Immune exhaustion contributes to treatment failure after allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. Immune checkpoint blockade, including programmed ...cell death protein-1 (PD-1) blockade, is a promising strategy to improve the antitumor effect of allogeneic HSCT with high rates of response reported in patients treated for disease relapse. However, severe and sometimes fatal Graft- vs.-Host-Disease (GvHD) has been reported as a complication. Little is known about the dynamics of PD-1 expression on immune effector cells after allogeneic HSCT. In the present study, we analyzed PD-1 expression on T cell subpopulations isolated from 105 allogeneic HSCT recipients. Our analysis revealed a significant increase in proportions of PD-1-expressing CD4 and CD8 T cells early after allogeneic HSCT followed by a progressive normalization of PD-1 expression at CD8 but not CD4 T cell surface. Analysis of co-expression of two other exhaustion markers, 2B4 and CD160, revealed a preferential expansion of PD-1-single positive cells. Moreover, the analysis of granzyme B and perforin expression in PD-1+ and PD-1- CD8 T cells from HSCT recipients did not reveal any impairment in cytotoxic molecules production by PD-1-expressing CD8 T cells. Analyzing the association between clinical factors and the expression of PD-1 on T cells, we identified the use of
and/or
T-cell depletion as the factor most strongly associated with elevated PD-1 levels on T cells. Our results extend our knowledge of the regulation of PD-1 expression at T cell surface after allogeneic HSCT, a crucial information for the optimization of post-transplantation PD-1 blocking therapies.
Sorafenib significantly improves survival of FLT3-ITD mutated AML patients when used as a post-allogeneic HSCT maintenance. Importantly, clinical trials reported a low rate of toxicities requiring ...sorafenib discontinuation. The aim of our analysis was to evaluate the real-world experience in patients treated with post-allogeneic HSCT sorafenib maintenance therapy for FLT3-ITD AML with a particular focus on tolerability and toxicity-related treatment interruption. We conducted a single-center retrospective study on 30 FLT3-ITD AML patients undergoing allogeneic HSCT in complete remission between 2017 and 2020 and who received sorafenib maintenance. 26 patients (87%) experienced toxicities leading to dose reduction (n=9) or direct interruption (n=17). Average time on sorafenib was 125 days (range 1-765). Most common toxicities were skin, gastrointestinal, and hematologic. Among patients who had a dose reduction, 4 eventually interrupted the drug and 5 were able to continue. Among patients who interrupted sorafenib because of toxicities, 7 were re-challenged with good tolerance in 3 cases. Overall, 18 patients (60% of the entire cohort) definitively discontinued sorafenib because of toxicities. 14 patients were thereafter switched to midostaurin. Importantly, with a median follow-up of 12 months, the median overall survival was not reached suggesting a positive impact of sorafenib maintenance despite the high rates of treatment interruption. In conclusion, our real-world analysis reveals high rates of toxicity-related interruption of sorafenib maintenance after allogeneic HSCT. Interestingly, our results suggest the feasibility of re-challenging with sorafenib and/or of switching to other maintenance approaches in case of intolerance.
Impaired immune reconstitution after allogeneic hematopoietic stem cell transplantation (HSCT) contributes to increased risk of cancer relapse and infection resulting in significant morbidity and ...mortality. Unfortunately, effective strategies to functionally assess the quality of immune reconstitution are still missing. Quantification of
replication of the ubiquitous, non-pathogenic virus Torque Teno Virus (TTV) has been reported in small series as a test to functionally evaluate the quality of post-transplant immune reconstitution. In the present study, we analyzed by quantitative PCR TTV titers in plasma samples from a large cohort of 168 allogeneic HSCT recipients. Our analysis confirms that TTV titers peaked at 100 days post-transplant, followed by progressive normalization thereafter. Negative correlation of TTV titers with T cell absolute numbers during the first year post-transplant points to the restoration of an active anti-TTV immunity. Univariable and multivariable linear regression analysis demonstrated that donor CMV positive serostatus, donor type and immune suppression resulting from GVHD treatment affected the restoration of anti-TTV immunity. Importantly, higher TTV titers at 100 days after transplantation were associated with worse overall survival and higher risk of acute GVHD and infections. Our results provide new insights into the factors affecting the dynamics of TTV replication and indicate that TTV is a potentially useful biomarker to assess immune reconstitution and to predict complications and outcomes of allogeneic HSCT.
Human pegivirus-1 (HPgV-1) is a so-called commensal virus for which no known associated organ disease has been found to date. Yet, it affects immune-reconstitution as previously studied in the HIV ...population, in whom active co-infection with HPgV-1 can modulate T and NK cell activation and differentiation leading to a protective effect against the evolution of the disease. Little is known on the effect of HPgV-1 on immune-reconstitution in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, a patient population in which we and others have previously reported high prevalence of HPgV-1 replication. The aim of this study was to compare the immune reconstitution after allo-HSCT among HPgV-1-viremic and HPgV-1-non-viremic patients.
Within a cohort study of 40 allo-HSCT patients, 20 allo-HSCT recipients positive in plasma sample for HPgV-1 by rRT-PCR during the first year (1, 3, 6, 12 months) after transplantation were matched with 20 allo-HSCT recipients negative for HPgV-1. T and NK cell reconstitution was monitored by flow cytometry in peripheral blood samples from allo-HSCT recipients at the same time points.
We observed no significant difference in the absolute number and subsets proportions of CD4 and CD8 T cells between patient groups at any analysed timepoint. We observed a significantly higher absolute number of NK cells at 3 months among HPgV-1-viremic patients. Immunophenotypic analysis showed a significantly higher proportion of CD56
NK cells mirrored by a reduced percentage of CD56
NK cells in HPgV-1-positive patients during the first 6 months after allo-HSCT. At 6 months post-allo-HSCT, NK cell phenotype significantly differed depending on HPgV-1, HPgV-1-viremic patients displaying NK cells with lower CD16 and CD57 expression compared with HPgV-1-negative patients. In accordance with their less differentiated phenotype, we detected a significantly reduced expression of granzyme B in NK cells in HPgV-1-viremic patients at 6 months.
Our study shows that HPgV-1-viremic allo-HSCT recipients displayed an impaired NK cell, but not T cell, immune-reconstitution compared with HPgV-1-non-viremic patients, revealing for the first time a potential association between replication of the non-pathogenic HPgV-1 virus and immunomodulation after allo-HSCT.
Mesenchymal stromal cells (MSCs) are potent immunomodulators that have successfully been used to circumvent various types of inflammations, including steroid-resistant graft-versus-host disease. ...Although initially believed to be restricted to multipotent MSCs, this immunoregulatory function is shared with differentiated cells from the mesenchymal lineage such as skin fibroblasts (SFs). Mesenchymal cell-induced immunoregulation is so potent that it may allow the reactivation of dormant malignancies, a fact that would preclude using such cells as therapeutic agents. Because NK cells are pivotal effectors controlling tumor cell containment we investigated the effect of allogenic MSCs and SFs on NK cell function in vitro. When NK cells were incubated with IL-15 and MSCs or SFs for 6 days, their proliferation and cytotoxic activity were significantly decreased compared to NK cells cultured with IL-15 alone or with human venous endothelial cells. Cytotoxic activity inhibition reached 86% when assayed on MHC-I+ allogenic primary hematopoietic blasts, and was associated with a significant decrease in cytolytic granule exocytosis and in perforin release. Stromal cell-mediated inhibition was effective only if cell–cell proximity was long lasting: when NK cells were activated with IL-15 in the absence of MSCs and assayed for cytotoxicity in their presence no inhibition occurred. MSC inhibition was ultimately mediated by a soluble factor generated upon incubation with NK cells activated by IL-15 or IL-2. The indoleamine 2,3 dioxygenase was activated in MSCs and SFs because L-kynurenine was detected in inhibitory supernatants, but its blockade did not restore NK cell functions. The profound inhibition of cytotoxic activity directed against allogenic hematopoietic blasts exerted by MSCs and SFs on NK cells may be a concern. Should this occur in vivo it may induce the inability of NK cells to control residual or dormant malignant diseases after infusion of therapeutic MSCs.
Small-molecule immunosuppressive drugs (ISD) prevent graft rejection mainly by inhibiting T lymphocytes. Therapeutic immunoglobulins (IVIg) are used for substitution, antibody-mediated rejection ...(AbMR) and HLA-sensitized recipients by targeting distinct cell types. Since the effect of ISD and IVIg on natural killer (NK) cells remains somewhat controversial in the current literature, the aim of this comparative study was to investigate healthy donor's human NK cell functions after exposure to ISD and IVIg, and to comprehensively review the current literature. NK cells were incubated overnight with IL2/IL12 and different doses and combinations of ISD and IVIg. Proliferation was evaluated by
H-thymidine incorporation; phenotype, degranulation and interferon gamma (IFNγ) production by flow cytometry and ELISA; direct NK cytotoxicity by standard
Cr-release and non-radioactive DELFIA assays using K562 as stimulator and target cells; porcine endothelial cells coated with human anti-pig antibodies were used as targets in antibody-dependent cellular cytotoxicity (ADCC) assays. We found that CD69, CD25, CD54, and NKG2D were downregulated by ISD. Proliferation was inhibited by methylprednisolone (MePRD), mycophenolic acid (MPA), and everolimus (EVE). MePRD and MPA reduced degranulation, MPA only of CD56
NK cells. MePRD and IVIg inhibited direct cytotoxicity and ADCC. Combinations of ISD demonstrated cumulative inhibitory effects. IFNγ production was inhibited by MePRD and ISD combinations, but not by IVIg. In conclusion, IVIg, ISD and combinations thereof differentially inhibit NK cell functions. The most potent drug with an effect on all NK functions was MePRD. The fact that MePRD and IVIg significantly block NK cytotoxicity, especially ADCC, has major implications for AbMR as well as therapeutic strategies targeting cancer and immune cells with monoclonal antibodies.
The treatment of acute myeloid leukemia (AML) remains a challenge especially among the elderly. The Bcl-2 inhibitor venetoclax recently showed significant survival benefits in AML patients when ...combined to low-dose cytarabine or azacitidine. Bcl-2 inhibition initiate mitochondrial apoptosis, but also respiration and cellular ATP production in AML. AMP-Activated Protein Kinase (AMPK) is a central energy sensor activated by increased AMP:ATP ratio to restore the cellular energy balance. Unexpectedly, we observed that venetoclax inhibited AMPK activity through caspase-dependent degradation of AMPK subunits in AML cells. On the other hand, genetic models of AMPK invalidation and re-expression suggested that AMPK participated to the early stages of apoptotic response through a negative regulation of multi-domain anti-apoptotic effectors such as Mcl-1 or Bcl-xL. Together our results suggested a new link between AMPK and Bcl-2-dependent mitochondrial apoptosis that participated to the anti-leukemic activity of venetoclax in AML.
Peripheral natural killer (NK) cells upregulate T-bet and downregulate Eomes, the key transcription factors regulating NK cell maturation and function during the last maturation steps toward ...terminally differentiated effector cells. During this process, NK cells acquire killer immunoglobulin-like receptors (KIR) and effector functions, such as cytotoxicity and target cell-induced cytokine production. Inhibitory KIR are pivotal in the control of effector functions, but whether they also modulate T-bet/Eomes expression is unknown. We have measured T-bet/Eomes levels, KIR expression, and effector functions of maturing CD94(neg)CD56(dim)NK cells using CD57 as surface marker for maturation. Our cohort consisted of 23 healthy blood donors (HBD) homozygous for the KIR A haplotype that contains only inhibitory KIR2DL1 (ligand HLA-C2), KIR2DL3 (ligand HLA-C1), and KIR3DL1 (ligand HLA-Bw4). We confirm that during maturation of NK cells, the number of KIR increases, levels of T-bet/Eomes are modulated, and that cells acquire effector functions, such as cytotoxicity (CD107) and target cell-induced cytokine production (TNF-α). Because maturation was associated with the increase of the number of KIR as well as with the modulation of T-bet/Eomes, the number of KIR correlated with the extent of T-bet/Eomes modulation. However, whether the KIR were triggered by their cognate HLA ligands or not had no impact on T-bet and Eomes expression, indicating that modulation of T-box transcription factors during NK cell maturation does not depend on signals conveyed by KIR. We discuss the relevance of this finding in the context of models of NK cell maturation while cautioning that results obtained in a perhaps quite heterogeneous cohort of HBD are not necessarily conclusive.
Recent reports give insights into the role of the T-box transcription factors, T-bet and Eomesodermin (Eomes), in NK cell biology. In this mini-review, we recapitulate the initial reports that ...delineate T-bet and Eomes as master regulators of NK cell development, maturation, and function. We discuss how T-bet and Eomes expression is regulated during NK cell development and peripheral maturation. Furthermore, we summarize the current literature on the role of T-bet and Eomes in the transcriptional regulation of NK cell function and review possible effects of T-box transcription factor anomalies during aging, infection, cancer, and after hematopoietic stem cell transplantation. We discuss how the current data argue in favor of a model of T-bet and Eomes synergy in transcriptional regulation of NK cell function and identify T-box transcription factors as potential targets for therapeutic interventions.
PDF
