HLA-DR Expression Research Articles

Immunomodulatory and Anti-Inflammatory Effects of Vitamin A and Tryptophan on Monocyte-Derived Dendritic Cells Stimulated with Gliadin in Celiac Disease Patients.

Celiac Disease (CeD) is an autoimmune disorder with various symptoms upon gluten exposure. Dendritic cells (DCs) play a crucial role in gliadin-induced inflammation. Vitamin A (retinol; Ret) and its metabolite, retinoic acid (RA), along with tryptophan (Trp) and its metabolite, kynurenic acid (KYNA), are known to influence the immune function of DCs and enhance their tolerogenicity. This research aims to assess the impact of gliadin on DC maturation and the potential of vitamin A and tryptophan to induce immune tolerance in DCs. The monocyte cells obtained from peripheral blood mononuclear cells (PBMCs) of celiac disease patients were differentiated into DCs in the absence or presence of Ret, RA, Trp, KYNA, and then stimulated with peptic and tryptic (PT) digested of gliadin. We used flow cytometry to analyze CD11c, CD14, HLA-DR, CD83, CD86, and CD103 expression. ELISA was carried out to measure TGF-β, IL-10, IL-12, and TNF-α levels. qRT-PCR was used to assess the mRNA expression of retinaldehyde dehydrogenase 2 (RALDH2) and integrin αE (CD103). The mRNA and protein levels of Indoleamine 2, 3-dioxygenase (IDO) was analyzed by qRT-PCR and Western blot assays, respectively. Our findings demonstrate that PT-gliadin enhances the expression of maturation markers, i.e. CD83, CD86 and HLA-DR and promote the secretion of TNF-α and IL-12 in DCs. Interestingly, vitamin A, tryptophan, and their metabolites increase the expression of CD103, while limiting the expression of HLA-DR, CD83, and CD86. They also enhance RALDH2 and IDO expression and promote the secretion of TGF-β and IL-10, while limiting IL-12 and TNF-α secretion. These findings suggest that vitamin A and tryptophan have beneficial effects on PT-gliadin-stimulated DCs, highlighting their potential as therapeutic agents for celiac disease. However, further research is needed to fully understand their underlying mechanisms of action in these cells.

Dupilumab‐associated ocular surface disease or atopic keratoconjunctivitis not improved by dupilumab? Upadacitinib may clarify the dilemma: A case report

AbstractDupilumab‐associated ocular surface disease is a common clinical sign appearing in patients with atopic dermatitis (AD) just few months after dupilumab treatment start, developing in about 25% of patients. Atopic keratoconjunctivitis (AKC) is a well‐identified clinical entity, defined as a chronic inflammatory disease of eye that affects 25%–40% of patients with AD. Most clinical signs of ocular involvement in AD patients treated with dupilumab overlaps the AKC symptoms and signs. We supposed that Dupilumab‐associated ocular surface disease and AKC represent the same disease but differently called by dermatologists and ophthalmologists. AKC‐like disease may develop during dupilumab therapy as a consequence of alternative cytokines pathway activation (e.g. IL33) secondary to IL‐4/13 pathway block. The novel upadacitinib drug may bypass ILs pathway through Janus Kinases selective inhibition, avoiding positive or negative ILs feedback at the ocular surface level. In this case report, molecular analysis on conjunctival samples showed a lower ocular surface inflammation (lower expression of HLADR) although higher levels of IL4 and IL13 in a patient with AD and AKC during upadacitinib therapy, compared to prior dupilumab treatment. Target therapies in patients suffering from AD may prevent ocular and dermatological comorbidities improving quality of life before quality of skin and vision.

Minor population of NK lymphocytes with CD19 coexpression

Single reports were published concerning a minor subpopulation of NK cells with weak coexpression of the B cell antigen CD19 in the patients’ blood and bone marrow. The frequency and relative number of CD56+CD19+dim cells is virtually not assessed, and there is no data on their phenotypic characteristics, as well as the connection of this subpopulation with any disease state. The purpose of the present study was to assess the frequency, relative quantity and phenotypic characteristics of CD56+CD19+dim lymphocytes in blood of patients referred for assessment of the lymphocyte subpopulation profile. Peripheral blood of immunocompromised individuals was taken, and subpopulation composition of lymphocytes was determined using eight-color flow cytometry (markers: CD3, CD4, CD8, CD19, CD25, CD45, CD56, HLA-DR). To estimate incidence of the CD56+CD19+dim subpopulation, we have carried out a retrospective analysis of LMD files on 1210 studies for 935 patients (average age, 39.8±14.7 years old) including 84 children under 18 years old. The study was performed repeatedly for some patients. Phenotyping of CD56+CD19+dim cells was performed using a panel of antibodies to B cell, T/NK cell antigens. The occurrence of blood samples containing CD56+CD19+dim was 1.2%, with a relative content of 2.1±1.9% among total lymphocyte population (0.8±0.6% of leukocytes). Long-term persistence of the subpopulation was noted in the patients throughout the entire observation period. The comparison of specific marker expression by NK CD56+CD19+dim, and CD56+CD19- cells revealed high expression of CD2, CD57, reduced expression density of CD7, CD16, CD38. The phenotype of the studied NK cell subpopulation was as follows: CD56+dimCD19+dimCD2+brightCD7+dimCD11c+CD16+dimCD38+dimCD45RA+CD57+CD94+dimNKG2D+CD3-CD4-CD5-CD20-CD21-CD25-CD45R0-CD62L-CD79b-CD117-, with variable expression of CD8 and HLA-DR. The phenotype is consistent with activated terminally differentiated adaptive NK associated with cytomegalovirus infection. The individuals with CD56+CD19+dim had a history of CMV-infection and reactivation of chronic EBV-infection at the time of the study. A probable cause of CD19 coexpression may be trogocytosis of B cell membrane fragments by natural killer cells during active EBV-infection. CD56+CD19+dim lymphocytes can reach noticeable values thus altering the results of studies performed by flow cytometry. The errors are most likely to occur upon assessing the minimal residual disease levels in acute B cell leukemias. The minor CD56+CD19+dimNK subpopulation may be detected in routine immunological analysis. Its functional features and association with certain disorders require further studies.

Evidence for immune activation in pathogenesis of the HLA class II associated disease, podoconiosis

Available evidences suggest that podoconiosis is triggered by long term exposure of bare feet to volcanic red clay soil particles. Previous genome-wide studies in Ethiopia showed association between the HLA class II region and disease susceptibility. However, functional relationships between the soil trigger, immunogenetic risk factors and the immunological basis of the disease are uncharted. Therefore, we aimed to characterise the immune profile and gene expression of podoconiosis patients relative to endemic healthy controls. Peripheral blood immunophenotyping of T cells indicated podoconiosis patients had significantly higher CD4 and CD8 T cell surface HLA-DR expression compared to healthy controls while CD62L expression was significantly lower. The levels of the activation markers CD40 and CD86 were significantly higher on monocytes and dendritic cell subsets in patients compared to the controls. RNA sequencing gene expression data indicated higher transcript levels for activation, scavenger receptors, and apoptosis markers while levels were lower for histones, T cell receptors, variable, and constant immunoglobulin chain in podoconiosis patients compared to healthy controls. Our finding provides evidence that podoconiosis is associated with high levels of immune activation and inflammation with over-expression of genes within the pro-inflammatory axis. This offers further support to a working hypothesis of podoconiosis as soil particle-driven, HLA-associated disease of immunopathogenic aetiology.

Effects of ageing and frailty on circulating monocyte and dendritic cell subsets

Ageing is associated with dysregulated immune responses, resulting in impaired resilience against infections and low-grade inflammation known as inflammageing. Frailty is a measurable condition in older adults characterized by decreased health and physical impairment. Dendritic cells (DCs) and monocytes play a crucial role in initiating and steering immune responses. To assess whether their frequencies and phenotypes in the blood are affected by ageing or frailty, we performed a flow cytometry study on monocyte and DC subsets in an immune ageing cohort. We included (n = 15 in each group) healthy young controls (HYC, median age 29 years), healthy older controls (HOC, 73 years) and Frail older controls (76 years). Monocyte subsets (classical, intermediate, non-classical) were identified by CD14 and CD16 expression, and DC subsets (conventional (c)DC1, cDC2, plasmacytoid (p)DC) by CD11c, CD1c, CD141 and CD303 expression. All subsets were checked for TLR2, TLR4, HLA-DR, CD86, PDL1, CCR7 and CD40 expression. We observed a lower proportion of pDCs in HOC compared to HYC. Additionally, we found higher expression of activation markers on classical and intermediate monocytes and on cDC2 in HOC compared to HYC. Frail participants had a higher expression of CD40 on classical and non-classical monocytes compared to the HOC group. We document a substantial effect of ageing on monocytes and DCs. Reduced pDCs in older people may underlie their impaired ability to counter viral infections, whereas enhanced expression of activation markers could indicate a state of inflammageing. Future studies could elucidate the functional consequences of CD40 upregulation with frailty.

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.

Novel Presentation of Major Histocompatibility Complex Class II Deficiency with Hemophagocytic Lymphohistiocytosis

PurposeMajor histocompatibility complex (MHC) class II deficiency is one of the combined immune deficiency disorders caused by defects in the MHC class II regulatory genes leading to abnormal T cells development and function. Therefore, patients mainly present with increased susceptibility to infections, diarrhea, and failure to thrive. In this report, we present one MHC class II deficient patient with a novel presentation with Hemophagocytic Lymphohistiocytosis (HLH).MethodsImmunophenotyping of lymphocyte subpopulations and HLA-DR expression was assess by flow cytometry. Gene mutational analysis was performed by whole exome and Sanger sequencing.ResultsWe reported a 7-year-old girl, who was diagnosed at age of 2 years with MHC class II deficiency by genetic testing and flow cytometry. Two years later, she developed disseminated BCGitis which was treated with proper antimicrobial agents. At the age of 7 years, she presented with clinical features fulfilling 6 diagnostic criteria of HLH including evidence of hemophagocytic activity in bone marrow aspiration. Accordingly, the diagnosis of HLH was established and the patient was started on IV Dexamethasone, Anakinra and IVIG. Eventually, patient started to improve and was discharged in good condition. Few months later, the patient was readmitted with severe pneumonia and sepsis leading to death.ConclusionPatients with MHC class II deficiency might present with disseminated BCGitis especially if the patient has severe T cell lymphopenia. Additionally, this immune defect might be added to the list of inborn errors of immunity that can be complicated with HLH.

Effect of cryopreservation on CD4+ T cell subsets in foreskin tissue.

Voluntary medical male circumcision (VMMC) reduces HIV acquisition by at least 60%, but the determinants of HIV susceptibility in foreskin tissues are incompletely understood. Flow cytometry is a powerful tool that helps us understand tissue immune defenses in mucosal tissue like the inner foreskin, but foreskin flow cytometry has only been validated using fresh tissue samples. This restricts immune analyses to timepoints immediately after surgical acquisition and hinders research in this area. We compared fresh analysis with whole tissue cryopreservation and later thawing and digestion to analyze CD4+ T cell populations relevant to HIV susceptibility (CCR5, CD25, CD127, CCR4, CXCR3, CCR6, CCR10, HLA-DR, and CD38). Eight foreskin samples from HIV-negative males aged >18 years were collected after VMMC. For each sample, half the foreskin was immediately cryopreserved for later digestion and flow cytometry analysis, while the remaining tissues were analyzed fresh. We demonstrate no significant impact of cryopreservation on CD4+ T cell expression of CD25, CCR4, CCR6, HLA-DR, CCR10, or CD127. Although expression levels of CCR5, CD38, and CXCR3 were increased after cryopreservation, the relative ranking of participants was retained. In conclusion, cryopreserved foreskin tissues may be suitable for subsequent digestion and flow cytometry phenotyping of HIV-susceptible T cell populations.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Association of immune cell traits with Parkinson's disease: a Mendelian randomization study.

Immunity and neuroinflammation play crucial roles in the pathogenesis of Parkinson's disease (PD). Nonetheless, prior investigations into the correlation between immune inflammation and PD have produced varying results. Identifying specific immune cell phenotypes that are truly associated with PD is challenging, and the causal relationship between immune cells and PD remains elusive. This study conducted a comprehensive two-sample Mendelian randomization (MR) analysis, employing five distinct analytical approaches, to clarify the causal connection between immune cell characteristics and the risk of PD. Utilizing GWAS data, we investigated the causal relationship between 731 immune cell traits and PD. These immune cell phenotypes encompass absolute cell (AC) counts, median fluorescence intensity (MFI), and relative cell (RC) counts for B cells, cDCs, mature stage T cells, monocytes, myeloid cells, TBNK (T cells, B cells, and natural killer cells), and Tregs, as well as the logistic parameter (MP) for cDCs and TBNK. The inverse variance weighted (IVW) analysis indicated that Myeloid DCs (p = 0.004), HVEM expression on CD45RA- CD4+ T cells (p = 0.007), CD62L- CD86+ Myeloid DCs (p = 0.015), and HLA DR expression on monocytes (p = 0.019) were associated with a reduced risk of PD. CD14+ CD16+ monocytes (p = 0.005), HLA DR+ NK cells within CD3- lymphocytes (p = 0.023), and CD28 expression on activated & secreting Tregs (p = 0.032) were associated with an increased risk of PD. This study establishes a causal link between immune cell phenotype and the pathogenesis of PD, identifying several specific immune cell characteristics associated with PD. This could inspire researchers to delve into the pathogenesis of PD at the cellular subtype level, and aid in the identification of potential pharmacological protein targets for PD.

Increased percentage of HLA-DR T cells in untreated juvenile dermatomyositis

This study investigates HLA-DR expression on activated T cells and serum neopterin levels in Juvenile Dermatomyositis (JDM) children pre- and post-treatment. Sixty-nine JDM children (less than 18 years) were included. Elevated HLA-DR+ T cells (>7 %) were observed in 19 % of untreated cases. Post-treatment, mean HLA-DR+ T cells decreased from 5.1 to 2.9 (P < 0.001), and serum neopterin levels declined from 19.3 to 9.1 nmol/L (P < 0.0001). A positive correlation between serum neopterin and HLA-DR T cell percentage was observed (r = 0.39, P = 0.01). Intravenous steroid treatment exhibited a 47.4 % improvement in HLA-DR+ T cells and a 50.5 % reduction in serum neopterin levels, in contrast to 14.8 % and 34.1 % in the oral steroid group. In conclusion, treatment, particularly with IV steroids, significantly improved HLA-DR+ T cells percentage and neopterin levels. A correlation between HLA-DR+ T cells percentage and serum neopterin was noted in untreated JDM patients.

Along with PaO2/FiO2 ratio and lymphopenia, low HLA-DR monocytes are the only additional parameter that independently predicts the clinical course of undifferentiated SARS-CoV-2 patients in emergency departments.

Because one-third of patients deteriorate after their admission to the emergency department, assessing the prognosis of COVID-19 patients is of great importance. However, to date, only lymphopenia and the partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) ratio have been reported as partly predictive of COVID-19-related further deterioration, and their association has not been evaluated. We asked whether other key biomarkers of SARS-CoV-2 immunologic defects-increase in circulating immature granulocytes, loss of monocyte HLA-DR (mHLA-DR) expression, and monocyte differentiation blockade-could also predict further COVID-19 deterioration. A series of 284 consecutive COVID-19 patients, with the sole inclusion criterion of being an adult, were prospectively enrolled at emergency department admission (day 0) of 2 different hospitals: 1 for the exploratory cohort (180 patients) and 1 for the confirmatory cohort (104 patients). Deterioration was assessed over the next 7 days. Neither increased immature granulocyte levels nor monocyte differentiation blockade predicted patient worsening. Among more than 30 clinical, biological, and radiological parameters, the value of decreased P/F ratio and lymphopenia for prediction of further COVID-19 deterioration was strongly confirmed, and the loss of mHLA-DR was the only additional independent marker. Combined together in a simple OxyLymphoMono score, the 3 variables perfectly predicted patients who did not worsen and correctly predicted worsening in 59% of cases. By highlighting lymphocyte and monocyte defects as preceding COVID-19 deterioration, these results point on early immunosuppression in COVID-19 deterioration. Combining P/F ratio, lymphopenia, and loss of mHLA-DR together in a simple and robust score could offer a pragmatic method for COVID-19 patient stratification.

Flow cytometric analysis of lymphocyte subsets, monocytes, and HLA-DR expressions on these cells in patients with COVID-19

Abstract Objectives We aimed to investigate the lymphocyte subsets and monocytes by flow cytometry and the correlations between their HLA-DR expressions and inflammatory markers in patients with COVID-19. Methods The study included 49 patients with COVID-19 and 42 healthy controls. Blood samples were taken into EDTA tubes. WBC counts were analyzed by the Sysmex/XN-1000i device, and lymphocyte subsets and monocytes were analyzed by flow cytometry. The percentage of HLA-DR expression on cells and median fluorescence intensity (MFI) values were recorded to detect activation. Lymphocyte counts were calculated using the dual-platform method. Correlations between antigen expression and ferritin, CRP, and D-dimer levels were analyzed. Results The patient group had lower WBC and lymphocyte counts but significantly higher monocyte counts and neutrophil/lymphocyte ratios compared to controls (p=0.009, p=0.045, respectively). The patient group had significantly lower T lymphocyte counts (p=0.008). B lymphocyte counts and percentages were lower (p&lt;0.001, p=0.004) in the patient group. There was no significant difference between the two groups in terms of NK cells. T helper and T cytotoxic lymphocyte counts were significantly lower, but there was no change in CD4/CD8 ratios. The percentage of HLA-DR expression on T lymphocytes, HLA-DR MFI values of T cytotoxic cells, and HLA-DR MFI values of CD16+ monocytes were significantly increased in the patient group (p=0.001, p=0.004, p&lt;0.001, respectively). CRP was positively correlated with HLA-DR expression on T lymphocytes (r=0.501, p&lt;0.001). Conclusions HLA-DR MFI values may be an important marker for demonstrating the function of both T cytotoxic cells and CD16+ monocytes in COVID-19.

Hyperactivation and enhanced cytotoxicity of reduced CD8+ gamma delta T cells in the intestine of patients with Crohn’s disease correlates with disease activity

Background and aimsWe aimed to investigate the immune characteristics of intestinal CD8+ gamma delta T (CD8+ γδ T) cells in Crohn’s disease (CD) and their correlation with disease activity.MethodsThe study cohorts included 21 CD patients and 21 healthy individuals. CD8+ γδ T cells were isolated from human ileal mucosa for detection by flow cytometry. The activation or inhibition status of cells was detected by detecting the expression of activation marker HLA-DR and the immunosuppressive molecule PD-1 on cells. The cytotoxicity of cells was assessed by detecting the expression of cytotoxic molecules (Perforin, Granzyme B, and TRAIL) in cells. Ratios of investigated cells were calculated as prediction factors by receiver operating characteristic curve (ROC) analysis.ResultsThe study revealed a reduction in intestinal CD8+ γδT cells among active CD patients, with a more pronounced reduction observed in moderately active patients compared to mildly active patients. Moreover, active CD patients exhibited heightened activation levels in their intestinal CD8+ γδT cells, whereas the activation was comparatively weakened in moderately active patients compared with mildly active patients. Additionally, the cytotoxicity of intestinal CD8+ γδT cells was enhanced solely in mildly active patients, while it was impaired in moderately active patients compared with mildly active patients. Furthermore, HLA-DR+ CD8+ γδT cell ratio, CD8+ γδT ratio, and CD8+ γδT count were identified as indicators in the diagnosis of active CD. Meanwhile, the ratios of Granzyme B+ CD8+ γδT cell and Perforin+ CD8+ γδT cell were identified as indicators that distinguish mildly moderately active CD cases.ConclusionsIntestinal CD8+ γδT was reduced in active CD patients, but their activation and cytotoxicity were enhanced. However, with increased disease activity, intestinal CD8+ γδ T cells became dysfunctional. CD-specific perturbations observed in various phenotypic markers in CD8+ γδ T cells can be used as indicators to assist in diagnosing CD patients.

An algorithm based on immunotherapy discontinuation and liver biopsy spares corticosteroids in two thirds of cases of severe checkpoint inhibitor-induced liver injury.

There are few data on corticosteroids (CS)-sparing strategies for checkpoint inhibitor (ICI)-induced liver injury (ChILI). We aimed to assess the performance of a 2-step algorithm for severe ChILI, based on ICI temporary discontinuation (step-1) and, if lack of biochemical improvement, CS based on the degree of necroinflammation at biopsy (step-2). Prospective study that included all subjects with grade 3/4 ChILI. Peripheral extended immunophenotyping was performed. Indication for CS: severe necroinflammation; mild or moderate necroinflammation with later biochemical worsening. From 111 subjects with increased transaminases (January 2020 to August 2023), 44 were diagnosed with grade 3 (N = 35) or grade 4 (N = 9) ChILI. Main reason for exclusion was alternative diagnosis. Lung cancer (13) and melanoma (12) were the most common malignancies. ICI: 23(52.3%) anti-PD1, 8(18.2%) anti-PD-L1, 3(6.8%) anti-CTLA-4, 10(22.7%) combined ICI. Liver injury pattern: hepatocellular (23,52.3%) mixed (12,27.3%) and cholestatic (9,20.5%). 14(32%) presented bilirubin >1.2 mg/dL. Overall, 30(68.2%) patients did not require CS: 22(50.0%) due to ICI discontinuation (step-1) and 8/22 (36.4%) based on the degree of necroinflammation (step-2). Biopsy mainly impacted on grade 3 ChILI, sparing CS in 8 out of 15 (53.3%) non-improvement patients after ICI discontinuation. CD8+ HLA-DR expression (p = 0.028), central memory (p = 0.046) were lower in CS-free managed subjects, but effector-memory cells (p = 0.002) were higher. Time to transaminases normalisation was shorter in those CS-free managed (overall: p < 0.001, grade 3: p < 0.001). Considering our results, a strategy based on ICI discontinuation and biopsy for grade 3 ChILI is proposed. An algorithm based on temporary immunotherapy discontinuation and biopsy allows CS avoidance in two thirds of cases of severe ChILI.

Immunomodulatory activity of heat-killed Lacticaseibacillus paracaseiMCC1849 based on the activation of plasmacytoid dendritic cells in the peripheral blood of healthy adults.

Probiotics are widely used in food for their health benefits to the host. Inactivated probiotics also reportedly improve the intestinal environment and immune regulation. Our previous studies showed that heat-killed Lacticaseibacillus paracasei MCC1849 (hk-MCC1849) effectively induced IL-12 production in mouse spleen cells and significantly reduced cold symptoms in clinical trial subjects. To further elucidate the mechanism of host immune regulation by hk-MCC1849, human peripheral blood mononuclear cells (PBMCs) were cocultured with hk-MCC1849. The Toll-like receptor 9 ligands CpG-ODN 2216 and hk-MCC1849 and the heat-killed Lacticaseibacillus rhamnosus ATCC53103 were used as positive and negative controls, respectively. The results showed that, compared with the control, hk-MCC1849 significantly increased the expression of the plasmacytoid dendritic cell (pDC) marker CD86 (p < .0001) and the pDC marker HLA-DR (p < .001) in PBMCs. The expression levels of the IL-12p40, IFNα, IFNα1, IFNγ, and ISG15 genes were significantly increased after coculture with hk-MCC1849 (p < .05, p < .05, p < .05, p < .05, and p < .05, respectively, vs. control). Furthermore, to confirm whether hk-MCC1849 directly interacted with pDCs, DCs were enriched with PBMCs following 24 h of coculture with hk-MCC1849. Phagocytosis of fluorescently labeled hk-MCC1849 by pDCs was observed, and there were significant increases in CD86 (p < .05) and HLA-DR (p < .0001) expression in pDCs. These results suggest that hk-MCC1849 exerts a potential immunomodulatory effect on the host through the activation of peripheral pDCs.

Actives from the Micro-Immunotherapy Medicine 2LMIREG® Reduce the Expression of Cytokines and Immune-Related Markers Including Interleukin-2 and HLA-II While Modulating Oxidative Stress and Mitochondrial Function.

Micro-immunotherapy (MI) is a therapeutic option employing low doses (LD) and ultra-low doses (ULD) of cytokines and immune factors to help the organism at modulating the immune responses. In an overpowering inflammatory context, this strategy may support the restoration of the body's homeostasis, as the active ingredients of MI medicines' (MIM) could boost or slow down the physiological functions of the immune cells. The aim of the study is to evaluate for the first time the in vitro anti-inflammatory properties of some actives employed by the MIM of interest in several human immune cell models. In the first part of the study, the effects of the actives from the MIM of interest were assessed from a molecular standpoint: the expression of HLA-II, interleukin (IL)-2, and the secretion of several other cytokines were evaluated. In addition, as mitochondrial metabolism is also involved in the inflammatory processes, the second part of the study aimed at assessing the effects of these actives on the mitochondrial reactive oxygen species (ROS) production and on the mitochondrial membrane potential. We showed that the tested actives decreased the expression of HLA-DR and HLA-DP in IFN-γ-stimulated endothelial cells and in LPS-treated-M1-macrophages. The tested MIM slightly reduced the intracellular expression of IL-2 in CD4+ and CD8+ T-cells isolated from PMA/Iono-stimulated human PBMCs. Additionally, while the secretion of IL-2, IL-10, and IFN-γ was diminished, the treatment increased IL-6, IL-9, and IL-17A, which may correspond to a "Th17-like" secretory pattern. Interestingly, in PMA/Iono-treated PBMCs, we reported that the treatment reduced the ROS production in B-cells. Finally, in PMA/Iono-treated human macrophages, we showed that the treatment slightly protected the cells from early cell death/apoptosis. Overall, these results provide data about the molecular and functional anti-inflammatory effects of several actives contained in the tested MIM in immune-related cells, and their impact on two mitochondria-related processes.

Cord Blood-Derived Multipotent Stem Cells Ameliorate in Vitro/in Vivo Alloreactive Responses, and This Effect Is Associated with Exosomal Microvesicles in Vitro

Human cord blood derived-multipotent stem cells (CB-SCs) have been found to have immunomodulatory capabilities that can result in inhibition of immune activation. Clinically, when used to interact with apheresed peripheral blood mononuclear cells (PBMCs) before reinfusion, they can counteract inflammation and restore immune balance in patients with autoimmune diseases, including alopecia areata and type 1 diabetes. The present study aimed to explore the potential application of CB-SCs to control donor alloreactive responses involved in allogeneic hematopoietic cell transplantation, which often results in acute graft-versus-host disease (GVHD). Phenotypically, we demonstrated that CB-SCs express CD45, CD11b, and CD9 markers on the cell surface; express Oct3/4, a transcription factor for embryonic stem cells; are negative for CD3, CD14, and CD34 expression; and have low expression of HLA-DR. In an allogeneic mixed lymphocyte culture (MLC) using human CD4 T cell enriched PBMCs and allogeneic myeloid derived dendritic cells, direct coculture with CB-SCs decreased CD4 T cell proliferation and activation, as evidenced by a marked decrease in the expression of the late activation markers CD25 and HLA-DR and a reduction of the PKH26 cell proliferation membrane lipophilic marker. Cytokine profiling of MLC supernatants revealed decreased concentrations of inflammatory proteins, including IFN-γ, IL-17, IL-13, IL-2, IL-6, and MIP1-α, along with marked increases in IL-1RA, IP-10, and MCP-1 concentrations in the presence of CB-SCs. Furthermore, transwell MLC experiments revealed that a soluble component was partially responsible for the immunomodulatory effects of CB-SCs. In this regard, exosomal microvesicles (EVs) positive for CD9, CD63, and CD81 were found in CB-SC-derived, ultrafiltered, and ultracentrifuged culture supernatants. CB-SC-EVs inhibited T cell proliferation in allogeneic MLC, suggesting a potential mode of action in allogeneic responses. Finally, CB-SCs were evaluated for their cellular therapy potential in vivo and found to ameliorate the development of GVHD responses in a xenogeneic human PBMC-induced NSG mouse model. Taken together, our results indicate that CB-SCs can directly and indirectly attenuate alloreactive CD4 T cell activation and proliferation in vitro with a potentially related EV mode of action and may have potential as a cellular therapy to control donor T cell-mediated GVHD responses in vivo.

Clinical Analysis of Pediatric Acute Megakaryocytic Leukemia With CBFA2T3-GLIS2 Fusion Gene.

CBFA2T3-GLIS2 is the most frequent chimeric oncogene identified to date in non-Down syndrome acute megakaryocytic leukemia (AMKL), which is associated with extremely poor clinical outcome. The presence of this fusion gene is associated with resistance to high-intensity chemotherapy, including hematopoietic stem cell transplantation (HSCT), and a high cumulative incidence of relapse frequency. The clinical features and clinical effects of China Children's Leukemia Group-acute myeloid leukemia (AML) 2015/2019 regimens and haploidentical HSCT (haplo-HSCT) for treatment of 6 children harboring the CBFA2T3-GLIS2 fusion gene between January 2019 and December 2021 were retrospectively analyzed. The 6 patients included 4 boys and 2 girls with a median disease-onset age of 19.5 months (range: 6-67mo) who were diagnosed with AMKL. Flow cytometry demonstrated CD41a, CD42b, and CD56 expression and lack of HLA-DR expression in all 6 patients. All the children were negative for common leukemia fusion genes by reverse transcription polymerase chain reaction, but positive for the CBFA2T3-GLIS2 fusion gene by next-generation sequencing and RNA sequencing. All patients received chemotherapy according to China Children's Leukemia Group-AML 2015/2019 regimens, and 4 achieved complete remission. Four children underwent haplo-HSCT with posttransplant cyclophosphamide-based conditioning; 3 had minimal residual disease negative (minimal residual disease <0.1%) confirmed by flow cytometry at the end of the follow-up, with the remaining patient experiencing relapse at 12 months after transplantation. Transcriptome RNA sequencing is required for the detection of the CBFA2T3-GLIS2 fusion gene and for proper risk-based allocation of pediatric patients with AML in future clinical strategies. Haplo-HSCT with posttransplant cyclophosphamide-based conditioning may improve survival in children with AMKL harboring the fusion gene.

Emergency Myelopoiesis Distinguishes Multisystem Inflammatory Syndrome in Children From Pediatric Severe Coronavirus Disease 2019.

Multisystem inflammatory syndrome in children (MIS-C) is a hyperinflammatory condition caused by recent infection with severe acute respiratory syndrome coronavirus 2, but the underlying immunological mechanisms driving this distinct syndrome are unknown. We utilized high-dimensional flow cytometry, cell-free (cf) DNA, and cytokine and chemokine profiling to identify mechanisms of critical illness distinguishing MIS-C from severe acute coronavirus disease 2019 (SAC). Compared to SAC, MIS-C patients demonstrated profound innate immune cell death and features of emergency myelopoiesis (EM), an understudied phenomenon observed in severe inflammation. EM signatures were characterized by fewer mature myeloid cells in the periphery and decreased expression of HLA-DR and CD86 on antigen-presenting cells. Interleukin 27 (IL-27), a cytokine known to drive hematopoietic stem cells toward EM, was increased in MIS-C, and correlated with immature cell signatures in MIS-C. Upon recovery, EM signatures decreased and IL-27 plasma levels returned to normal levels. Despite profound lymphopenia, we report a lack of cfDNA released by adaptive immune cells and increased CCR7 expression on T cells indicative of egress out of peripheral blood. Immune cell signatures of EM combined with elevated innate immune cell-derived cfDNA levels distinguish MIS-C from SAC in children and provide mechanistic insight into dysregulated immunity contributing toward MIS-C, offering potential diagnostic and therapeutic targets.