Accréditation de la mesure de l'expression des molécules HLA-DR à la surface des monocytes (mHLA-DR) par cytométrie en flux.

INTRODUCTION: Le niveau d'expression des molécules HLA-DR à la surface des monocytes (mHLA-DR) est un marqueur diagnostique utilisé pour évaluer l'immunité des patients en réanimation (choc septique, polytraumatisés, brulures, greffe et plus récemment Covid-19). Il est également utilisé comme un outil de stratification dans les essais cliniques utilisant des thérapies immunostimulantes chez ces patients. L'objectif de cette étude était d'évaluer les performances analytiques d'une méthode de cytométrie en flux pour mesurer mHLA-DR afin de répondre aux exigences de la norme NF EN ISO 15189 dans le cadre de l'accréditation des laboratoires de biologie médicale. Matériels et méthodes. L'évaluation (performances de la technique, étendue de la mesure, comparaison de méthode) a été menée en suivant le SH GTA 04, guide recommandé par le Comité français d'accréditation (COFRAC). En complément, certaines conditions pré analytiques ont été ré-évaluées. Résultats. L'ensemble des coefficients de variation évaluant les performances étaient inférieurs à 10 % (répétabilité, reproductibilité, variabilité interopérateur). Les limites de quantification et de linéarité étaient adaptées à l'utilisation clinique du paramètre. Les résultats étaient identiques quel que soit le type et le fournisseur de cytomètre en flux. Les contraintes de conservation pré-analytiques des échantillons ont été confirmées. CONCLUSION: Les résultats étaient conformes aux exigences de qualité recommandées par le COFRAC. Ils permettent l'accréditation de la mesure de mHLA-DR par cytométrie en flux et son utilisation en soins courants.

Lymphocyte HLA-DR/CD-38 co-expression correlates with Hodgkin lymphoma cell cytotoxicity in vitro independent of PD-1/PD1-L pathway.

The interactions between Hodgkin and Reed Sternberg cells and tumor microenvironment, the changes that occur with therapy and, in particular, checkpoint inhibition are not fully understood. Understanding these is key to optimizing outcomes for patients with Hodgkin lymphoma (HL). We evaluated the immunophenotypic characteristics of cytotoxic, helper T and NK lymphocytes upon in vitro stimulation, cell-mediated cytotoxicity against HL cells, HDLM-2 and KM-H2, and the association with effector cell activation state, as well as changes in cytotoxicity following PD-1 or PDL-1 blockade. Higher HLA-DR/CD38 expression on effector cells was associated with increased cytotoxicity against HL cells. All effector cell types were cytotoxic of HL cells, though achieved maximum activation and cytotoxicity at variable timepoints. HLA-DR/CD38 co-expression correlated with cytotoxicity, but PD-1 expression did not. There was no significant change in cell-mediated cytotoxicity following PD-1/PDL-1 blockade. The mechanism of action of checkpoint inhibitors may not be limited to direct PD-1/PDL-1 blockade.

HLA-DR Expression Level in CD8+ T Cells Correlates With the Severity of Children With Acute Infectious Mononucleosis.

Background: This study aimed to assess the host immune signatures associated with EBV infection and its clinical value in indicating the severity of children with acute infectious mononucleosis (IM). Methods: Twenty-eight pediatric patients with IM aged 3-8 years were enrolled. The immune phenotypes and cytokine secretion capability of T cells were detected. Results: The percentages and absolute numbers of CD3+ and CD8+ T cells were significantly increased in IM patients compared with HCs. The percentages of Naïve CD4+ and CD8+ T cells were decreased but with increased percentages of memory CD4+ and CD8+ T subsets. Our results showed the upregulation of active marker HLA-DR, TCR-αß, and inhibitory receptors PD-1, TIGIT in CD8+ T cells from IM patients, which suggested that effective cytotoxic T cells were highly against EBV infection. However, EBV exposure impaired the cytokine (IFN-γ, IL-2, and TNF-α) secretion capability of CD4+ and CD8+ T cells after stimulation with PMA/ionomycin in vitro. Multivariate analysis revealed that the percentage of HLA-DR+ CD8+ T cells was an independent prognostic marker for IM. The percentage of HLA-DR+ CD8+ T cells was significantly correlated with high viral load and abnormal liver function results. Conclusion: Robust expansion and upregulation of HLA-DR in CD8+ T cells, accompanied with impaired cytokine secretion, were typical characteristics of children with acute IM. The percentage of HLA-DR+ CD8+ T cells might be used as a prominent marker not only for the early diagnosis but also for indicating the severity of IM.

Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion.

Objective: The development of advanced single-cell technologies to decipher inter-cellular heterogeneity has enabled the dynamic assessment of individual cells behavior over time, overcoming the limitation of traditional assays. Here, we evaluated the feasibility of an advanced microfluidic assay combined to fluorescence microscopy to address the behavior of circulating monocytes from septic shock patients. Methods: Seven septic shock patients and ten healthy volunteers were enrolled in the study. Using the proposed microfluidic assay we investigated the production over time of LPS-elicited TNFα by single monocytes encapsulated within droplets. Cellular endocytic activity was assessed by internalization of magnetic nanoparticles. Besides, we assessed HLA-DR membrane expression and LPS-induced TNFα production in monocytes through classical flow cytometry assays. Results: Consistent with the flow cytometry results, the total number of TNFα molecules secreted by encapsulated single monocytes was significantly decreased in septic shock patients compared to healthy donors. TNFα production was dampened as soon as 30 and 60 minutes after LPS stimulation in monocytes from septic patients. Furthermore, the microfluidic assay revealed heterogeneous individual behavior of monocytes from septic shock patients. Of note, monocytes from both healthy donors and patients exhibited similar phagocytic activities over time. Conclusion: The microfluidic assay highlights the functional heterogeneity of monocytes, and provides in-depth resolution in assessing the hallmark monocyte deactivation encountered in post-septic immunosuppression.

Phenotypic and Functional Consequences of PLT Binding to Monocytes and Its Association with Clinical Features in SLE.

Platelets (PLTs) can modulate the immune system through the release of soluble mediators or through interaction with immune cells. Monocytes are the main immune cells that bind with PLTs, and this interaction is increased in several inflammatory and autoimmune conditions, including systemic lupus erythematosus (SLE). Our aim was to characterize the phenotypic and functional consequences of PLT binding to monocytes in healthy donors (HD) and in SLE and to relate it to the pathogenesis of SLE. We analyzed the phenotypic and functional features of monocytes with non-activated and activated bound PLTs by flow cytometry. We observed that monocytes with bound PLTs and especially those with activated PLTs have an up-regulated HLA-DR, CD86, CD54, CD16 and CD64 expression. Monocytes with bound PLTs also have an increased capacity for phagocytosis, though not for efferocytosis. In addition, monocytes with bound PLTs have increased IL-10, but not TNF-α, secretion. The altered phenotypic and functional features are comparable in SLE and HD monocytes and in bound PLTs. However, the percentages of monocytes with bound PLTs are significantly higher in SLE patients and are associated with undetectable levels of anti-dsDNA antibodies and hematuria, and with normal C3 and albumin/creatinine levels. Our results suggest that PLTs have a modulatory influence on monocytes and that this effect may be highlighted by an increased binding of PLTs to monocytes in autoimmune conditions.

Monocytic Human Leukocyte Antigen DR Expression in Young Infants Undergoing Cardiopulmonary Bypass.

BACKGROUND: Monocytic human leukocyte antigen DR (mHLA-DR) expression levels have been reported to be a marker of immunosuppression and a predictor of sepsis and mortality. There are, however, scant data regarding mHLA-DR monitoring in young infants after cardiopulmonary bypass. Our objectives were to investigate the kinetics of mHLA-DR expression and to determine whether mHLA-DR levels are associated with healthcare-associated infection (HAI) after cardiopulmonary bypass in young infants. METHODS: mHLA-DR levels were analyzed by flow cytometry using a standardized method in 49 infants (<3 months old) with congenital heart disease before and after cardiopulmonary bypass. Results are expressed as the number of anti-HLA-DR antibodies per cell (AB/c). RESULTS: Postoperative mHLA-DR expression was reduced in all infants. Eleven patients (22%) developed HAI, and 4 patients (8%) died during the 30-day follow-up. mHLA-DR expression was significantly lower on postoperative day 4 in the HAI group compared with those who without HAI (3768 AB/c [range, 1938-6144] vs 13,230 AB/c [range, 6152-19,130], P = .014). Although mHLA-DR expression was associated with postoperative severity, mHLA-DR ≤4500 AB/c in the first 72 hours among patients with higher postoperative severity (extracorporeal membrane oxygenation and/or corticoids and/or delayed closure of sternum) was associated with occurrence of HAI in the univariate analysis (odds ratio, 6.3; 95% confidence interval, 1.0-38.7; P = .037). CONCLUSIONS: Cardiopulmonary bypass induces a profound decrease in mHLA-DR expression in young infants. Among patients with higher postoperative severity, low level of mHLA-DR in the early postoperative period is associated with the development of HAI.

Reduced Monocytic Human Leukocyte Antigen-DR Expression Indicates Immunosuppression in Critically Ill COVID-19 Patients.

BACKGROUND: The cellular immune system is of pivotal importance with regard to the response to severe infections. Monocytes/macrophages are considered key immune cells in infections and downregulation of the surface expression of monocytic human leukocyte antigen-DR (mHLA-DR) within the major histocompatibility complex class II reflects a state of immunosuppression, also referred to as injury-associated immunosuppression. As the role of immunosuppression in coronavirus disease 2019 (COVID-19) is currently unclear, we seek to explore the level of mHLA-DR expression in COVID-19 patients. METHODS: In a preliminary prospective monocentric observational study, 16 COVID-19-positive patients (75% male, median age: 68 [interquartile range 59-75]) requiring hospitalization were included. The median Acute Physiology and Chronic Health Evaluation-II (APACHE-II) score in 9 intensive care unit (ICU) patients with acute respiratory failure was 30 (interquartile range 25-32). Standardized quantitative assessment of HLA-DR on monocytes (cluster of differentiation 14+ cells) was performed using calibrated flow cytometry at baseline (ICU/hospital admission) and at days 3 and 5 after ICU admission. Baseline data were compared to hospitalized noncritically ill COVID-19 patients. RESULTS: While normal mHLA-DR expression was observed in all hospitalized noncritically ill patients (n = 7), 89% (8 of 9) critically ill patients with COVID-19-induced acute respiratory failure showed signs of downregulation of mHLA-DR at ICU admission. mHLA-DR expression at admission was significantly lower in critically ill patients (median, [quartiles]: 9280 antibodies/cell [6114, 16,567]) as compared to the noncritically ill patients (30,900 antibodies/cell [26,777, 52,251]), with a median difference of 21,508 antibodies/cell (95% confidence interval [CI], 14,118-42,971), P = .002. Reduced mHLA-DR expression was observed to persist until day 5 after ICU admission. CONCLUSIONS: When compared to noncritically ill hospitalized COVID-19 patients, ICU patients with severe COVID-19 disease showed reduced mHLA-DR expression on circulating CD14+ monocytes at ICU admission, indicating a dysfunctional immune response. This immunosuppressive (monocytic) phenotype remained unchanged over the ensuing days after ICU admission. Strategies aiming for immunomodulation in this population of critically ill patients should be guided by an immune-monitoring program in an effort to determine who might benefit best from a given immunological intervention.

Expansion and characterization of cells from surgically removed intervertebral disc fragments in xenogen-free medium.

Low back pain due to degeneration of intervertebral disc (IVD) is a major health problem resulting in significant disability as well as adding to the economic burden. Discectomy is a very common procedure done worldwide to relieve this pain. At present all the surgically removed disc tissue is mostly discarded. However, there are reports that state that progenitor cells in the IVD can be grown ex vivo and have the potential to be used for IVD repair and regeneration. We report here that viable cells can be harvested from surgically removed, herniated disc tissue and can be potentially used in cell based therapy. Further, we have successfully replaced xenogenic supplements such as foetal bovine serum with either autologous serum or human platelet lysate for culturing IVD cells from patient's surgically removed disc tissue, without loss of any cell characteristics, including cell surface markers, growth factor secretion in the conditioned medium and osteogenic and chondrogenic differentiation potential in vitro. The present work will not only contribute to overcoming some of the major barriers in carrying out human clinical trials, but also provide a cheap, alternate source of proteins and growth factors for growing IVD cells ex vivo for therapy.

Interferon-γ-induced HLA Class II expression on endothelial cells is decreased by inhibition of mTOR and HMG-CoA reductase.

In organ transplantation, donor-specific HLA antibody (DSA) is considered a major cause of graft rejection. Because DSA targets primarily donor-specific human leukocyte antigen (HLA) expressed on graft endothelial cells, the prevention of its expression is a possible strategy for avoiding or salvaging DSA-mediated graft rejection. We examined the effect of various clinically used drugs on HLA class II expression on endothelial cells. Interferon-γ (IFN-γ)-induced HLA class II DR (HLA-DR) was downregulated by everolimus (EVR, 49.1% ± 0.8%; P < 0.01) and fluvastatin (FLU, 33.8% ± 0.6%; P < 0.01). Moreover, the combination of EVR and FLU showed a greater suppressive effect on HLA-DR expression. In contrast, cyclosporine, tacrolimus, mycophenolic acid, and prednisolone did not exhibit any significant suppressive effect. FLU, but not EVR, suppressed mRNA of HLA-DR. Imaging analysis revealed that HLA-DR expressed in cytosol or on the cell surface was repressed by EVR (cytosol: 58.6% ± 4.9%, P < 0.01; cell surface: 80.9% ± 4.0%, P < 0.01) and FLU (cytosol: 19.0% ± 3.4%, P < 0.01; cell surface: 48.3% ± 4.8%, P < 0.01). These data indicated that FLU and EVR suppressed IFN-γ-induced HLA-DR expression at the transcriptional and post-translational level, respectively, suggesting a potential approach for alleviating DSA-related issues in organ transplantation.

Disruption of Monocyte and Macrophage Homeostasis in Periodontitis.

Monocytes and macrophages are major cellular components of the innate immunity that play essential roles in tissue homeostasis. The contribution of different subsets of monocytes/macrophages to periodontal health and disease has not been fully elucidated. Type 2 diabetes mellitus (T2DM) is a risk factor for periodontitis. We hypothesized that the monocyte/macrophage signaling is perturbed in periodontitis-affected sites versus periodontally healthy sites and that this perturbation plays a critical role in the pathogenesis of periodontitis. Pairs of gingival tissue samples (each from a periodontally healthy and a periodontitis-affected site of the same patient) were harvested from 27 periodontitis patients, with and without T2DM. Each sample was processed to form a single-cell suspension, and a flow-cytometry panel was designed and validated to study monocyte and macrophage phenotypes. In separate experiments, the transcriptional changes associated with a pro-inflammatory phenotype were also examined in monocyte/macrophage subsets obtained from peripheral blood of patients with T2DM versus diabetes-free controls. A significantly higher proportion of intermediate (CD14+CD16+) monocytes was observed in periodontitis-affected tissues compared to healthy tissues. These monocytes overexpressed HLA-DR and PDL1 molecules, suggesting their activated inflammatory status. PDL1 increase was specific to intermediate monocytes. The ratio of M1/M2 macrophages was also significantly higher in periodontally affected sites, signifying an imbalance between inflammatory and repair mechanisms. We found a significantly higher expression of PDL1 in overall monocytes and M1 macrophages in periodontitis-affected sites compared to controls. Importantly, we identified a subpopulation of M1 macrophages present in periodontally affected tissues which expressed high levels of CD47, a glycoprotein of the immunoglobulin family that plays a critical role in self-recognition and impairment of phagocytosis. Analysis of the transcriptional landscape of monocytes/macrophages in gingival tissue of T2DM patients with periodontitis revealed a significant disruption in homeostasis toward a proinflammatory phenotype, elevation of pro-inflammatory transcription factors STAT1 and IRF1, and repression of anti-inflammatory JMJD3 in circulating monocytes. Taken together, our results demonstrate disruption of myeloid-derived cell homeostasis in periodontitis, with or without T2DM, and highlight a potentially significant role of these cell types in its pathogenesis. The impact of macrophage and monocyte signaling pathways on the pathobiology of periodontitis should be further evaluated.

Characterization of Circulating IL-10-Producing Cells in Septic Shock Patients: A Proof of Concept Study.

Sepsis is a worldwide health priority characterized by the occurrence of severe immunosuppression associated with increased risk of death and secondary infections. Interleukin 10 (IL-10) is a potent immunosuppressive cytokine which plasma concentration is increased in septic patients in association with deleterious outcomes. Despite studies evaluating IL-10 production in specific subpopulations of purified cells, the concomitant description of IL-10 production in monocytes and lymphocytes in septic patients' whole blood has never been performed. In this pilot study, we characterized IL-10 producing leukocytes in septic shock patients through whole blood intracellular staining by flow cytometry. Twelve adult septic shock patients and 9 healthy volunteers were included. Intracellular tumor necrosis factor-α (TNFα) and IL-10 productions after lipopolysaccharide stimulation by monocytes and IL-10 production after PMA/Ionomycine stimulation by lymphocytes were evaluated. Standard immunomonitoring (HLA-DR expression on monocytes, CD4+ T lymphocyte count) of patients was also performed. TNFα expression by stimulated monocytes was reduced in patients compared with controls while IL-10 production was increased. This was correlated with a reduced monocyte HLA-DR expression. B cells, CD4+, and CD4- T lymphocytes were the three circulating IL-10 producing lymphocyte subsets in both patients and controls. No difference in IL-10 production between patients and controls was observed for B and CD4- T cells. However, IL-10 production by CD4+ T lymphocytes significantly increased in patients in parallel with reduced CD4+ T cells number. Parameters reflecting altered monocyte (increased IL-10 production, decreased HLA-DR expression and decreased TNFα synthesis) and CD4+ T lymphocyte (increased IL-10 production, decreased circulating number) responses were correlated. Using a novel technique for intracellular cytokine measurement in whole blood, our results identify monocytes and CD4+ T cells as the main IL-10 producers in septic patients' whole blood and illustrate the development of a global immunosuppressive profile in septic shock. Overall, these preliminary results add to our understanding of the global increase in IL-10 production induced by septic shock. Further research is mandatory to determine the pathophysiological mechanisms leading to such increased IL-10 production in monocytes and CD4+ T cells.

Poor Outcome Could Be Predicted by Lower Monocyte Human Leukocyte Antigen-DR Expression in Patients with Complicated Intra-Abdominal Infections: A Review.

Complicated intra-abdominal infections (cIAIs) are still associated with high morbidity and mortality levels. Early prognostic evaluation is a great challenge, and a serious amount of resources have been used to find the perfect mortality predictor. Monocyte human leukocyte antigen-DR (mHLA-DR) expression has been studied as a biomarker in patients with sepsis and other infections. Our aim was to evaluate the potential prognostic performance of mHLA-DR in patients with cIAIs. Methods: We performed an electronic search of Google Scholar and PubMed databases for articles published before January 2019. The search terms were "HLA-DR," "monocyte HLA-DR," "intra-abdominal infections," "sepsis," "outcome," and "mortality." Results: A total of 12 studies with 761 patients met our inclusion criteria. In 10 studies, poor outcome was predicted by lower mHLA-DR expression, and two studies showed no prognostic value. Conclusion: This review found association between lower mHLA-DR expression and mortality. We concluded that mHLA-DR could be a reliable and meaningful predictor of poor outcome in patients with cIAIs. Nevertheless, more large prospective studies with surgical patients exclusively are needed before using this biomarker in a clinical setting.

PD-L1, LAG3, and HLA-DR are increasingly expressed during smoldering myeloma progression.

Symptomatic multiple myeloma (MM) is a plasma cell neoplasm that represents the final stage of a continuum of clinical conditions that start from monoclonal gammopathy of unknown significance (MGUS), then transits in the more advance, but still asymptomatic, smoldering MM (SMM), with a final evolution in symptomatic MM. To investigate SMM microenvironment modifications, we studied 16 patients diagnosed at our hospital. Eight of them (group A) developed MM within 2 years from diagnosis while the others (group B) had stable SMM. Samples were bone marrow biopsies at diagnosis and after 2 years (± 4 months) and were analyzed by immunohistochemical analysis. Firstly, we found a significant increase in both CD4+ cells (11 vs 17%, p < 0.01) and CD8+ cells (15 vs 18%, p < 0.01) between diagnosis and at follow-up samples (whole cohort). This was associated to an increase in the CD4+/CD8+ ratio (0.74 vs 0.93, p < 0.01). Secondly, we discovered an increased expression of T cell inhibitory molecules during SMM evolution. In fact, plasma cell PD-L1 and microenvironment cell LAG3 expression increased from 1 to 12% (p = 0.03) and 4 to 10% (p = 0.04), respectively, from diagnosis to follow-up. Also, plasma cells and microenvironment cells HLA-DR expression augmented during SMM evolution from 7 to 10% (p = 0.04) and 29 to 39% (p = 0.01), respectively. When comparing group A vs group B, we found an increased CD68-KP1+ cell infiltration in favor of group B at diagnosis (23 vs 28%, p = 0.01) and a greater plasma cell infiltration at follow-up (50 vs 26%, p < 0.01). Our findings suggest how immune escape mechanisms appear earlier during multiple myeloma evolution, and that LAG3 could be a possible immunologic target in this setting.

Patients with out-of-hospital cardiac arrest show decreased human leucocyte antigen-DR expression on monocytes and B and T lymphocytes after return of spontaneous circulation.

Immune disorders are an important feature of patients with out-of-hospital cardiac arrest (OHCA) after return of spontaneous circulation (ROSC). However, the precise immune alterations in patients with OHCA that occur immediately after ROSC are unclear. In this study, we investigated human leucocyte antigen-DR (HLA-DR) expression on circulatory monocytes and B and T lymphocytes. Sixty-eight consecutive patients with OHCA with ROSC >12 hours were enrolled. Clinical data and 28-day survival were recorded. Peripheral blood samples after ROSC days 1 and 3 were analysed to evaluate HLA-DR expression. Fifty healthy individuals were enrolled as controls. Compared with levels in healthy individuals, HLA-DR expression on monocytes and B lymphocytes, but not on T lymphocytes, decreased on days 1 and 3 after ROSC. No significant difference in HLA-DR expression was detected between survivors and non-survivors on day 1. For 41 patients with expression data for days 1 and 3, HLA-DR expression on monocytes and B lymphocytes in non-survivors was lower than that in survivors on day 3. In non-survivors, the mean fluorescence intensities of HLA-DR on B lymphocytes and percentages of HLA-DR+ T lymphocytes were lower on day 3 than on day 1. On days 1 and 3, there were significant correlations between HLA-DR expression on monocytes and B lymphocytes and clinical indicators, such as time to ROSC, adrenaline dose, acute physiology, chronic health evaluation II and the sequential organ failure assessment. The decreases in HLA-DR expression on circulatory monocytes and B and T lymphocytes after ROSC may be involved in the observed immunosuppression in patients with OHCA.

Dynamic changes in HLA-DR expression during short-term and long-term ibrutinib treatment in patients with chronic lymphocytic leukemia.

There is the first evidence of changes in the kinetics of B cell antigen receptor (BCR) internalisation of neoplastic cells in chronic lymphocytic leukemia (CLL) after the short-term and long-term administration of ibrutinib. We aimed to assess the influence of short-term and long-term ibrutinib treatment on the HLA-DR expression on CLL cells, T cells and monocytes. The immunophenotyping of CLL and immune cells in peripheral blood was performed on 16 high-risk CLL patients treated with ibrutinib. After early ibrutinib administration, the HLA-DR expression on CLL cells reduced (P = 0.032), accompanied by an increase in CLL cell counts in peripheral blood (P = 0.001). In vitro culturing of CLL cells with ibrutinib also revealed the reduction in the HLA-DR expression at protein and mRNA levels (P < 0.01). The decrease in HLA-DR on CLL cells after the first month was followed by the gradual increase of its expression by the 12th month (P = 0.001). A one-month follow-up resulted in elevated absolute counts of CD4+ (P = 0.002) and CD8+ (P < 0.001) T cells as well as CD4+ and CD8+ cells bearing HLA-DR (P < 0.01). The long-term administration of ibrutinib was associated with the increased numbers of CD4+ bearing HLA-DR (P = 0.006) and elevation of HLA-DR expression on all monocyte subsets (P ≤ 0.004). Our results provide the first evidence of the time-dependent immunomodulatory effect of ibrutinib on CLL and T cells and monocytes. The clinical consequences of time-dependent changes in HLA-DR expression in ibrutinib treated patients deserve further investigation.

Monocyte function in patients with myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a malignant hematopoietic stem cell disorder that frequently evolves into acute myeloid leukemia (AML). Patients with MDS are prone to infectious complications, in part due to the presence of severe neutropenia and/or neutrophil dysfunction. However, not all patients with neutropenia become infected, suggesting that other immune cells may compensate in these patients. Monocytes are also integral to immunologic defense; however, much less is known about monocyte function in patients with MDS. In the current study, we monitor the composition of peripheral blood monocytes and several aspects of monocyte function in MDS patients, including HLA-DR expression, LPS-induced inflammatory cytokine production, and phagocytosis. We find that monocytes from MDS patients exhibit relatively normal innate immune functions compared to monocytes from healthy control subjects. We also find that HLA-DR expression is moderately increased in monocytes from MDS patients. These results suggest that monocytes could compensate for other immune deficits in MDS patients to help fight infection. We also find that the range of immune functions in monocytes from MDS patients correlates with several key clinical parameters, including blast cell count, monocyte count, and revised International Prognostic Scoring System score, suggesting that disease severity impacts monocyte function in MDS patients.

Human Immunodeficiency Virus Type-1 Elite Controllers Maintain Low Co-Expression of Inhibitory Receptors on CD4+ T Cells.

Human immunodeficiency virus type-1 (HIV-1) elite controllers (ELCs) represent a unique population that control viral replication in the absence of antiretroviral therapy (cART). It is well established that expression of multiple inhibitory receptors on CD8+ T cells is associated with HIV-1 disease progression. However, whether reduced co-expression of inhibitory receptors on CD4+ T cells is linked to natural viral control and slow HIV-1 disease progression remains undefined. Here, we report on the expression pattern of numerous measurable inhibitory receptors, associated with T cell exhaustion (programmed cell death-1, CTLA-4, and TIGIT), on different CD4+ T cell memory populations in ELCs and HIV-infected subjects with or without long-term cART. We found that the co-expression pattern of inhibitory receptors was significantly reduced in ELCs compared with HIV-1 cART-treated and viremic subjects, and similar to healthy controls. Markers associated with T cell exhaustion varied among different memory CD4+ T cell subsets and highest levels were found mainly on transitional memory T cells. CD4+ T cells co-expressing all inhibitory markers were positively correlated to T cell activation (CD38+ HLA-DR+) as well as the transcription factors Helios and FoxP3. Finally, clinical parameters such as CD4 count, HIV-1 viral load, and the CD4/CD8 ratio all showed significant associations with CD4+ T cell exhaustion. We demonstrate that ELCs are able to maintain lower levels of CD4+ T cell exhaustion despite years of ongoing viral replication compared with successfully cART-treated subjects. Our findings suggest that ELCs harbor a "healthy" state of inhibitory receptor expression on CD4+ T cells that might play part in maintenance of their control status.

Staining of HLA-DR, Iba1 and CD68 in human microglia reveals partially overlapping expression depending on cellular morphology and pathology.

HLA-DR, Iba1 and CD68 are widely used microglia markers in human tissue. However, due to differences in gene regulation, they may identify different activation stages of microglia. Here, we directly compared the expression of HLA-DR, Iba1 and CD68 in microglia with different phenotypes, ranging from ramified to amoeboid, to foamy phagocytizing macrophages, in adjacent sections immunocytochemically double stained for two of the markers. Material was used from patients diagnosed with multiple sclerosis (MS) and Alzheimer's disease (AD) patients and control subjects because together they contain all the microglia activation stages in an acute and a chronic inflammatory setting. We found a similar, yet not identical, overall expression pattern. All three markers were expressed by ramified/amoeboid microglia around chronic active MS lesions, but overlap between HLA-DR and Iba1 was limited. Foamy macrophages in the demyelinating rims of active MS lesions of MS expressed more HLA-DR and CD68 than Iba1. All markers were expressed by small microglia accumulations (nodules) in MS NAWM. Dense core AD plaques in the hippocampus were mostly associated with microglia expressing HLA-DR. Diffuse AD plaques were not specifically associated with microglia at all. These results indicate that microglia markers have different potential for neuropathological analysis, with HLA-DR and CD68 reflecting immune activation and response to tissue damage, and Iba1 providing a marker more suited for structural studies in the absence of pathology.

Enterovirus but not Parvovirus B19 is associated with idiopathic dilated cardiomyopathy and endomyocardial CD3, CD68, or HLA-DR expression.

We assessed Enterovirus (EV) &Parvovirus B19 (PVB19) genomes and CD3, CD68&HLA-DR detection in dilated cardiomyopathies (DCM). EV&PVB19 genomes and CD3, CD68&HLA-DR were detected by PCR and immunohistochemistry assays in 115 endomyocardial biopsies obtained in 13 idiopathic DCM (iDCM) and 10 explained DCM (eDCM) patients. Results were compared with those of 47 atrial surgical samples (47 surgery controls) and 22 autoptic cardiac samples (11 healthy heart controls) (2008-2014, Reims, France). EV was detected in 23.1% of iDCM patients but not in eDCM and controls (P = 0.003) (viral load 803 copies/µg). PVB19 was detected in 76.9%, 80.0%, 63.6% and 78.2% of iDCM, eDCM, healthy heart and surgery controls (P = 0.99) with a mean viral load of 413, 346, 1,428, and 71 copies/µg. CD3, CD68 or HLA-DR were detected in 100 and 50% of EV and PVB19 "mono-infected" iDCM patients. EV was exclusively detected in iDCM cases in association with CD3, CD68, or HLA-DR indicating that EV could be an etiological cause in a subset of iDCM cases. By contrast the equal frequent detection of PVB19 in iDCM cases and controls without association with CD3, CD68, or HLA-DR suggested that PVB19 could be a bystander in many DCM cases. J. Med. Virol. 89:55-63, 2017. © 2016 Wiley Periodicals, Inc.

Potential specific immunological indicators for stroke associated infection are partly modulated by sympathetic pathway activation.

BACKGROUND: Evidence has led to the consideration of immunodepression after stroke as an important contributor to stroke associated infection (SAI). However, so far no specific immunological indicator has been identified for SAI, and the underlying mechanism remains poorly understood. RESULTS: SAI patients had significantly higher IL-6 and IL-10 levels and lower HLA-DR levels than no-infection patients within 48h after stroke onset. NA significantly increased IL-10 levels, reduced HLA-DR expression, and decreased IL-6 expression by increasing ß-arrestin2 expression which reduced the activation of the NF-κB pathway. Propranolol reversed this effect of NA by reducing ß-arrestin2 expression. MATERIALS AND METHODS: A systematic search for eligible clinical studies was applied to pool the differences in peripheral cytokine levels between infection and no-infection stroke patients. The underlying mechanism behind these differences was investigated in vitro by applying norepinephrine (NA) and lipopolysaccharide (LPS) to simulate sympathetic pathway activation and sepsis respectively in THP-1 cells. Propranolol was applied to determine the effect of reversing the activation of the sympathetic pathway. Immunological indicators were also detected to assess the immune activation of THP-1 cells and measurements of the expression of ß-arrestin2, NF-κB, IκBα and phosphor-IκBα were performed to assess the activation of the sympathetic pathway. CONCLUSION: IL-6, IL-10 and HLA-DR are good candidate biomarkers for SAI. The activation of the sympathetic pathway could partly account for the specific immunological alterations found in SAI patients including HLA-DR decrease and IL-10 increase, which both could be reversed by propranolol. However, the mechanism underlying IL-6 increase still needs further exploration.