Multifactorial determinants of NK cell repertoire organization: insights into age, sex, KIR genotype, HLA typing, and CMV influence.

Introduction: Polymorphisms in the KIR and HLA genes contribute to the diversity of the NK cell repertoire. Extrinsic factors also play a role in modifying this repertoire. The best example is cytomegalovirus, which promotes the expansion of memory-like NK cells. However, the mechanisms governing this phenotypic structure are poorly understood. Furthermore, the influence of age and sex has been understudied. Methods: In this study, we examined these parameters in a cohort of 200 healthy volunteer blood donors, focusing on the major inhibitory KIR receptors and CD94/NKG2A, as well as the differentiation marker CD57 and the memory-like population marker NKG2C. Flow cytometry and two joint analyses, unsupervised and semi-supervised, helped define the impact of various intrinsic and extrinsic markers on the phenotypic structure of the NK cell repertoire. Results: In the KIR NK cell compartment, the KIR3DL1 gene is crucial, as unexpressed alleles lead to a repertoire dominated by KIR2D interacting only with HLA-C ligands, whereas an expressed KIR3DL1 gene allows for a greater diversity of NK cell subpopulations interacting with all HLA class I ligands. KIR2DL2 subsequently favors the KIR2D NK cell repertoire specific to C1/C2 ligands, whereas its absence promotes the expression of KIR2DL1 specific to the C2 ligand. The C2C2Bw4+ environment, marked by strong -21T motifs, favors the expansion of the NK cell population expressing only CD57, whereas the absence of HLA-A3/A11 ligands favors the population expressing only NKG2A, a population highly represented within the repertoire. The AA KIR genotype favors NK cell populations without KIR and NKG2A receptors, whereas the KIR B+ genotypes favor populations expressing KIR and NKG2A. Interestingly, we showed that women have a repertoire enriched in CD57- NK cell populations, while men have more CD57+ NK cell subpopulations. Discussion: Overall, our data demonstrate that the phenotypic structure of the NK cell repertoire follows well-defined genetic rules and that immunological history, sex, and age contribute to shaping this NK cell diversity. These elements can contribute to the better selection of hematopoietic stem cell donors and the definition of allogeneic NK cells for cell engineering in NK cell-based immunotherapy approaches.cters are displayed correctly.

[Update for cord blood unit selection in hematopoietic stem cell transplantation (workshop SFGM-TC)]. / Mise à jour des recommandations pour le choix des unités de sang placentaire en greffe de cellules souches hématopoïétiques (atelier SFGM-TC).

Changing practices and the limited use of cord blood units as a source of cells for allogeneic hematopoietic stem cell transplants (HSC) led us to reconsider the recommendations established in 2011 and 2012, and to propose an update incorporating recent bibliographic data. If HLA compatibility was until now established at low resolution for HLA-A and B loci, and at high resolution for HLA-DRB1, the recent papers are converging towards an increase in the level of resolution, making way for a compatibility now defined in high resolution for all the considered loci, and the inclusion of the HLA-C locus, in order to establish a level of HLA compatibility on 8 alleles (HLA-A, B, C and DRB1). The CD34+ dose is a determining factor in hematopoietic reconstitution but it is not correlated with the total nucleated cells content. This is why we recommend taking these two data into account when choosing a cord blood unit. The recommendations established by our group are presented as a flow chart taking into account the characteristics of the underlying pathology (malignant or non-malignant), the cell dose and the HLA compatibility criteria, as well as criteria linked to the banks in which units are stored.

[Relapse with HLA loss after hematopoietic stem cell transplantation with non-HLA identical donor: Guidelines from the Francophone society of bone marrow transplantation and cellular therapy (SFGM-TC)]. / Perte d'hétérozygotie HLA dans les rechutes après greffes de cellules souches hématopoïétiques avec donneur non HLA identique : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

Loss of heterozygosity or HLA loss is a genomic-type escape mechanism highlighted in certain types of relapses after allogeneic hematopoietic stem cell transplantation with a non-HLA identical donor, and especially after haplo-identical transplantation. The diagnosis must be made with certainty because the result conditions the therapy. In this article, the different mechanisms and techniques that can be used for the diagnosis of loss of heterozygosity, as well as the therapeutic options are reviewed, making it possible to establish clinico-biological recommendations for the diagnosis confirmation and management of the patients in relapse.

Characterization of the novel HLA-B*15:648 allele by next-generation sequencing.

HLA-B*15:648 differs from HLA-B*15:02:01:01 by one nucleotide substitution in codon 77 in exon 2.

Characterization of 43 novel HLA-H alleles.

Forty-three novel HLA-H alleles, including 32 identified in cohorts of French donors for Hematopoietic Stem Cell Transplantation, have been characterized by Next-Generation Sequencing (NGS) using a long range PCR approach. A phylogenetic study confirms three main HLA-H clades.

Characterization of the novel HLA-A*33:246 allele using next-generation sequencing.

HLA-A*33:246 differs from HLA-A*33:01:01:01 allele by one nucleotide substitution in codon 135 in exon 3.

Kidney allograft rejection is associated with an imbalance of B cells, regulatory T cells and differentiated CD28-CD8+ T cells: analysis of a cohort of 1095 graft biopsies.

Introduction: The human immune system contains cells with either effector/memory or regulatory functions. Besides the well-established CD4+CD25hiCD127lo regulatory T cells (Tregs), we and others have shown that B cells can also have regulatory functions since their frequency and number are increased in kidney graft tolerance and B cell depletion as induction therapy may lead to acute rejection. On the other hand, we have shown that CD28-CD8+ T cells represent a subpopulation with potent effector/memory functions. In the current study, we tested the hypothesis that kidney allograft rejection may be linked to an imbalance of effector/memory and regulatory immune cells. Methods: Based on a large cohort of more than 1000 kidney graft biopsies with concomitant peripheral blood lymphocyte phenotyping, we investigated the association between kidney graft rejection and the percentage and absolute number of circulating B cells, Tregs, as well as the ratio of B cells to CD28-CD8+ T cells and the ratio of CD28-CD8+ T cells to Tregs. Kidney graft biopsies were interpreted according to the Banff classification and divided into 5 biopsies groups: 1) normal/subnormal, 2) interstitial fibrosis and tubular atrophy grade 2/3 (IFTA), 3) antibody-mediated rejection (ABMR), 4) T cell mediated-rejection (TCMR), and 5) borderline rejection. We compared group 1 with the other groups as well as with a combined group 3, 4, and 5 (rejection of all types) using multivariable linear mixed models. Results and discussion: We found that compared to normal/subnormal biopsies, rejection of all types was marginally associated with a decrease in the percentage of circulating B cells (p=0.06) and significantly associated with an increase in the ratio of CD28-CD8+ T cells to Tregs (p=0.01). Moreover, ABMR, TCMR (p=0.007), and rejection of all types (p=0.0003) were significantly associated with a decrease in the ratio of B cells to CD28-CD8+ T cells compared to normal/subnormal biopsies. Taken together, our results show that kidney allograft rejection is associated with an imbalance between immune cells with effector/memory functions and those with regulatory properties.

Very Low Dose Anti-Thymocyte Globulins Versus Basiliximab in Non-Immunized Kidney Transplant Recipients.

The choice between Basiliximab (BSX) or Anti-Thymocyte Globulin (ATG) as induction therapy in non-immunized kidney transplant recipients remains uncertain. Whilst ATG may allow steroid withdrawal and a decrease in tacrolimus, it also increases infectious complications. We investigated outcomes in non-immunized patients receiving a very low dosage of ATG versus BSX as induction. Study outcomes were patient/graft survival, cumulative probabilities of biopsy proven acute rejection (BPAR), infectious episode including CMV and post-transplant diabetes (PTD). Cox, logistic or linear statistical models were used depending on the studied outcome and models were weighted on propensity scores. 100 patients received ATG (mean total dose of 2.0 mg/kg) and 83 received BSX. Maintenance therapy was comparable. Patient and graft survival did not differ between groups, nor did infectious complications. There was a trend for a higher occurrence of a first BPAR in the BSX group (HR at 1.92; 95%CI: [0.77; 4.78]; p = 0.15) with a significantly higher BPAR episodes (17% vs 7.3%, p = 0.01). PTD occurrence was significantly higher in the BSX group (HR at 2.44; 95%CI: [1.09; 5.46]; p = 0.03). Induction with a very low dose of ATG in non-immunized recipients was safe and associated with a lower rate of BPAR and PTD without increasing infectious complications.

Case Report: Long-term observations from the tacrolimus weaning randomized clinical trial depicts the challenging aspects for determination of low-immunological risk patients.

Whilst calcineurin inhibitors (CNI) are the cornerstone of immunosuppressive maintenance therapy in kidney transplantation, several studies have investigated the safety of CNI withdrawal in order to avoid their numerous side effects. In this context, we performed several years ago a clinical randomized trial evaluating CNI weaning in stable kidney transplant recipients without anti-HLA immunization. The trial was interrupted prematurely due to a high number of de novo DSA (dnDSA) and biopsy proven acute rejection (BPAR) in patients who underwent tacrolimus weaning, resulting in treatment for rejection and resumption of tacrolimus. We report here the long-term outcomes of patients included in this clinical trial. Ten years after randomization, all patients are alive with a functional allograft. They all receive tacrolimus therapy except one with recurrent cutaneous neoplasia issues. Long-term eGFR was comparable between patients of the two randomized groups (46.4 ml/min vs 42.8 ml/min). All dnDSA that occurred during the study period became non-detectable and all rejections episodes were reversed. The retrospective assessment of HLA DQ single molecule epitope mismatching determined that a majority of patients who developed dnDSA after tacrolimus withdrawal would have been considered at high immunological risk. Minimization of immunosuppression remains a challenging objective, mainly because of the issues to properly select very low immunological risk patients. Valuable improvements have been made the last decade regarding evaluation of the allograft rejection notably through the determination of numerous at-risk biomarkers. However, even if the impact of such tools still need to be clarify in clinical routine, they may permit an improvement in patients' selection for immunosuppression minimization without increasing the risk of allograft rejection.

Antibody-mediated allograft rejection is associated with an increase in peripheral differentiated CD28-CD8+ T cells - Analyses of a cohort of 1032 kidney transplant recipients.

BACKGROUND: CD28-CD8+ T cells represent a differentiated CD8+ T cell subset that is found to be increased in various conditions associated with chronic antigenic stimulation such as aging, chronic viral infections, autoimmune diseases, cancers, and allotransplantation. METHODS: Using multivariate models, we analyzed a large cohort of 1032 kidney transplant patients in whom 1495 kidney graft biopsies were performed concomitant with a peripheral blood leukocyte phenotyping by flow cytometry. We investigated the association between the level of CD28-CD8+ T cells in the blood and the diagnosis of graft rejection according to the recent Banff classification of renal allograft pathology. FINDINGS: We found that antibody-mediated rejection (ABMR) was associated with a significant increase in the percentage as well as the absolute number of CD28-CD8+ T cells in the peripheral blood of kidney transplant patients at the time of biopsy. The confounder-adjusted mean difference of log percentage and log absolute value between the ABMR group and the normal/subnormal histology group were 0.29 (p=0.0004) and 0.38 (p=0.0004), respectively. Moreover, we showed that CD28-CD8+ T cells from the patients diagnosed with ABMR responded more rigorously to TCR and FcγRIIIA (CD16) engagement compared to their CD28+ counterparts as evidenced by an increase in the expression of IFNγ, TNFα, and CD107a. INTERPRETATION: Collectively, our data suggest that differentiated CD28-CD8+ T cells, with increased frequency, number, and function, may participate in the pathobiology of ABMR. Further studies are warranted to clarify the immunological role of this T cell subset in kidney graft rejection. FUNDING: Agence nationale de la recherche (France).

The MHC class I MICA gene is a histocompatibility antigen in kidney transplantation.

The identity of histocompatibility loci, besides human leukocyte antigen (HLA), remains elusive. The major histocompatibility complex (MHC) class I MICA gene is a candidate histocompatibility locus. Here, we investigate its role in a French multicenter cohort of 1,356 kidney transplants. MICA mismatches were associated with decreased graft survival (hazard ratio (HR), 2.12; 95% confidence interval (CI): 1.45-3.11; P < 0.001). Both before and after transplantation anti-MICA donor-specific antibodies (DSA) were strongly associated with increased antibody-mediated rejection (ABMR) (HR, 3.79; 95% CI: 1.94-7.39; P < 0.001; HR, 9.92; 95% CI: 7.43-13.20; P < 0.001, respectively). This effect was synergetic with that of anti-HLA DSA before and after transplantation (HR, 25.68; 95% CI: 3.31-199.41; P = 0.002; HR, 82.67; 95% CI: 33.67-202.97; P < 0.001, respectively). De novo-developed anti-MICA DSA were the most harmful because they were also associated with reduced graft survival (HR, 1.29; 95% CI: 1.05-1.58; P = 0.014). Finally, the damaging effect of anti-MICA DSA on graft survival was confirmed in an independent cohort of 168 patients with ABMR (HR, 1.71; 95% CI: 1.02-2.86; P = 0.041). In conclusion, assessment of MICA matching and immunization for the identification of patients at high risk for transplant rejection and loss is warranted.

Occurrence of De novo Donor-Specific Antibodies After COVID-19 in Kidney Transplant Recipients Is Low Despite Immunosuppression Modulation.

Introduction: Decreased immunosuppression has been proposed for kidney transplant recipients infected with coronavirus disease 2019 (COVID-19), but the impact on the alloreactive immune response during and after infection has been poorly investigated. We evaluated the occurrence of antihuman leukocyte antigen (HLA) donor-specific antibodies (DSAs) (post-COVID-19) and rejection episodes after COVID-19 with particular focus on immunosuppression modulation. Methods: Kidney transplant recipients from 2 French institutions had anti-HLA antibody screening before and after COVID-19. Management of immunosuppression, rejection episodes, COVID-19 severity, inflammatory markers, and antiviral therapies were recorded. Results: From 251 recruited patients, 72 were excluded because of COVID-19-related death (n = 25) and incomplete immunologic follow-up (n = 47). Among the remaining 179 included patients, almost half were hospitalized (49.2%). Antimetabolites were interrupted in 47% of patients (82% in hospitalized, median time of resumption of 23 days and in 15% nonhospitalized, median time of resumption of 7 days). Calcineurin inhibitors were interrupted in 12% of patients (all hospitalized, median time of resumption of 11 days). The incidence of post-COVID-19 DSA was 4% (8% and 0% in hospitalized and nonhospitalized, respectively). Allograft rejection occurred in 3 patients (1.7%) and all were hospitalized. Younger age, transplantation <1 year, and preexisting DSA were more frequently observed in patients with post-COVID-19 DSA, whereas inflammatory markers, lymphopenia, and use of antiviral therapies were not. Conclusion: The incidence of post-COVID-19 DSA among COVID-19-positive kidney transplant recipients was low (4%) despite a significant decrease in immunosuppression and was mainly restricted to high-risk immunologic patient's status. COVID-19 severity was not associated with post-COVID-19 DSA and/or rejection.

HLA-C*01:224N, a novel null HLA-C allele characterized by two next-generation sequencing methods.

We describe here the novel HLA-C*01:224N allele which has a premature stop codon in exon 3.

Characterization of the novel HLA-C*06:312 allele by two next-generation sequencing methods.

The novel HLA-C*06:312 allele was characterized using two next generation sequencing technologies.

Characterization of the novel HLA-A*03:425 allele by two next-generation sequencing methods.

The novel HLA-A*03:425 allele was characterized using two next-generation sequencing technologies.

Characterization of the novel HLA-C*02:207 allele by two next-generation sequencing methods.

The novel HLA-C*02:207 allele was characterized using two next generation sequencing technologies.

Characterization of the novel HLA-B*15:10:08 allele by two next-generation sequencing methods.

The novel HLA-B*15:10:08 allele was characterized using two next generation sequencing technologies.

Characterization of the novel HLA-B*40:02:35 allele by two next-generation sequencing methods.

The novel HLA-B*40:02:35 allele was characterized using two next generation sequencing technologies.

Characterization of the novel HLA-C*07:01:105 allele by two next-generation sequencing methods.

The novel HLA-C*07:01:105 allele was characterized using two next generation sequencing technologies.

Characterization of the novel HLA-B*42:01:05 allele by two next-generation sequencing methods.

The novel HLA-B*42:01:05 allele was characterized using two next generation sequencing technologies.