Impact of calcineurin inhibitor-free immunosuppression on de novo donor-specific antibody formation in liver transplant recipients.

BACKGROUND & AIMS: Low calcineurin inhibitor (CNI) levels expose liver transplant recipients to rejection episodes and potentially to antibody-mediated rejection. There are little data on the impact of CNI-free immunosuppression on de novo donor-specific HLA antibody (dnDSA) development. Here we evaluated the prevalence of dnDSA in liver transplant recipients on CNI-free maintenance regimens and their associations with histopathological abnormalities of allografts. METHODS: Seven hundred and twenty-seven liver transplant recipients underwent a first liver transplant between 2000 and 2018 in three French transplant centres and had protocolized follow-up with dnDSA screening and allograft biopsy 1, 5 and 10 years after transplantation. RESULTS: CNIs were withdrawn in 166 (22.8%) patients with or without conversion to mammalian target of rapamycin inhibitors and/or maintenance with mycophenolic acid. DSA were present after withdrawal in 30.1% (50/166) patients on CNI-free immunosuppression compared with 16% (90/561) on CNI maintenance therapy (p < 0.001). The cumulative incidence of dnDSA 10 years after transplant was 20% in the CNI group versus 28% in the CNI-free group (p < 0.01). dnDSAs were associated with histological graft abnormalities (significant allograft fibrosis or rejection) (HR 2.24, 95% CI 1.2-4.1; p = 0.01). In univariate Cox regression analysis, being on a CNI-free regimen did not impact graft histology. CONCLUSIONS: Patients on a CNI-free IS regimen have a higher prevalence of dnDSA than patients on a standard IS regimen. dnDSAs but not CNI-free immunosuppression were associated with abnormal allograft histology.

Tumor-induced natural killer cell dysfunction is a rapid and reversible process uncoupled from the expression of immune checkpoints.

Natural killer (NK) cells often become dysfunctional during tumor progression, but the molecular mechanisms underlying this phenotype remain unclear. To explore this phenomenon, we set up mouse lymphoma models activating or not activating NK cells. Both tumor types elicited type I interferon production, leading to the expression of a T cell exhaustion-like signature in NK cells, which included immune checkpoint proteins (ICPs). However, NK cell dysfunction occurred exclusively in the tumor model that triggered NK cell activation. Moreover, ICP-positive NK cells demonstrated heightened reactivity compared to negative ones. Furthermore, the onset of NK cell dysfunction was swift and temporally dissociated from ICPs induction, which occurred as a later event during tumor growth. Last, NK cell responsiveness was restored when stimulation was discontinued, and interleukin-15 had a positive impact on this reversion. Therefore, our data demonstrate that the reactivity of NK cells is dynamically controlled and that NK cell dysfunction is a reversible process uncoupled from the expression of ICPs.

Bicentric evaluation of stabilizing sampling tubes for assessment of monocyte HLA-DR expression in clinical samples.

BACKGROUND: Diminished expression of human leukocyte antigen DR on circulating monocytes (mHLA-DR), measured by standardized flow cytometry procedure, is a reliable indicator of immunosuppression in severely injured intensive care unit patients. As such, it is used as stratification criteria in clinical trials evaluating novel immunostimulating therapies. Preanalytical constraints relative to the short delay between blood sampling and flow cytometry staining have nevertheless limited its use in multicentric studies. The objective of the present work was to compare mHLA-DR expression between whole blood samples simultaneously drawn in EDTA or Cyto-Chex BCT tubes. METHODS: In two university hospitals, mHLA-DR was assessed in fresh whole blood from septic patients (n = 12) and healthy donors (n = 6) simultaneously sampled on EDTA and Cyto-Chex BCT tubes. Staining was performed immediately after sampling and after blood storage at room temperature. RESULTS: We confirmed that samples collected in Cyto-Chex tube had substantially enhanced stability for mHLA-DR results (48-72 h) over those collected in EDTA. On baseline values, despite good correlation between tubes (r = 0.98, p < 0.001), mHLA-DR expression was systematically lower with Cyto-Chex BCT. CONCLUSION: The present reports confirms the potential of Cyto-Chex BCT tubes to stabilize mHLA-DR expression before staining and extends the work of Quadrini et al. [Cytometry B 2021;100:103-114]. In centers without rapid access to flow cytometry facilities, it enables to tolerate delays in mHLA-DR staining. However, a 30% gap exists between results obtained with EDTA and Cyto-Chex BCT tubes. As current thresholds for clinical decisions were obtained with EDTA samples, further studies are needed to confirm clinical thresholds with Cyto-Chex BCT tubes.

Recombinant human interleukin-7 reverses T cell exhaustion ex vivo in critically ill COVID-19 patients.

BACKGROUND: Lymphopenia is a hallmark of severe coronavirus disease 19 (COVID-19). Similar alterations have been described in bacterial sepsis and therapeutic strategies targeting T cell function such as recombinant human interleukin 7 (rhIL-7) have been proposed in this clinical context. As COVID-19 is a viral sepsis, the objectives of this study were to characterize T lymphocyte response over time in severe COVID-19 patients and to assess the effect of ex vivo administration of rhIL-7. RESULTS: Peripheral blood mononuclear cells from COVID-19 patients hospitalized in intensive care unit (ICU) were collected at admission and after 20 days. Transcriptomic profile was evaluated through NanoString technology. Inhibitory immune checkpoints expressions were determined by flow cytometry. T lymphocyte proliferation and IFN-γ production were evaluated after ex vivo stimulation in the presence or not of rhIL-7. COVID-19 ICU patients were markedly lymphopenic at admission. Mononuclear cells presented with inhibited transcriptomic profile prevalently with impaired T cell activation pathways. CD4 + and CD8 + T cells presented with over-expression of co-inhibitory molecules PD-1, PD-L1, CTLA-4 and TIM-3. CD4 + and CD8 + T cell proliferation and IFN-γ production were markedly altered in samples collected at ICU admission. These alterations, characteristic of a T cell exhaustion state, were more pronounced at ICU admission and alleviated over time. Treatment with rhIL-7 ex vivo significantly improved both T cell proliferation and IFN-γ production in cells from COVID-19 patients. CONCLUSIONS: Severe COVID-19 patients present with features of profound T cell exhaustion upon ICU admission which can be reversed ex vivo by rhIL-7. These results reinforce our understanding of severe COVID-19 pathophysiology and opens novel therapeutic avenues to treat such critically ill patients based of immunomodulation approaches. Defining the appropriate timing for initiating such immune-adjuvant therapy in clinical setting and the pertinent markers for a careful selection of patients are now warranted to confirm the ex vivo results described so far. Trial registration ClinicalTrials.gov identifier: NCT04392401 Registered 18 May 2020, http:// clinicaltrials.gov/ct2/show/NCT04392401.

Inverted direct allorecognition triggers early donor-specific antibody responses after transplantation.

The generation of antibodies against donor-specific major histocompatibility complex (MHC) antigens, a type of donor-specific antibodies (DSAs), after transplantation requires that recipient's allospecific B cells receive help from T cells. The current dogma holds that this help is exclusively provided by the recipient's CD4+ T cells that recognize complexes of recipient's MHC II molecules and peptides derived from donor-specific MHC alloantigens, a process called indirect allorecognition. Here, we demonstrated that, after allogeneic heart transplantation, CD3ε knockout recipient mice lacking T cells generate a rapid, transient wave of switched alloantibodies, predominantly directed against MHC I molecules. This is due to the presence of donor CD4+ T cells within the graft that recognize intact recipient's MHC II molecules expressed by B cell receptor-activated allospecific B cells. Indirect evidence suggests that this inverted direct pathway is also operant in patients after transplantation. Resident memory donor CD4+ T cells were observed in perfusion liquids of human renal and lung grafts and acquired B cell helper functions upon in vitro stimulation. Furthermore, T follicular helper cells, specialized in helping B cells, were abundant in mucosa-associated lymphoid tissue of lung and intestinal grafts. In the latter, more graft-derived passenger T cells correlated with the detection of donor T cells in recipient's circulation; this, in turn, was associated with an early transient anti-MHC I DSA response and worse transplantation outcomes. We conclude that this inverted direct allorecognition is a possible explanation for the early transient anti-MHC DSA responses frequently observed after lung or intestinal transplantations.

Allograft recognition by recipient's natural killer cells: Molecular mechanisms and role in transplant rejection.

The current transplant immunology dogma defends that allograft rejection is initiated by recipient's adaptive immune system. In this prevalent model, innate immune cells in general, and natural killer (NK) cells in particular, are merely considered as downstream effectors which participate in the destruction of the graft only upon recruitment by adaptive effectors: alloreactive T cells or donor-specific antibodies (DSA). Challenging this vision, recent data demonstrated that recipients' NK cells are capable of a form of allorecognition because they can sense the absence of self HLA class I molecules on the surface of graft endothelial cells. Missing-self triggers mTORC1-dependent activation of NK cells, which in turn promote the development of graft microvascular inflammation and detrimentally impact graft survival. The fact that some patients develop chronic vascular rejection in absence of DSA or genetically-predicted missing self suggests that other molecular mechanisms could underly NK cell allorecognition. This review provides an overview of these proven and putative molecular mechanisms and discusses future research directions in this emerging field in organ transplant immunology.

Predictive value of HLAMatchmaker and PIRCHE-II scores for de novo donor-specific antibody formation after adult and pediatric liver transplantation.

Production of de novo DSA (dnDSA) is associated with an increased risk of antibody mediated rejection after liver transplantation. Antibodies not only recognize the entire antigen but are able to bind specific functional epitopes present on the HLA molecule surface. The HLAMatchmaker and the PIRCHE-II (predicted indirectly recognizable HLA epitopes) algorithms are able to determine predictive epitope mismatches scores and de novo DSA (dnDSA) synthesis based on alloreactive eplets' identification. The aim of the present study was to assess, for the first time in liver transplantation, the complementarity between these two algorithms. We retrospectively analyzed a cohort of 407 adult and 133 pediatric liver transplant patients without preformed DSA, transplanted between 1991 and 2019 in Lyon and Montpellier. HLA antibodies were detected by single antigen bead assay. HLA typing of the donor-recipient pair was achieved by serological and/or DNA-based techniques. PIRCHE-II and HLAMatchmaker algorithms were then applied on both groups. During follow-up, 27.3% of adults and 38.3% of children developed dnDSA. HLA-DRB1 and DQB1-PIRCHE-II and HLAMatchmaker scores were significantly higher in dnDSA group compared to no DSA group for both pediatric and adult patients (except for PIRCHE-II HLA-DRB1 locus score in pediatrics). ROC curves allowed determining score thresholds classifying patients in low- and high-risk of dnDSA synthesis. The two algorithms' Kaplan-Meier curves showed a predicted incidence of dnDSA 20 years after transplantation significantly lower in the low-risk group compare with the high-risk group (log rank <0.05), in both cohorts, with a good negative predictive value. In conclusion, HLAMatchmaker and PIRCHE-II algorithms both are effective tools to identify anti-HLA immunization risk and to predict dnDSA formation after liver transplantation.