T-cell defect in diffuse large B-cell lymphomas involves expansion of myeloid-derived suppressor cells.

In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils represents an independent prognostic factor. These cell subsets include monocytic and granulocytic myeloid-derived suppressor cells (M- and G-MDSCs) defined by their ability to suppress T-cell responses. MDSCs are a heterogeneous population described in inflammatory and infectious diseases and in numerous tumors including multiple myeloma, chronic lymphocytic leukemia, and DLBCL. However, their mechanisms of action remain unclear. We broadly assessed the presence and mechanisms of suppression of MDSC subsets in DLBCL. First, a myeloid suppressive signature was identified by gene expression profiling in DLBCL peripheral blood. Accordingly, we identified, in a cohort of 66 DLBCL patients, an increase in circulating G-MDSC (Lin(neg)HLA-DR(neg)CD33(pos)CD11b(pos)) and M-MDSC (CD14(pos)HLA-DR(low)) counts. Interestingly, only M-MDSC number was correlated with the International Prognostic Index, event-free survival, and number of circulating Tregs. Furthermore, T-cell proliferation was restored after monocyte depletion. Myeloid-dependent T-cell suppression was attributed to a release of interleukin-10 and S100A12 and increased PD-L1 expression. In summary, we identified expanded MDSC subsets in DLBCL, as well as new mechanisms of immunosuppression in DLBCL.

CD10 delineates a subset of human IL-4 producing follicular helper T cells involved in the survival of follicular lymphoma B cells.

In follicular lymphoma (FL), follicular helper T cells (TFH) have been depicted as one of the main components of the malignant B-cell niche and a promising therapeutic target. Although defined by their capacity to sustain FL B-cell growth together with specific gene expression and cytokine secretion profiles, FL-TFH constitute a heterogeneous cell population. However, specific markers reflecting such functional heterogeneity are still lacking. In this study, we demonstrate that CD10 identifies a subset of fully functional germinal center TFH in normal secondary lymphoid organs. Importantly, this subset is amplified in the FL context, unlike in other B-cell lymphomas with a follicular growth pattern. Furthermore, whereas FL-TFH produce high levels of interleukin (IL)-21 and low levels of IL-17 irrespectively of their CD10 expression, CD10(pos) FL-TFH specifically exhibit an IL-4(hi)IFN-γ(lo)TNF-α(hi) cytokine profile associated with a high capacity to sustain directly and indirectly malignant B-cell survival. Altogether, our results highlight the important role of this novel functional subset in the FL cell niche.

Drug transporters are implicated in the diffusion of tacrolimus into the T lymphocyte in kidney and liver transplant recipients: Genetic, mRNA, protein expression, and functionality.

Because of a narrow therapeutic index and a wide inter- and intra-patient variability, therapeutic drug monitoring of the immunosuppressant drug tacrolimus (TAC) based on whole-blood concentrations (Cblood) is mandatory in solid organ transplant recipients. Using peripheral blood mononuclear cells concentrations (CPBMC) could improve patient outcomes. The poor correlation between Cblood and CPBMC makes hypothesize that drug transporters are implicated in the intracellular accumulation of TAC. The aim of this work was therefore to clinically study: i) the role of genetic variants and ii) the effect of mRNA and protein expression of 4 drug transporters on the TAC CPBMC/blood ratio. In addition, functional in vitro experiments were performed to mechanistically validate the clinical observations. Genetic variants of ABCB1/P-gp and SLC28A3/CNT3 did not influence TAC CPBMC in liver transplant recipients (LTR). ABCC2/MRP2 at the mRNA level; ABCB1/P-gp, SLC28A3/CNT3 and SLC29A1/ENT1 at the protein level; correlated with the CPBMC/blood in kidney and LTR. In vitro results suing transporter-expressing cells confirmed that TAC is substrate of P-gp but not MRP2, whereas experiments remained inconclusive for CNT3 and ENT1. In conclusion, the genetic-transcription-protein-functional approach presented in this work provides new insights in the understanding of TAC transport at the T lymphocyte plasma membrane.

Tacrolimus diffusion across the peripheral mononuclear blood cell membrane: impact of drug transporters.

Measuring tacrolimus (TAC) concentration in peripheral blood mononuclear cells (PBMCs) could better reflect the drug effect on its target (calcineurin (CaN) in lymphocytes) than whole blood concentrations. Mechanisms influencing TAC diffusion into PBMC are not well characterized. This work aimed at describing, ex vivo, TAC diffusion kinetics into PBMC and investigating the contribution of membrane transporters to regulate TAC intracellular concentration as well as the impact on CaN activity. PBMCs were incubated with TAC for 5 min to 4 h and under several experimental conditions: 37 °C (physiological conditions), 4 °C (inhibition of influx and efflux active transport), 37 °C + transporter inhibitors (verapamil, carvedilol, and probenecid and bromosulfophthalein, respectively, inhibitors of P-gp, OAT, and OATP). TAC concentration and CaN activity were measured in PBMC using liquid chromatography coupled with mass spectrometry. TAC intra-PBMC concentration was maximal after 1 h of incubation. Mean TAC PMBC concentrations were significantly lower in samples incubated at 4 °C compared to the 37 °C groups. Addition of verapamil slightly increased TAC accumulation in PBMC while other inhibitors had no effect. A significant correlation was found between TAC intra-PBMC concentration and the level of inhibition of CaN. Using an ex vivo cellular model, these results suggest that P-gp is involved in the drug efflux from PBMC while influx active transporters likely to regulate TAC intra-PBMC disposition remain to be identified. TAC concentration in PBMC is correlated with its pharmacodynamic effect. Then, TAC intra-PBMC concentration appears to be a promising biomarker to refine TAC therapeutic drug monitoring.

Pan-HDAC Inhibitors Restore PRDM1 Response to IL21 in CREBBP-Mutated Follicular Lymphoma.

PURPOSE: Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). We explored the relation that exists between the differentiation arrest of follicular lymphoma cells and loss-of-function of CREBBP acetyltransferase.Experimental Design: The study used human primary cells obtained from either follicular lymphoma tumors characterized for somatic mutations, or inflamed tonsils for normal germinal center B cells. Transcriptome and functional analyses were done to decipher the B- and T-cell crosstalk. Responses were assessed by flow cytometry and molecular biology including ChIP-qPCR approaches. RESULTS: Conversely to normal B cells, follicular lymphoma cells are unable to upregulate the transcription repressor, PRDM1, required for plasma cell differentiation. This defect occurs although the follicular lymphoma microenvironment is enriched in the potent inducer of PRDM1 and IL21, highly produced by Tfhs. In follicular lymphoma carrying CREBBP loss-of-function mutations, we found a lack of IL21-mediated PRDM1 response associated with an abnormal increased enrichment of the BCL6 protein repressor in PRDM1 gene. Moreover, in these follicular lymphoma cells, pan-HDAC inhibitor, vorinostat, restored their PRDM1 response to IL21 by lowering BCL6 bound to PRDM1. This finding was reinforced by our exploration of patients with follicular lymphoma treated with another pan-HDAC inhibitor. Patients showed an increase of plasma cell identity genes, mainly PRDM1 and XBP1, which underline the progression of follicular lymphoma B cells in the differentiation process. CONCLUSIONS: Our data uncover a new mechanism by which pan-HDAC inhibitors may act positively to treat patients with follicular lymphoma through the induction of the expression of plasma cell genes.

Pharmacokinetics and pharmacodynamics of tacrolimus in liver transplant recipients: inside the white blood cells.

OBJECTIVES: Despite improvements in patient management and extensive use of therapeutic drug monitoring (TDM), the rate of acute cellular rejection (ACR) remains high in patients treated with tacrolimus (TAC). Moreover, some patients experienced ACR while their whole-blood (WB) concentrations were maintained within the therapeutic range meaning that TDM in WB misrepresents the drug effect. Thus, monitoring TAC directly inside of its effect compartment (intracellular concentrations) or monitoring directly the inhibitory effect on the target protein (calcineurin activity) could be more relevant. The aim of the present study was to explore, in 10 de novo liver transplant recipients, the relationship between TAC whole-blood concentrations, TAC intracellular concentrations and TAC-induced intracellular calcineurin inhibition at day 1 and day 7 after treatment initiation. DESIGN AND METHODS: Prospective monocentric observational pharmacokinetic (WB and intracellular concentrations)-pharmacodynamic (calcineurin activity) study. RESULTS: Full intracellular TAC pharmacokinetic as well as calcineurin activity steady-state profiles is presented in the study. The main result of this study is the lack of relationship between TAC pharmacokinetics (WB and leukocytes) and calcineurin activity in leukocytes at day 1 and day 7 after the graft implantation. CONCLUSIONS: Drug monitoring of TAC intracellular concentrations and determination of the calcineurin activity are among future potential biomarkers of acute rejection in transplant recipients. A better knowledge of the relationship between TAC whole blood and intracellular concentrations and calcineurin activity appears necessary before planning clinical trials to evaluate their potential interest as predictive biomarkers.

Acute alcohol exposure has an independent impact on C-reactive protein levels, neutrophil CD64 expression, and subsets of circulating white blood cells differentiated by flow cytometry in nontrauma patients.

Acute and massive alcohol exposure (blood alcohol concentration of ≥1 g/L) is a common way to consume alcohol. In a prospective study performed in critically ill nontrauma patients, we compared C-reactive protein (CRP) values, circulating subsets of white blood cells, and neutrophil CD64 indexes recorded at admission to the intensive care unit between abstinent or moderate drinkers (n = 173), patients with acute or chronic alcohol exposure (n = 32), and patients with acute exposure but not chronically exposed to alcohol (n = 27). Values for CRP (P < 0.001), circulating neutrophils (P < 0.001), and neutrophil CD64 indexes (P < 0.001) were significantly lower in patients acutely exposed compared with the other patients, whereas values for B lymphocytes (P < 0.001) and cytotoxic (P < 0.001) and noncytotoxic T lymphocytes (P < 0.001) were significantly higher. After multiple regression analysis, alcohol exposure remained independently associated with values of CRP, neutrophils CD4 indexes, cytotoxic and noncytotoxic T lymphocytes, and CD16-negative and -positive monocytes. These results were not affected by the presence or absence of infection at admission. Our results suggest that in nontrauma critically ill patients, acute alcohol exposure diminishes inflammation and increases numbers of circulating B and T lymphocytes.

Evaluation of FMH QuikQuant for the detection and quantification of fetomaternal hemorrhage.

BACKGROUND: The Kleihauer-Betke test (KBT) is the most widely used assay for fetomaternal hemorrhage (FMH) detection in rhesus D negative women. In the current study, we sought to evaluate the performance of a flow cytometry (FCM) kit (FMH QuikQuant) using an anti-HbF antibody. METHODS: Eighty-three pregnant women, 58 umbilical cord blood (UCB) dilutions in adult blood, and 6 control samples were tested in parallel with FCM and KBT. RESULTS: Firstly, we compared for each assay, on the 58 UCB preparations, results obtained to dilutions prepared. FCM results showed an excellent correlation (r = 0.97), a high reproducibility with a coefficient of variation lower than 20% for values reaching 10 fetal RBCs. KBT values were correlated (r = 0.94) but exhibited a poor reproducibility. Then, we compared both techniques on all samples. FCM showed a good correlation with KBT (r = 0.87) but the KBT exhibited a systematic overestimation of the FMH. For 8 out of 83 pregnant women, KBT was positive. Five were concordant with FCM results (KBT+/FCM+). On the three discordant (KBT+/FCM-), 2 were finally classified as false positive of the KBT because a second control sample was negative and additionally, FCM identified an increased rate of F cells. One discordant case (KBT+/FCM-) remained unexplained. By receiver operating characteristic analysis, we found a threshold at 4.5 RBCs for FCM with a sensitivity of 89.8% and a specificity of 93.2%. CONCLUSIONS: The FMH QuikQuant kit is a reliable and highly reproductive FCM method for FMH quantification.