Multimodal Approach in Dry Eye Disease Combining In Vivo Confocal Microscopy and HLA-DR Expression.

Purpose: The purpose of this study was to determine the association between corneal images provided by in vivo confocal microscopy (IVCM) with clinical parameters and conjunctival expression of HLA-DR antigen in patients with dry eye disease (DED). Methods: Two hundred fourteen eyes of 214 patients with DED were analyzed, consisting of 2 groups of patients - 63 with autoimmune dry eye disease (AIDED) and 151 with non-autoimmune dry eye disease (NAIDED). Patients underwent a full clinical examination, including symptom screening, using the Ocular Surface Disease Index (OSDI) questionnaire, and objective analysis of DED signs by Schirmer's testing, tear break-up time (TBUT), Oxford's test, and IVCM corneal imaging. The IVCM scoring criteria were based on corneal sub-basal nerve density (ND), nerve morphology (NM), and inflammatory cell (IC) density. Quantification of conjunctival HLA-DR antigen was performed by flow cytometry. Results: The total IVCM score (T-IVCM) as well as the IVCM-IC subscore (sc) were positively correlated with HLA-DR levels with r = 0.3, P < 0.001 and r = 0.3, P < 0.01, respectively in the total population of patients with DED. The IVCM-NDsc was negatively correlated with TBUT in patients with AIDED (r = -0.2, P < 0.05) and with the Schirmer's test in patients with NAIDED (r = -0.24, P < 0.05). However, the IVCM-NMsc was positively correlated with the Oxford score only in patients with AIDED (r = 0.3, P < 0.05). Conclusions: The proposed IVCM scoring system showed significant correlations with clinical parameters along with conjunctival HLA-DR quantification in patients with DED. Translational Relevance: The IVCM grading score represents an interesting point of commonality among clinical parameters, imaging, and molecular investigation of the ocular surface.

Dual roles of CD11b+CD33+HLA-DR-/lowCD14- myeloid-derived suppressor cells with a granulocytic morphology following allogeneic hematopoietic stem cell transplantation: from inflammation promoters to immune suppressors within 90 days.

Introduction: Granulocytic myeloid-derived suppressor cells (G-MDSCs) show fast recovery following allogeneic hematopoietic stem cell transplantation (allo-HSCT) constituting the major part of peripheral blood in the early phase. Although G-MDSCs mediate immune suppression through multiple mechanisms, they may also promote inflammation under specific conditions. Methods: G-MDSCs were isolated from 82 patients following allo-HSCT within 90 days after allo-HSCT, and their interactions with autologous CD3+ T-cells were examined. T-cell proliferation was assessed by flow cytometry following CFSE staining, while differentiation and interferon-γ secretion were characterized using chemokine receptor profiling and ELISpot assays, respectively. NK cell cytotoxicity was evaluated through co-culture with K562 cells. An aGVHD xenogeneic model in humanized mice was employed to study the in vivo effects of human leukocytes. Furthermore, transcriptional alterations in G-MDSCs were analyzed via RNA sequencing to investigate functional transitions. Results: G-MDSCs promoted inflammation in the early-stage, by facilitating cytokine secretion and proliferation of T cells, as well as their differentiation into pro-inflammatory T helper subsets. At day 28, patients with a higher number of G-MDSCs exhibited an increased risk of developing grades II-IV aGvHD. Besides, adoptive transfer of G-MDSCs from patients at day 28 into humanized mice exacerbated aGvHD. However, at day 90, G-MDSCs led to immunosuppression, characterized by upregulated expression of indoleamine 2,3-dioxygenase gene and interleukin-10 secretion, coupled with the inhibition of T cell proliferation. Furthermore, transcriptional analysis of G-MDSCs at day 28 and day 90 revealed that 1445 genes were differentially expressed. These genes were associated with various pathways, revealing the molecular signatures of early post-transplant differentiation in G-MDSCs. In addition, genes linked to the endoplasmic reticulum stress were upregulated in patients without aGvHD. The acquisition of immunosuppressive function by G-MDSCs may depend on the activation of CXCL2 and DERL1 genes. Conclusion: Our findings revealed the alteration in the immune characteristics of G-MDSCs within the first 90 days post-allo-HSCT. Moreover, the quantity of G-MDSCs at day 28 may serve as a predictive indicator for the development of aGvHD.

Mediating role of chiro-inositol metabolites on the effects of HLA-DR-expressing CD14 + monocytes in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD), a chronic inflammatory condition, is caused by several factors involving aberrant immune responses. Genetic factors are crucial in IBD occurrence. Mendelian randomization (MR) can offer a new perspective in understanding IBD's genetic background. METHODS: Single nucleotide polymorphisms (SNPs) were considered instrumental variables (IVs). We analyzed the relationship between 731 immunophenotypes, 1,400 metabolite phenotypes, and IBD. The total effect was decomposed into indirect and direct effects, and the ratio of the indirect effect to the total effect was calculated. RESULTS: We identified the causal effects of HLA-DR-expressing CD14 + monocytes on IBD through MR analysis. The phenotype "HLA-DR expression on CD14 + monocytes" showed the strongest association among the selected 48 immune phenotypes. Chiro-inositol metabolites mediated the effect of CD14 + monocytes expressing HLA-DR on IBD. An increase in Chiro-inositol metabolites was associated with a reduced risk of IBD occurrence, accounting for 4.97%. CONCLUSION: Our findings revealed a new pathway by which HLA-DR-expressing CD14 + monocytes indirectly reduced the risk of IBD occurrence by increasing the levels of Chiro-inositol metabolites. The results provided a new perspective on the immunoregulatory mechanisms underlying IBD, laying a theoretical foundation for developing new therapeutic targets in the future.

Evaluation of Monocyte Subpopulations in Patients with Systemic Sclerosis and its Association with Clinical Manifestations of the Disease: a Cross-sectional Controlled Study.

Background: Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized not only by fibrosis and vasculopathy but also by inflammation. Previous studies have demonstrated monocyte involvement in SSc development, suggesting a role for immune dysfunction in SSc pathogenesis. Objective: To investigate the relationship between SSc's clinical manifestations and altered levels of monocyte subpopulations. Methods: Twenty-six patients meeting the ACR/EULAR SSc criteria along with twenty healthy individuals as the control group, were enrolled in the study. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood samples of both the SSc patients and the control group. Subpopulations of monocytes were assessed based on HLA-DR, CD14, and CD16 expression using multi-color flow cytometry. The one-way ANOVA, Student's t-test, and Mann-Whitney U test were employed for normally and non-normally distributed data. The Spearman correlation test was utilized to identify correlations between the variables. Results: The SSc patients showed a significant increase in the number of circulating peripheral blood monocytes (p<0.001). The percentage of CD16+ monocyte subpopulations was higher in the SSc cases compared to the control group. A significant decrease in the ratio of classic to non-classic monocytes was observed in SSc cases (7.43%) compared to the control group (52.09%, p<0.001). No association was observed between monocyte subpopulations and clinical characteristics of SSC. Conclusion: Our results showed an increase in the level of CD16+ monocytes in patients with SSc compared to healthy individuals. Further investigation is required to determine the clinical significance of this alteration.

HLA-DR Expression in Natural Killer Cells Marks Distinct Functional States, Depending on Cell Differentiation Stage.

HLA-DR-positive NK cells, found in both healthy individuals and patients with different inflammatory diseases, are characterized as activated cells. However, data on their capacity for IFNγ production or cytotoxic response vary between studies. Thus, more precise investigation is needed of the mechanisms related to the induction of HLA-DR expression in NK cells, their associations with NK cell differentiation stage, and functional or metabolic state. In this work, HLA-DR-expressing NK cell subsets were investigated using transcriptomic analysis, metabolic activity assays, and analysis of intercellular signaling cascades. We demonstrated that HLA-DR+CD56bright NK cells were characterized by a proliferative phenotype, while HLA-DR+CD56dim NK cells exhibited features of adaptive cells and loss of inhibitory receptors with increased expression of MHC class II trans-activator CIITA. The activated state of HLA-DR-expressing NK cells was confirmed by higher levels of ATP and mitochondrial mass observed in this subset compared to HLA-DR- cells, both ex vivo and after stimulation in culture. We showed that HLA-DR expression in NK cells in vitro can be induced both through stimulation by exogenous IL-2 and IL-21, as well as through auto-stimulation by NK-cell-produced IFNγ. At the intracellular level, HLA-DR expression depended on the activation of STAT3- and ERK1/2-mediated pathways, with subsequent activation of isoform 3 of the transcription factor CIITA. The obtained results broaden the knowledge about HLA-DR-positive NK cell appearance, diversity, and functions, which might be useful in terms of understanding the role of this subset in innate immunity and assessing their possible implications in NK cell-based therapy.

Expression of CD39 is associated with T cell exhaustion in ovarian cancer and its blockade reverts T cell dysfunction.

Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

Research and Analysis of Molecules such as CD28, CD45RA, CD45RO, CD38, HLA-DR, and CD57 on T Cells in Multiple Myeloma.

BACKGROUND: For many years it has been postulated that the immune system controls the progress of multiple myeloma (MM). However, the phenotypes of T cells in MM remain to be elucidated. In this study, we compared the phenotypes of T cells, which were obtained from the peripheral blood, in MM patients with those in healthy donors (HD). The expression of CCR7, CD57, CD28, HLA-DR, CD38, CD45RA, and CD45RO were assessed on T cells from MM patients and HDs using multicolor flow cytometry (MFC). METHODS: For this study, 17 newly diagnosed MM patients were selected, and 20 healthy people were selected as a control group. MFC was used to detect the markers on T cells. RESULTS: We detected significant increases in the expression levels of HLA-DR, CD38, and CD57on CD8+ T cells, significant decreases in the expression levels of CD28 and CD45RA on CD8+ T cells, and a decrease of CD4+ effec-tor T cells in MM patients, compared to the HD group. CONCLUSIONS: Our study shows that the accumulation of peripheral CD8+CD57+T cells, CD8+CD38high T cells, and CD8+HLA-DR+CD38high T cells is reflective of an ongoing antitumor T cell response and a progressive immune dysfunction in MM. During chemotherapy, the recovery of immune function can be monitored by detecting the proportion of activated molecules of T lymphocytes.

Augmentation of tumor expression of HLA-DR, CXCL9, and CXCL10 may improve olfactory neuroblastoma immunotherapeutic responses.

BACKGROUND: Olfactory neuroblastoma is a rare malignancy of the anterior skull base typically treated with surgery and adjuvant radiation. Although outcomes are fair for low-grade disease, patients with high-grade, recurrent, or metastatic disease oftentimes respond poorly to standard treatment methods. We hypothesized that an in-depth evaluation of the olfactory neuroblastoma tumor immune microenvironment would identify mechanisms of immune evasion in high-grade olfactory neuroblastoma as well as rational targetable mechanisms for future translational immunotherapeutic approaches. METHODS: Multispectral immunofluorescence and RNAScope evaluation of the tumor immune microenvironment was performed on forty-seven clinically annotated olfactory neuroblastoma samples. A retrospective chart review was performed and clinical correlations assessed. RESULTS: A significant T cell infiltration was noted in olfactory neuroblastoma samples with a stromal predilection, presence of myeloid-derived suppressor cells, and sparse natural killer cells. A striking decrease was observed in MHC-I expression in high-grade olfactory neuroblastoma compared to low-grade disease, representing a mechanism of immune evasion in high-grade disease. Mechanistically, the immune effector stromal predilection appears driven by low tumor cell MHC class II (HLA-DR), CXCL9, and CXCL10 expression as those tumors with increased tumor cell expression of each of these mediators correlated with significant increases in T cell infiltration. CONCLUSION: These data suggest that immunotherapeutic strategies that augment tumor cell expression of MHC class II, CXCL9, and CXCL10 may improve parenchymal trafficking of immune effector cells in olfactory neuroblastoma and augment immunotherapeutic responses.

Regulatory effects of microRNAs on monocytic HLA-DR surface expression.

Decreased monocytic HLA-DR expression is the most studied biomarker of immune competency in critically ill and autoimmune disease patients. However, the underlying regulatory mechanisms remain largely unknown. One probable HLA-DR dysregulation is through microRNAs. The aim of this study was to investigate the effects of specific microRNAs on HLA-DR expression in human monocytic cells. Four up- and four down-HLA-DR-regulating microRNAs were identified, with hsa-miR-let-7f-2-3p showing the most significant upregulation and hsa-miR-567 and hsa-miR-3972 downregulation. Anti-inflammatory glucocorticoid medication Dexamethasone-decreased HLA-DR was significantly restored by hsa-miR-let-7f-2-3p and hsa-miR-5693. Contrarily, proinflammatory cytokines IFN-γ and TNF-α-increased HLA-DR were significantly reversed by hsa-miR-567. Clinically, paired plasma samples from patients before and one day after cardiac surgery revealed up-regulated expression of hsa-miR-5693, hsa-miR-567, and hsa-miR-3972, following the major surgical trauma. In silico approaches were applied for functional microRNA-mRNA interaction prediction and candidate target genes were confirmed by qPCR analysis. In conclusion, novel monocytic HLA-DR microRNA modulators were identified and validated in vitro. Moreover, both the interaction between the microRNAs and anti- and proinflammatory molecules and the up-regulated microRNAs identified in cardiac surgery highlight the potential clinical relevance of our findings.

Structural insights into human MHC-II association with invariant chain.

The loading of processed peptides on to major histocompatibility complex II (MHC-II) molecules for recognition by T cells is vital to cell-mediated adaptive immunity. As part of this process, MHC-II associates with the invariant chain (Ii) during biosynthesis in the endoplasmic reticulum to prevent premature peptide loading and to serve as a scaffold for subsequent proteolytic processing into MHC-II-CLIP. Cryo-electron microscopy structures of full-length Human Leukocyte Antigen-DR (HLA-DR) and HLA-DQ complexes associated with Ii, resolved at 3.0 to 3.1 Å, elucidate the trimeric assembly of the HLA/Ii complex and define atomic-level interactions between HLA, Ii transmembrane domains, loop domains, and class II-associated invariant chain peptides (CLIP). Together with previous structures of MHC-II peptide loading intermediates DO and DM, our findings complete the structural path governing class II antigen presentation.

Expression of HLA-DR and KLRG1 enhances the cytotoxic potential and cytokine secretion capacity of CD3+ T cells in tuberculosis patients.

BACKGROUND: Human T cells play an important role in immunity against tuberculosis (TB) infection. Activating receptor HLA-DR and inhibitory receptor KLRG1 are critical regulators of T cell function during viral infection and tumorigenesis, but they have been less studied in TB infection. METHODS: In this study, we explored the relationship between CD3+ T cell expression of HLA-DR and KLRG1 receptors and function against TB infection. Flow cytometry was conducted to assess the immunomodulatory effects of HLA-DR and KLRG1 receptors on CD3+ T cells in patients with different TB infection status. RESULTS: We found activating receptors HLA-DR, NKG2C, CD57 and NKP46, and inhibitory receptors KLRG1 and KIR on CD3+ T cells in different TB infection status showed different distribution patterns; the cytotoxic potential and cytokine secretion capacity of CD3+ T cells after Mtb-specific antigen stimulation were significantly enhanced in TB infection groups. Further studies revealed HLA-DR+ T and KLRG1+ T cells expressed higher activating and inhibitory receptors than the negative population. In addition, the expression of cytotoxic potential and cytokine secretion capacity of HLA-DR+ T and KLRG1+ T cells was significantly higher than that of HLA-DR- T and KLRG1- T cells. CONCLUSIONS: Expression of HLA-DR and KLRG1 enhances the cytotoxic potential and cytokine secretion capacity of CD3+ T cells in TB patients, suggesting CD3+ T cells expressing HLA-DR and KLRG1 are important effector cell phenotypes involved in the host anti-TB infection. HLA-DR and KLRG1 expressed by CD3+ T cells may be potential predictive markers of TB disease progression and clinical immune assessment.

Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Artery Disease.

Monocytes are associated with human cardiovascular disease progression. Monocytes are segregated into three major subsets: classical (cMo), intermediate (iMo), and nonclassical (nMo). Recent studies have identified heterogeneity within each of these main monocyte classes, yet the extent to which these subsets contribute to heart disease progression is not known. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects within the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using the Gensini Score (GS). We employed high-dimensional single-cell transcriptome and protein methods to define how human monocytes differ in subjects with low to severe coronary artery disease. We analyzed 487 immune-related genes and 49 surface proteins at the single-cell level using Antibody-Seq (Ab-Seq). We identified six subsets of myeloid cells (cMo, iMo, nMo, plasmacytoid DC, classical DC, and DC3) at the single-cell level based on surface proteins, and we associated these subsets with coronary artery disease (CAD) incidence based on Gensini score (GS) in each subject. Only frequencies of iMo were associated with high CAD (GS > 32), adj.p = 0.024. Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r = 0.314, p = 0.014) and further showed a robust sex-dependent positive correlation in female subjects (r = 0.663, p = 0.004). cMo frequencies did not correlate with CAD severity. Key gene pathways differed in iMo among low and high CAD subjects and between males and females. Further single-cell analysis of iMo revealed three iMo subsets in human PBMC, distinguished by the expression of HLA-DR, CXCR3, and CD206. We found that the frequency of immunoregulatory iMo_HLA-DR+CXCR3+CD206+ was associated with CAD severity (adj.p = 0.006). The immunoregulatory iMo subset positively correlated with GS in both females (r = 0.660, p = 0.004) and males (r = 0.315, p = 0.037). Cell interaction analyses identified strong interactions of iMo with CD4+ effector/memory T cells and Tregs from the same subjects. This study shows the importance of iMo in CAD progression and suggests that iMo may have important functional roles in modulating CAD risk, particularly among females.

CD14-CD10-CD45+HLA-DR-SSC+ neutrophils may be granulocytic myeloid-derived suppressor cell-like cells and relate to disease progression in non-Hodgkin's lymphoma patients.

We explored the frequency of CD14-CD10-CD45+HLA-DR-SSC++ neutrophils (CD10- neutrophils) in patients with non-Hodgkin's lymphoma (NHL), and their immunologic characteristics and clinical significance. Patients with NHL who were newly diagnosed (NDP; n = 33), in remission (RMP; n = 28) and relapsed (RLP; n = 29) were included, and 47 volunteers were recruited as healthy controls (HCs). The frequency of CD10- neutrophils in the peripheral blood from HC and patients with NHL was detected. CD10- and CD10+ neutrophils were sorted, and their cytology was analyzed. CD3+ T cells were also isolated and cultured with the autologous CD10- or CD10+ neutrophils, after which the proliferation and death rates of T cells were determined. The levels of arginase-1 (Arg-1) and reactive oxygen species (ROS) in CD10+ or CD10- neutrophils were examined. Few CD10- neutrophils were detected in HCs but were significantly elevated in patients with NHL, especially in NDP and RLP. In addition, CD10- neutrophils in NDP with advanced stage and high risk were markedly higher than those in NDP with limited stage and low risk. In RMP and RLP, the relapse-free survival and overall survival in patients with high CD10- neutrophils were shorter than those with low CD10- neutrophils. CD10- neutrophils from patients with NHL, which mainly consist of immature neutrophils, inhibit T-cell proliferation and facilitate T-cell death. Furthermore, a significant increase was observed in Arg-1 expression, along with an increase to a certain extent in ROS. CD10- neutrophils in patients with NHL have characteristics of myeloid-derived suppressor cells and may be related to disease progression and poor prognosis.

Impaired monocyte-derived dendritic cell phenotype in prostate cancer patients: A phenotypic comparison with healthy donors.

BACKGROUND: Dendritic cells (DCs) play a crucial role in immunity. Research on monocyte-derived DCs (Mo-DCs) cancer vaccines is in progress despite limited success in clinical trials. This study focuses on Mo-DCs generated from prostate cancer (PCA) patients, comparing them with DCs from healthy donors (HD-DCs). METHODS: Mo-DCs were isolated from PCA patient samples, and their phenotype was compared to HD-DCs. Key parameters included monocyte count, CD14 expression, and the levels of maturation markers (HLA-DR, CD80, CD86) were assessed. RESULTS: PCA samples exhibited a significantly lower monocyte count and reduced CD14 expression compared to healthy samples (p ⟨ 0.0001). Additionally, PCA-DCs expressed significantly lower levels of maturation markers, including HLA-DR, CD80, and CD86, when compared to HD-DCs (p = 0.123, p = 0.884, and p = 0.309, respectively). CONCLUSION: The limited success of DC vaccines could be attributed to impaired phenotypic characteristics. These observations suggest that suboptimal characteristics of Mo-DCs generated from cancer patient blood samples might contribute to the limited success of DC vaccines. Consequently, this study underscores the need for alternative strategies to enhance the features of Mo-DCs for more effective cancer immunotherapies.

Alterations in epidermal stem cells within the pilosebaceous unit in atrophic acne scars.

BACKGROUND: Atrophic acne scarring is a common sequela of inflammatory acne, causing significant problems for affected patients. Although prolonged inflammation and subsequent aberrant tissue regeneration are considered the underlying pathogenesis, the role of epidermal stem cells, which are crucial to the regeneration of pilosebaceous units, remains unknown. OBJECTIVES: To examine the changes occurring in epidermal stem cells in atrophic acne scars. METHODS: Changes in collagen, elastic fibre and human leukocyte antigen (HLA)-DR expression were analysed in normal skin and inflammatory acne lesions at days 1, 3 and 7 after development. The expression of epidermal stem cell markers and proliferation markers was compared between normal skin and mature atrophic acne scar tissue. RESULTS: In acne lesions, inflammation had invaded into pilosebaceous units over time. Their normal structure had been destructed and replaced with a reduced amount of collagen and elastic fibre. Expression of stem cell markers including CD34, p63, leucine-rich repeat-containing G protein-coupled receptor (LGR)6 and LGR5, which are expressed in the interfollicular epidermis, isthmus and bulge of hair follicles, significantly decreased in atrophic acne scar tissue compared to normal skin. Epidermal proliferation was significantly reduced in scar tissue. CONCLUSIONS: These findings suggest that as inflammatory acne lesions progress, inflammation gradually infiltrates the pilosebaceous unit and affects the resident stem cells. This disruption impedes the normal regeneration of the interfollicular epidermis and adnexal structures, resulting in atrophic acne scars.

Could we use PD-1 and PD-L1 expression on lymphocytes and monocytes as predictive markers for prognosis of acute biliary pancreatitis?

PURPOSE: This study aimed to assess the significance of immunophenotyping and serum cytokines in predicting the clinical progression of acute biliary pancreatitis (ABP). MATERIALS AND METHODS: Cytokine levels, T-helper, cytotoxic T, natural killer (NK) cells, monocytes, HLA-DR, and PD-1, as well as PDL-1 immune checkpoints, were measured in ABP patients at the time of diagnosis and compared with results from healthy volunteers. The study also compared leukocyte counts, hematocrit, immunophenotyping results, cytokine statuses, and PD-1, PDL-1 expression between healthy volunteers and ABP subgroups categorized by pancreatitis severity. RESULTS: The study included 65 ABP patients and 20 healthy volunteers. Significant differences were observed between groups in hematocrit, leukocyte counts, total monocytes, lymphocytes, CD3+ total T cells, CD4+ Th cells, PD-1 expression on CD4+ and CD8+T lymphocytes, HLA-DR expression on CD14+ monocytes, NK cells, PD-L1 expression on CD14+ monocytes, classical and intermediate monocytes, as well as levels of IL-6, IL-8, IL-10, IL-18, and IL-33 cytokines. Moderate correlations were found with lymphocyte counts, PD-1+CD4+ cells, PD-L1+CD14+ cells, and strong correlations with HLA-DR+CD14+ cells. Hematocrit, CD3+ total T cells, NK cells, CD4+PD-1 + T cells, and CD8+PD-1 + T cells showed independent associations with the severity of ABP. Lymphocyte counts, CD14+HLA-DR+ cells, CD14+PD-L1+ cells, CD4+PD-1 + T cells, classical, and intermediate monocytes exhibited the highest Area Under the Curve rates in determining organ failure. CONCLUSIONS: Hematocrit, lymphocyte counts, CD14+HLA-DR+ cells, CD14+PD-L1+ cells, and intermediate monocytes emerged as parameters most closely associated with the severity and these parameters could be useful in predicting the severity of ABP.

Association between Plasma HLA-DR+ Placental Vesicles and Preeclampsia: A Pilot Longitudinal Cohort Study.

(1) Background: Preeclampsia (PE) usually presents with hypertension and proteinuria, related to poor placentation. Reduced maternal-fetal immunological tolerance is a possible trigger of inadequate placentation. Aberrant antigen expression of HLA-DR has been observed in the syncytiotrophoblast of PE patients. In this study, we analyzed plasma levels of Human Leukocyte Antigen (HLA)-DR+ syncytiotrophoblast-derived extracellular vesicles (STEVs) during the three trimesters of pregnancy in relation to PE onset. (2) Methods: Pregnant women underwent venous blood sampling during the three trimesters. STEVs were collected from plasma via ultracentrifugation (120,000 g) and characterized by Western blot, nanotracking analysis and flow cytometry for the expression of Placental Alkaline Phosphatase (PLAP), a placental-derived marker, and HLA-DR. (3) Results: Out of 107 women recruited, 10 developed PE. STEVs were detected in all three trimesters of pregnancy with a zenith in the second trimester. A significant difference was found between the non-PE and PE groups in terms of plasma levels of HLA-DR+ STEVs during all three trimesters of pregnancy. (4) Conclusions: More research is needed to investigate HLA-DR+ as a potential early marker of PE.

Immunohistochemical characterization of the immune cell response during chlamydial infection in the male and female koala (Phascolarctos cinereus) reproductive tract.

Chlamydiosis is one of the main causes of the progressive decline of koala populations in eastern Australia. While histologic, immunologic, and molecular studies have provided insights into the basic function of the koala immune system, the in situ immune cell signatures during chlamydial infection of the reproductive tract in koalas have not been investigated. Thirty-two female koalas and 47 males presented to wildlife hospitals with clinical signs suggestive of Chlamydia infection were euthanized with the entire reproductive tract collected for histology; immunohistochemistry (IHC) for T-cell (CD3ε, CD4, and CD8α), B-cell (CD79b), and human leukocyte antigen (HLA)-DR markers; and quantitative real-time polymerase chain reaction (rtPCR) for Chlamydia pecorum. T-cells, B-cells, and HLA-DR-positive cells were observed in both the lower and upper reproductive tracts of male and female koalas with a statistically significant associations between the degree of the inflammatory reaction; the number of CD3, CD4, CD79b, and HLA-DR positive cells; and the PCR load. CD4-positive cells were negatively associated with the severity of the gross lesions. The distribution of immune cells was also variable according to the location within the genital tract in both male and female koalas. These preliminary results represent a step forward towards further exploring mechanisms behind chlamydial infection immunopathogenesis, thus providing valuable information about the immune response and infectious diseases in free-ranging koalas.

A reactive monocyte subset characterized by low expression of CD91 is expanded during sterile and septic inflammation.

OBJECTIVES: This study was undertaken to assess CD91 expression on monocytes and changes in monocyte subset distribution during acute tissue damage and bloodstream infection (BSI). METHODS: We investigated blood specimens from healthy individuals, trauma and cardiac surgery patients as a model of tissue damage, and patients with BSI, by flow cytometry using a panel of antibodies comprising CD45, HLA-DR, CD14, CD16 and CD91 for the identification of monocyte subsets. RESULTS: While infrequent in healthy subjects, CD91low/neg monocyte levels were markedly high in BSI, trauma and after cardiac surgery. This monocyte subset expanded up to 15-fold in both patient cohorts, whereas CD14+CD16+ inflammatory monocytes were multiplied by a factor of 5 only. CD14+CD91low monocytes displayed a significantly lower density of HLA-DR and markedly reduced expression of CD300e, compared to the other subsets. They also expressed high levels of myeloperoxidase and showed robust phagocytic and oxidative burst activity. CONCLUSIONS: Expansion of CD91low monocytes is a sensitive marker of acute inflammatory states of infectious and non-infectious etiology.

New light on the HLA-DR immunopeptidomic landscape.

The set of peptides processed and presented by major histocompatibility complex class II molecules defines the immunopeptidome, and its characterization holds keys to understanding essential properties of the immune system. High-throughput mass spectrometry (MS) techniques enable interrogation of the diversity and complexity of the immunopeptidome at an unprecedented scale. Here, we analyzed a large set of MS immunopeptidomics data from 40 donors, 221 samples, covering 30 unique HLA-DR molecules. We identified likely co-immunoprecipitated HLA-DR irrelevant contaminants using state-of-the-art prediction methods and unveiled novel light on the properties of HLA antigen processing and presentation. The ligandome (HLA binders) was enriched in 15-mer peptides, and the contaminome (nonbinders) in longer peptides. Classification of singletons and nested sets showed that the first were enriched in contaminants. Investigating the source protein location of ligands revealed that only contaminants shared a positional bias. Regarding subcellular localization, nested peptides were found to be predominantly of endolysosomal origin, whereas singletons shared an equal distribution between the cytosolic and endolysosomal origin. According to antigen-processing signatures, no significant differences were observed between the cytosolic and endolysosomal ligands. Further, the sensitivity of MS immunopeptidomics was investigated by analyzing overlap and saturation between biological MS replicas, concluding that at least 5 replicas are needed to identify 80% of the immunopeptidome. Moreover, the overlap in immunopeptidome between donors was found to be very low both in terms of peptides and source proteins, the latter indicating a critical HLA bias in the antigen sampling in the HLA antigen presentation. Finally, the complementarity between MS and in silico approaches for comprehensively sampling the immunopeptidome was demonstrated.