. Binding of Mage3 to the HLA‐A2 molecule can be modeled by a standard receptor‐ligand interaction characterized by an affinity constant. This model enables the measurement of the affinity of other immunogenic peptides for HLA‐A2 by a competition test and the calculation of the density of complexes stabilized at the T2 cell surface for all peptide concentrations. Quantification of the HLA‐A2/Mage3 complexes at target cell surfaces was used to estimate the number of complexes required to reach cytotoxicity ED50 of human T cell clones sorted from an unprimed repertoire. We confirm with this antibody the direct relationship between clone avidity and TCR affinity, and the moderate contribution of the CD8 co‐receptor in the reinforcement of TCR‐MHC/peptide contact. Nevertheless, CD8 plays a critical role in the amplification of the specific signal to establish an efficient T cell response at low specific complex densities found in physiological situations.

Risk factors and outcome of graft failure after HLA matched and mismatched unrelated donor hematopoietic stem cell transplantation: a study on behalf of SFGM-TC and SFHI.

Graft failure remains a severe complication of hematopoietic stem cell transplantation (HSCT). Several risk factors have already been published. In this study, we re-evaluated them in a large cohort who had the benefit of the recent experience in HSCT (2006–2012). Data from 4684 unrelated donor HSCT from 2006 to 2012 were retrospectively collected from centers belonging to the French Society for Stem Cell Transplantation. Among the 2716 patients for whom HLA typing was available, 103 did not engraft leading to a low rate of no engraftment at 3.8%. In univariate analysis, only type of disease and status of disease at transplant for malignant diseases remained significant risk factors (P=0.04 and P<0.0001, respectively). In multivariate analysis, only status of disease was a significant risk factor (P<0.0001). Among the 61 patients who did not engraft and who were mismatched for 1 HLA class I and/or HLA-DP, 5 donor-specific antibodies (DSAs) were detected but only 1 was clearly involved in graft failure, for the others their role was more questionable. Second HSCT exhibited a protective although not statistically significant effect on OS (hazard ratio=0.57 [0.32–1.02]). In conclusion, only one parameter (disease status before graft) remains risk factor for graft failure in this recent cohort.

Development of monoclonal antibodies directed against cocaine and cocaethylene: potential new tools for immunotherapy.

Cocaine abuse is a major health problem, with the number of overdose-related incidents on a constant increase. Monoclonal antibodies against cocaine and its major toxic metabolite cocaethylene, have been developed for immunotherapeutical neutralization in vivo. A series of monoclonal antibodies with high affinity for cocaethylene and cocaine were obtained. Clones DASm244-4D8A4A4 (4D8) and DASm244-5B3C3C6 (5B3) were selected and fully characterized. The antibodies secreted exhibited 1.40 x 10(8) and 3.69 x 10(7) M(-1) affinity constants for [3H]-cocaine and cocaethylene, respectively. In addition to cocaine, they bound to cocaethylene and did not recognize non-toxic cocaine metabolites. They did not bind to blood cells, indicating that they may be potential tools for cocaine neutralization in vivo in cases of overdose.

Investigation of the impact of HLA-DPB1 matching status in 10/10 HLA matched unrelated hematopoietic stem cell transplantation: Results of a French single center study

The impact of HLA-DPB1 mismatches after unrelated hematopoietic stem cell transplantation (HSCT) remains controversial. We retrospectively analyzed the impact of permissive/non-permissive HLA-DPB1 mismatches on the outcome of 141 patients who underwent 10/10 HLA allelic-matched unrelated HSCT. Each pair was classified according to the 3 (TCE3) and 4-group (TCE4) algorithm based on DPB1 alleles immunogenicity. Outcome analysis revealed that TCE3 and TCE4 non-permissive HLA-DPB1 disparities were not associated with worsened overall survival, relapse risk neither risk of acute GvHD. Overall, this single center retrospective study does not confirm the adverse prognostic of non-permissive HLA-DPB1 mismatches.

MICA Compatibility and Immunization in Third Kidney Transplantations

Significantly lower graft survival has been observed among recipients of a third (G3) compared with a first or second kidney transplantation. Because patients awaiting G3 are largely HLA immunized, they are usually transplanted with a high HLA match. Moreover, their rate of acute rejection episodes is similar to a first or second transplantation. Since major histocompatibility complex class I related chain A (MICA) molecules have been proposed as new targets for antibody recognition, we were interested to type donors and recipients for MICA alleles and to study MICA immunization of these patients. Forty-three pairs of donors and recipients were typed for MICA alleles using Luminex technology (LABtype RSSO). MICA alleles showed strong linkage disequilibrium with the B locus: some 4-digit alleles were preferentially associated with a given MICA allele. A greater frequency of patients with 2 MICA mismatches (MM) was observed among patients with rejection (40%), whereas all the graft losses were observed in patients with 0 or 1 MICA MM. MICA immunization was studied using sera from 52 patients collected on day 0 and after transplantation using a Luminex assay (LABScreen). MICA immunization was less frequent than HLA immunization, and MICA donor-specific antibody (DSA) was equally present in functional and failed grafts. These observations confirmed the potential role of MICA immunization in rejection, whereas the poor graft survival among third transplantations could not be explained by MICA incompatibility or immunization.

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.