Marker CD69 Research Articles

Minocycline Alleviates White Matter Injury following Intracerebral Hemorrhage by Regulating CD4+ T Cell Differentiation via Notch1 Signaling Pathway.

Neuroinflammation is a major reason for white matter injury (WMI) after intracerebral hemorrhage (ICH). Apart from microglia/macrophage activation, T cells also play an important role in regulating immune responses after ICH. In a previous study, we have revealed the role of minocycline in modulating microglia/macrophage activation after ICH. However, the exact mechanisms of minocycline in regulating T cells differentiation after ICH are still not well understood. Hence, this study explored the relationship between minocycline and CD4+ T cell differentiation after ICH. Piglet ICH model was used to investigate naive CD4+ T cell differentiation and T cells signal gene activation after ICH with immunofluorescence and whole transcriptome sequencing. Naive CD4+ T cells and primary oligodendrocyte coculture model were established to explore the effect and mechanism of minocycline in modulating CD4+ T cell differentiation after ICH. Flow cytometry was used to indicate CD4+ T cell differentiation after ICH. The mechanism of minocycline in modulating CD4+ T cell differentiation was demonstrated with immunofluorescence and western blot. Double immunostaining of representative CD4+ T cell marker CD3 and different subtype CD4+ T cell assisted proteins (IL17, IL4, Foxp3, and IFNγ) demonstrated naive CD4+ T cell differentiation in piglet after ICH. Whole transcriptome sequencing for perihematomal white matter sorted from piglet brains indicated T cell signal gene activation after ICH. The results of luxol fast blue staining, immunofluorescent staining, and electron microscopy showed that minocycline alleviated white matter injury after ICH in piglets. For our in vitro model, minocycline reduced oligodendrocyte injury and neuroinflammation by regulating CD4+ T cell differentiation after ICH. Moreover, minocycline increased the expression of NOTCH1, ACT1, RBP-J, and NICD1 in cultured CD4+ T cell when stimulated with hemoglobin. Hence, minocycline treatment could modulate naive CD4+ T cell differentiation and attenuate white matter injury via regulating Notch1 signaling pathway after ICH.

Thymic hypoplasia induced by copy number variations contributed to explaining sudden infant death based on forensic autopsies

Thymic hypoplasia is a primary cellular immunodeficiency that causes susceptibility to serious infections leading to sudden death in infants. Some genetic disorders in humans could result in the evident permanent hypoplasia or occasional aplasia of the thymus at birth. However, determining the genetic etiology of thymic hypoplasia is challenging for the sudden infant death due to primary cellular immunodeficiency. In this study, in order to find the fundamental reasons for sudden death of infants with thymic hypoplasia, 5 infants with suspected thymic hypoplasia and 10 control infants were assessed, and the immunohistochemistry and DNA analysis were used to investigate whether the infants with thymic hypoplasia had DiGeorge syndrome (DGS) with copy number variations (CNVs) in 22q11.2 and other chromosomes. The results showed that the weight of the thymus was significantly lower than the normal except the case 4, and that all the infants had hypocalcemia and a significant decrease or even absence of the markers CD1a, CD2, CD3, CD4 and CD8, which are related to T-cell maturation. In addition, multiplex ligation-dependent probe amplification (MLPA) analysis showed that these infants carried CNVs in 22q11.2 and other associated chromosomes with deletion and duplication of 25 genes. The results of thymus weight, histopathology, molecular pathology and MLPA analysis suggested that DGS predominantly with thymic hypoplasia induced by CNVs caused the sudden death of these infants under various infections or other unexplained reasons, which may provide new insights into the diagnosis of sudden infant death and could help the parents of deceased infants to attach more importance of genetic screening and thymus ultrasound to reduce the postnatal mortality of the infant, and demonstrates the value of genetic diagnosis in the forensic pathology.

γδ Tcells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ Tcells show that direct interaction between γδ Tcells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ Tcells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after invivo depletion of γδ Tcells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ Tcells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ Tcells an attractive target to treat autoimmunity.

Lactic acid bacteria derived-bioactive molecules condition an in vitro cell model into a tolerogenic dendritic-like cell phenotype

Abstract Retinoic acid (RA) is a vitamin A metabolite; CD103+ dendritic cells (DCs) acquire a tolerogenic phenotype through an RA-dependent mechanism, driving the differentiation of gut-homing regulatory T cells in the gut mucosa. Growing evidence shows bioactive molecules derived from lactic acid bacteria exhibit immunomodulatory activity by interacting with antigen presenting cells (APCs), stimulating their differentiation and altering their phenotype to further shape adaptive immunity. We utilized the monocytic THP-1 cell line as an in vitro model to examine the impact of the Lacticaseibacillus rhamnosus secretome (LrS) and interactions with RA on APC differentiation into a tolerogenic dendritic-like cell (DLC) phenotype. THP-1 monocytes were treated with Phorbol 12-myristate 13-acetate to drive differentiation into macrophages, then conditioned with the LrS and/or RA for 24, 48 or 72 hours. Expression of key DC markers CD11b, CD11c, DC-SIGN, CD83, CD80 and CD103 increased, while expression of proinflammatory marker CD64, co-stimulatory marker CD86 and MHC class II molecule HLA-DR was reduced. Cytokine profiling revealed LrS + RA co-treatment increased the production of immunoregulatory cytokines IL-10 and IL-1RA. Expression of NFKB1 and NFKB2, key genes involved in the activation of the NF-κB canonical and non-canonical pathways was induced by 48 hours of LrS conditioning, accompanied by increased expression of CD40 and IDO1, indicating an ability to drive DLC maturation and induce co-stimulatory capacity. Deciphering the mechanisms behind these types of microbiota-host interactions gives insight into how microbial metabolites can influence immune maturation and maintain immune homeostasis. Supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN-2017-05237

Levels of epigenetic regulators TET2 and DNMT1 correlate with virus-stimulated pDC differentiation from IFN-α producing cells to antigen presenting cells

Abstract Plasmacytoid dendritic cells (pDC) are potent producers of IFN-α in response to DNA and RNA viruses and subsequently mature into antigen presenting cells. We investigated the role of DNA methyltransferase 1 (DNMT1) and Ten-eleven translocation methyl-cytosine dioxygenase 2 (TET2), which are generally known as gene silencers and activators, respectively, in pDC activation. PBMC from healthy donors were stimulated with Herpes Simplex Virus (HSV) or Influenza-A virus (IAV) for 6- and 24h. PBMC were surface-stained for pDC markers CD123, HLA-DR and CD11C and co-stimulatory markers CD80 and CD86, then intracellularly for DNMT1, TET2 and IFN-α, and acquired by flow cytometry. mRNA expression of DNMT1 in purified pDC was determined by qRT-PCR. At 6h, DNMT1 protein was expressed at equivalent levels in pDC with and without stimulation and in IFN-α+ and IFN-α-pDC, and in unstimulated controls. In contrast, TET2 proteins were elevated in IFN-α+ but not IFN-α-pDC and controls at 6h. At 24h, DNMT1 increased while TET2 was reduced in stimulated pDC, correlating with high IFN-α levels at 6h and low levels at 24h. There was no change in DNMT1 gene expression in virus-stimulated vs. unstimulated controls. CD80 and CD86 were elevated at 24h compared to 6h in stimulated pDC, confirming pDC differentiation from IFN-α producing to antigen presenting cells. At the point of pDC differentiation, TET2 levels were decreased and DNMT1 increased. These results suggest that TET2 is demethylating and causing activation of genes associated with pDC IFN-α production, while DNMT1 methylates and silences genes in this pathway. This results in an initial induction of TET2 and IFN-α production followed by upregulation of co-stimulatory markers, DNMT1 and loss of IFN-α. Supported by 1. AAI trainee fellowship Careers in Immunology Fellowship Program AAI EIN #: 52-2317193 2. R01Al106125

Regulatory T cell coordination of the mucosal NK cell response during viral infection.

Abstract In many infections the mucosal barrier is the site of pathogen exposure, yet the regulation of mucosal immunity is not well understood. Herpes simplex virus-2 (HSV-2) is one of the most prevalent mucosal infections, making this disease an ideal model to study mucosal anti-viral immune response. My goal is to define how regulatory T cells (Tregs) shape anti-viral immune responses through modulation of natural killer (NK) cells during mucosal virus infection. To accomplish my goal, I utilized a mouse model of intravaginal HSV-2 infection. NK cell phenotypes were examined using high parameter flow cytometry. To define how transient Treg depletion during HSV-2 infection impacts NK cells, I utilized Foxp3DTR mice. Treg depletion led to increased maturation of NK cells in the vaginal tract (VT). KLRG1 is associated with NK cell maturation and is a marker of terminally mature NK cell subsets. I found a significant increase in the frequency and total number of KLRG1+ NK cells in the VT two days after HSV-2 infection in the absence of Tregs. I further analyzed NK cell maturation using the expression of the two surface markers CD11b and CD27 and found a significant increase in CD11b+CD27+ NK cells after Treg depletion. This subset of NK cells is known to be significant producers of pro-inflammatory cytokines. The cytokine IL-15 is necessary for NK cell development and maturation. I found that in the VT, CD11b+Ly6C+ monocyte-derived DCs display increased IL-15 transpresentation after Treg depletion. My data suggests that Tregs have a modulatory effect on NK cell development in the VT as Treg depletion leads to altered NK cell maturation and development. This demonstrates the role of Tregs in mediating an effective immune response while limiting immune pathology. Supported by grants from NIH (R01 AI141435-01, T32 AI 7509-21)

Human lung tissue-resident memory CD8+ T cells are transcriptionally, epigenetically, and phenotypically diverse

Abstract Tissue-resident memory CD8+ T cells (TRM) are strategically located in peripheral tissues, especially at mucosal surfaces, where they can provide protection against invading pathogenic microbes. In the lungs, TRM play a critical role in limiting disease and transmission of respiratory viruses; however, the heterogeneity present in TRM populations in the human lung remains largely unexplored. Here we show that human lungs harbor transcriptionally, epigenetically, and phenotypically distinct populations of memory CD8+ T cells expressing the tissue residency-associated markers CD69 and CD103. High-dimensional flow cytometry and single-cell RNA sequencing of memory CD8+ T cells isolated from human lungs shows that heterogeneity exists both between and within CD69+ CD103− and CD69+ CD103+ subsets, with transcriptional diversity among the CD69+ CD103− subset being most prominent. Single-cell ATACseq demonstrates that similar levels of epigenetic diversity exist between and within these different TRM subsets. However, flow cytometry and single-cell RNAseq of influenza- and SARS-CoV-2-specific lung TRM demonstrates that heterogeneity between CD69+ CD103− and CD69+ CD103+ subsets is largely eliminated when the cells are specific for a common antigen. Together, these data illuminate underappreciated and unexplored aspects of heterogeneity in TRM populations in humans. Supported by grants from NIH/NIAID (75N93019R00028) and NIH/NHLBI (R35 HL150803)

EMCN knockout leads to increased monocyte infiltration in kidney and albuminuria in mice

Endomucin (EMCN), a type I integral membrane glycoprotein, is specifically expressed in endothelium and enriched in kidney glomerular endothelium. We have previously reported that under resting conditions EMCN functions as an anti‐inflammatory molecule, preventing leukocyte‐endothelial cell adhesion and that EMCN plays an important role in modulating VEGFR2 signaling. The goal of this study is to characterize the phenotype of the kidney in the first‐reported EMCN knockout mouse model. EMCN−/−mice (with a EGFP tag for the knockout allele) were obtained by crossing EMCN‐floxed mice, generated in conjunction with Cyagen Biosciences, with the ROSA26‐Cre mice. The mRNA level of EMCN, CD31, CD34, E‐selection, VCAM‐1, and ICAM‐1 were quantified by reverse transcription PCR and semi‐quantitative PCR using total RNA isolated from the adult mouse kidneys. The mRNA level of EMCN in EMCN−/−mice were not detectable (n>4, p<0.0001) compared to EMCN+/+. Renal CD34 at mRNA level was decreased (fold change, 0.68±0.09 vs 1, n=4, p<0.05) and VCAM‐1 was increased (fold change, 2.2 ±0.45 vs 1, n=4, p<0.01) in EMCN−/− kidneys compared to wildtype (wt) controls. CD31, E‐selection, and ICAM‐1 mRNA levels were unchanged between EMCN−/−and wildtype kidneys. Kidneys from adult EMCN+/+ and EMCN−/− mice were collected, fixed, and sectioned. Expression and localization of EMCN, eGFP, CD45 and podocin in adult kidneys were examined by immunohistochemistry and quantified, which revealed a significant increase in CD45+ cell infiltration in EMCN−/− mice compared to EMCN+/+ mice (72.50±8.13 cells/mm2 vs 39.67±4.91 cells/ mm2, n=4, p<0.001). To characterize the renal inflammatory cell composition, freshly collected kidneys from EMCN+/+ and EMCN−/− were enzymatically digested and the leukocyte markers CD45, CD3, CD4, CD19, CD11b, Ly6G and Ly6C were examined by flow cytometry to identify the proportion of myeloid populations. Flow cytometry analysis suggested a trend of increased CD45+ cells in the kidney from EMCN−/−mice compared to wildtype (n=9, p=0.07), and revealed a significant increase in the percentage of Ly6GhighLy6Chighmyeloid cells in EMCN−/−compared to wildtype kidneys (1.6±0.23 vs 1.01±0.18, n=9, p<0.05). Sporadic albuminuria was detected through Coomassie staining in spot urine samples of EMCN−/− mice. Ultrastructural morphology of glomeruli, examined from both EMCN+/+ and EMCN−/− through transmission electron microscopy, showed the effacement of podocyte foot processes and disorganized endothelial cell fenestrations in glomerular capillaries of EMCN−/− mice compared to wt. The result indicates EMCN plays a key role in the prevention of inflammatory cells infiltration in kidney, presumably by blocking leukocyte adhesion, and is also involves in maintaining normal glomerular filtration barrier structure and function, potentially via its role in VEGFR2 signaling.

TRM and mucosal tissue sites are protected from age-associated phenotypic changes in secondary lymphoid organs

Abstract The persistence and maintenance of immunological memory is critical for long-term immunity generated in response to infection and vaccination. Aging of memory T cells in humans has predominantly been studied in the peripheral blood, and considerably less is known about the aging of memory T cells within tissues. Using tissues obtained from human organ donors, we investigated how tissue localization, expression of the CD103 integrin, and age influence the phenotype of tissue resident memory T cells (TRM). Using multi-parameter flow cytometry, we characterized diverse tissue sites from donors ages 9 to 93 years old and demonstrate that age-associated changes in the T cell compartment exhibit tissue- and subset- specific dynamics. CD8 T cells in blood-rich tissues spleen and lung have elevated expression of replicative senescence markers CD57 and KLRG1 relative to lung-associated lymph nodes (LLN), jejunum, and mesenteric lymph nodes (MLN). CD103+ TRM have the lowest expression of CD57 and KLRG1 across all tissue sites relative to both CD103− TRM and circulating TEM. CD8 T cells in lymphoid tissues spleen, LLN, and MLN exhibit the largest age-associated increases in expression of CD57, KLRG1, CD244, and PD-1. Together, these results demonstrate that age-associated phenotypic changes are most prominent in secondary lymphoid organs, while TRM and mucosal sites exhibit few senescent changes. Supported by the National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) and grants from NIH (P01 AI106697, U19 AI057266)

Phenotypical changes in peripheral NK cells and their impact on the clinical outcomes of patients with severe pandemic influenza A(H1N1)

Abstract NK cells are crucial antiviral effectors. However, their role against emerging respiratory pathogens has not been extensively addressed in humans. Here, we characterized the phenotype of peripheral NK cells in patients with severe pandemic influenza A(H1N1) and age- and sex-matched healthy controls (HC) using an extended panel of flow cytometry antibodies against markers of activation, cytotoxicity, cytokine production, immune memory, and surface receptors. Enrolled patients were grouped according to their clinical outcome (survival vs. death). The study included 42 influenza patients (73% men) with a median age of 48 years, from which eight (26%) died during the follow-up period. All patients were admitted to the intensive care unit and required mechanical ventilation. Individuals with influenza differentiated from HC by reduced numbers of total, CD56bright, CD56dimCD16+, CD27+ memory-like, NKp46+, and perforin+ NK cell subpopulations, but increased CD69+ NK cells. Notably, lower percentages of CD27+ memory-like NK cells correlated with increased alkaline phosphatase levels, a readout of lung damage in patients with acute respiratory distress syndrome. Strikingly, a higher expression of the senescence marker CD57 in CD27+ memory-like NK cells and expansion of double-positive CD27+CD57+ NK cells was characteristic of deceased influenza patients, but not survivors. Finally, despite being reduced in the overall influenza group with respect to HC, increased percentages of IFN-γ+ NK cells distinguished death vs. survival outcomes. Collectively, our results demonstrate that alterations in the phenotype of NK cells influence with the progression and outcomes of patients with severe pandemic influenza A(H1N1) infection. Supported by grants from CONACyT (FORDECyT/10SE/2020/05/14-06, FORDECyT/10SE/2020/05/14-07) and SECTEI CDMX (SECTEI/050/2020) to JZ. JAR-N received a scholarship from CONACyT (CVU-1097402) for achieving her M.S.

Eight Weeks of Daily Cannabidiol Ingestion Does Not Alter Peripheral Blood Mononuclear Cell Profile or Natural Killer Cell Cytotoxicity

ObjectiveThe purpose of this randomized placebo‐controlled trial was to determine the effects of an 8‐week cannabidiol (CBD) intervention on peripheral blood mononuclear cells (PBMCs) profile and natural killer cell (NKC) cytotoxicity.HypothesisFollowing an 8‐week CBD intervention, participants consuming CBD will have an increased percentage of NKC in peripheral blood, as well as decreased NKC cytotoxicity determined by a measure of K562 cell viability.MethodsPhysically active men and women (18‐45 y), currently meeting the American College of Sports Medicine (ACSM) guidelines for physical activity were randomly assigned to placebo (CN, n=5) or CBD (CB, n=9) groups. Participants consumed capsulized control; coconut derived medium chain triglycerides (MCT; 225mg/day) or CBD (50mg/day with 175mg of MCT) daily for 8 weeks. Participants completed measures of body size, body composition (BodPod) and fitness (VO2max), and peripheral blood was drawn before and after the intervention. Ficoll‐isolated PBMCs were co‐incubated for 4h with K562 human leukemia cells before cytotoxicity analysis by flow cytometry. Before co‐culture, PBMCs were stained with T cell marker CD3, and NKC marker, CD56, for immune profiling. K562 cells were pre‐stained with cell‐specific CD71 (transferrin receptor) and the cell viability dye Calcein AM. Data are presented as mean ± standard deviation with significance set at a=0.05. At the pre intervention time point, data was not normally distributed, so a Welch’s t‐test was preformed to determine group differences. A 2 (group) x 2 (time) analysis of variance (ANOVA) was used to identify any interactions or main effects.ResultsAt the pre‐intervention time points, there were no significant differences between groups with respect to participant characteristics (age: 27.1 ± 6.1 y; height: 169 ± 9.7 cm; weight: 73.8 ± 15.5 kg; lean body mass: 57.5 ± 12.0 kg; body fat: 21.5 ± 8.3%; VO2Peak: 45.2 ± 7.6 ml/kg/min). There were no significant differences in percentage of NKC (Pre: 8.8 ± 5.8%; Post:7.4 ± 5.7%), natural killer T‐cells (NKT; Pre: 1.7 ±1.1; Post: 1.5 ±1.1%), or T‐cells (Pre: 47.37±13.3; Post: 46.29±10.17%) when CN was compared to CB following the intervention. Furthermore, there were no differences in K562 cell mean or median fluorescence intensity of Calcein AM when CN was compared to CB following the intervention.ConclusionEight weeks of CBD (50mg/day) did not alter NKC, NKT, or T‐cell distribution in PBMCs. In line with these findings, NKC showed similar cytolytic function through no change in mean or median fluorescence intensity of Calcein AM expression of K562 human leukemia cells, indicating no change in cell viability following CBD supplementation. This preliminary study suggests that CBD does not alter the distribution of circulating mononuclear immune cells or NKC cytolytic function.

Abstract 217: PCSK6 Is A Key Regulator Of Immune Status In Mice And Its Ablation Increases Atherosclerotic Plaque Burden In A Bone Marrow Transplant Model

Introduction: Proprotein convertase subtilisins/kexins (PCSKs) activate cytokines and growth factors and have been implicated in various cancers. We have previously shown that PCSK6 is a key protease modulating smooth muscle cell response in atherosclerosis, but its expression also correlated positively with typical markers of T lymphocytes and macrophages in plaques and it was localised in the proximity of these cells. Here, we hypothesized that PCSK6 may be involved in modulating inflammatory responses and aimed to elucidate its role in a hyperlipidemic mice model. Methods: Detailed immunophenotyping using histology, FACS-, OLINK- and ELISA-based analyses and primary cell cultures was used to compare Pcsk6 -/- and littermate controls. Atherosclerosis was evaluated in Ldlr -/- mice upon bone marrow transplant with Pcsk6 -/- or wild-type bone-marrow. Results: At baseline Pcks6 -/- mice showed an enrichment of pro-inflammatory cytokines Ccl2, Ccl3, Ccl20, Cxcl1 and in particular Il17a and Il17f in plasma compared controls. Spleens of Pcsk6 -/- mice had an increased number of germinal centres and FACS analysis showed that they contained significantly more CD8+ T cells. Microarray analysis of spleens from Pcsk6 -/- vs. controls confirmed that T cell markers CD4, CD3E and CD3G were upregulated. In vitro , splenocytes isolated from Pcsk6 -/- mice secreted more IFN-γ, IL-2 and IL-10 than controls upon stimulation with α-CD3 and α-CD28 antibodies. Moreover, peritoneal macrophages from Pcsk6 -/- mice secreted more TNF-α, MCP-1, IL-6 and IL-10 compared to control mice upon LPS stimulation. Interestingly, bone marrow derived macrophages from Pcsk6 -/- mice were also more prone to lipid uptake. Finally, in vivo transplantation of Pcsk6 -/- bone marrow to Ldlr -/- mice led to increased atherosclerotic plaque burden compared to controls, as quantified in the aortic root. Conclusions: PCSK6 ablation led to increased number of CD8+ T cells, as well as macrophage and cytokine activation. Transplantation of Pcsk6 -/- bone marrow resulted in an increase in atherosclerotic plaque burden compared to controls. Taken together, these results indicate that PCSK6 is a key regulator of the immune system, though the exact mechanisms involved require further investigation.

Validation of dermatopathological criteria to diagnose cutaneous lesions of mastocytosis: importance of KIT D816V mutation analysis.

Cutaneous lesions of mastocytosis (CLM) are often subtle and may require biopsy. However, dermatohistopathological criteria for CLM remain undefined. To establish criteria for CLM by validating histological and molecular parameters. In skin samples from Caucasian patients with CLM and controls (atopic dermatitis, chronic urticaria, pruritus, tissue from tumor safety margin excisions), mast cell (MC) numbers, size, shape, distribution, immunostainability with a large panel of markers, pigmentation and presence of KIT D816V mutation were analysed. Forty-seven CLM patients (32 maculopapular cutaneous mastocytosis (MPCM), 15 mastocytomas) and 36 controls were included. Mastocytomas were easily identified by densely packed cuboidal MCs. In MPCM, skin MC density in CD117 stains was higher in CLM patients than in controls (P < 0.0001) and values correlated closely (r = 0.65, P < 0.0001) to results in tryptase stains. The optimized upper dermis cut-off number of 62 MC/mm2 had a sensitivity and specificity of 92% in both stainings, corresponding to approximately 12 MC/high power field (HPF). MC size was larger in MPCM than in controls (P = 0.01). Interstitial (= not perivascular or periadnexal) MCs and stronger basal pigmentation of the epidermis were indicative of MPCM (P < 0.0001 each) and clusters of >3 nucleated MC/HPF exclusively found in MCPM. Surface markers CD2, CD25 and CD30 stained T-lymphocytes, but only negligibly CLM MC. The KIT D816V mutation in formalin fixed paraffin embedded (FFPE) skin was evaluable in 87.5% of MCPM patients and had both 100% sensitivity and specificity. MPCM can be predicted by major and minor criteria combined in a scoring model. Presence of D816V mutation in FFPE skin and MC density > 27/HPF are >95%-specific major criteria for MPCM. MC densities 12/HPF, interstitial MC, clusters and basal pigmentation are minor criteria.

Immune effects of CDK4/6 inhibitors in patients with HR+/HER2- metastatic breast cancer: Relief from immunosuppression is associated with clinical response.

SummaryBackgroundCyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are innovative small target molecules that, in combination with endocrine therapy, have recently been employed in the treatment of patients with HR+/HER2− metastatic breast cancer (mBC). In this prospective study, we investigate the impact of CDK4/6i on the immune profile of patients with HR+/HER2− mBC.MethodsImmune cell subsets were analysed using flow cytometry of peripheral blood mononuclear cells (PBMCs) isolated from patients with HR+/HER2− mBC, both before and during treatment. Regulatory T cells (Tregs) were identified using the markers CD4, CD25, CTLA4, CD45RA, and intracellular FOXP3. Monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSCs and PMN-MDSCs) and other immune populations were analysed using CD45, CD14, CD66b, CD11c, HLA-DR, CD3, CD8, CD28, CD137, PD1, CD45RA, CCR7, and Ki67.FindingsThe percentage of circulating Tregs and M/PMN-MDSCs was significantly downregulated from baseline during CDK4/6i-treatment (p<0.0001 and p<0.05, respectively). In particular, the effector Treg subset (CD4+CD25+FOXP3highCD45RA−) was strongly reduced (p<0.0001). The decrease in Treg levels was significantly greater in responder patients than in non-responder patients. Conversely, CDK4/6i treatment was associated with increased levels of CD4+ T cells and anti-tumour CD137+CD8+ T cells (p<0.05).InterpretationCDK4/6i treatment results in downregulation of Tregs, M-MDSCs, and PMN-MDSCs, thus weakening tumour immunosuppression. This decrease is associated with response to treatment, highlighting the importance of unleashing immunity in cancer treatment efficacy. These results suggest a novel mechanism of immunomodulation in mBC and provide valuable information for the future design of novel treatments combining CDK4/6i with immunotherapy in other cancer settings.FundingSapienza University of Rome.

P231 Targeting the Rho/MRTF-A pathway inhibits growth factor and cytokine release but enhances efferocytosis in scleroderma macrophages

Abstract Background/Aims In the systemic sclerosis (SSc) microenvironment, accumulation of extracellular matrix (ECM) components induces changes in the matrix's mechanical properties, which triggers Rho/MRTFF-A activation and the downstream signalling cascades, inducing the expression of fibrotic and inflammatory genes in fibroblasts. However, macrophages also play a key role in tissue homeostasis and wound repair. It is possible that these cells are also influenced by ECM stiffness and therefore we investigated the effects of altering substrate stiffness and tested the effects of an inhibitor of the mechano-sensing Rho/MRTF-A pathway on the activation state of SSc macrophages. Methods SSc and healthy control macrophages (both n = 4 donors) were derived by culture of blood monocytes in the presence of M-CSF (4ng/ml) for 7 days. Soft substrates (4kPa) or stiff ECM substrates (50kPa) were used to model the effect of soft healthy skin tissue and stiff fibrotic SSc skin (Softwell, Matrigen, polymer gels, collagen I coated). Morphology, gene expression (qPCR for CD86 for M1-like, CD206 for M2-like, MerTK for efferocytosis), Multiplex assay, labelled E. Coli uptake assay and Rho/MRTF/SRF pathway inhibition (small molecule CCG-257081) were used to assess the polarisation and activation state of these cells. Results Morphology and CD86 did not vary with matrix stiffness, but CD206 expression was significantly lower in stiff compared to soft gels (Mean+/- SEM; soft 38.2+/- 4.2 vs stiff 16.2+/-3.7 relative expression, p value 0.0167). CCG-257081 strongly inhibited secretion of cytokines, growth factors and chemokines. Factors present at &amp;gt; 50 pg/ml suppressed by CCG-257081 include TNFα, GM-CSF, IL-1RA, IP-10, RANTES, MIP-1α, MIP-1β, MCP-1, MCP-3, PDGF-BB (all p &amp;lt; 0.05). SSc macrophages secreted at high levels on soft, and were attenuated by culture on stiff, whereas healthy control macrophages showed the opposite trend inducing more secretion on stiff, inhibited by CCG-257081. Although polarisation markers CD206 and CD86 did not alter with the CCG-257081 treatment, there was a significant increase in MerTK with treatment (p &amp;lt; 0.0001). Endocytosis was moderately yet significantly suppressed by the CCG treatment (basal endocytosis 80.2+/- 4.0 vs CCG-257081 treated 62+/-2.5; % endocytosis; p &amp;lt; 0.01), whereas cell viability was not significantly altered. Conclusion Although differences are observed amongst patients, this data indicates that stiffness of the matrices, as used in this study, did not greatly alter the morphology or polarisation of the macrophages. However, transfer to stiff matrices enhanced the secretion of certain cytokines by control macrophages. Strong effects were seen with the Rho/MRTF-A inhibitor, which appeared to block the secretion of multiple factors, including inflammatory cytokines, pro-fibrotic factors, as well as regulatory factors and to strongly induce MerTK, a marker of efferocytosis. One possibility is that the inhibitor converts these cells from secretory to resolving phenotype. Based on these findings, CCG-257081 has promising effects against activated macrophages, which could be utilised in inflammatory fibrotic diseases. Disclosure S. Lopez Garces: None. B. Ahmed Abdi: None. C. MacFadyen: None. R. Smillie: None. L. Nagib: None. S. Ghani: None. S. Shah: None. D. Abraham: None. C. Denton: None. R.J. Stratton: None.

The Evolution of Blood Cell Phenotypes, Intracellular and Plasma Cytokines and Morphological Changes in Critically Ill COVID-19 Patients.

Background: Severe coronavirus disease 2019 (COVID-19) causes a strong inflammatory response. To obtain an overview of inflammatory mediators and effector cells, we studied 25 intensive-care-unit patients during the timeframe after off-label chloroquine treatment and before an introduction of immunomodulatory drugs. Material and methods: Blood samples were weekly examined with flow cytometry (FCM) for surface and intracytoplasmic markers, cytokine assays were analyzed for circulating interleukins (ILs), and blood smears were evaluated for morphological changes. Samples from healthy volunteers were used for comparison. Organ function data and 30-day mortality were obtained from medical records. Results: Compared to that of the healthy control group, the expression levels of leukocyte surface markers, i.e., the cluster of differentiation (CD) markers CD2, CD4, CD8, CD158d, CD25, CD127, and CD19, were lower (p < 0.001), while those of leukocytes expressing CD33 were increased (p < 0.05). An aberrant expression of CD158d on granulocytes was found on parts of the granulocyte population. The expression levels of intracellular tumor necrosis factor alpha (TNFα) and IL-1 receptor type 2 in leukocytes were higher (p < 0.001) as well as plasma levels of TNFα, IL-2, IL-6, IL-8, IL-10 (p < 0.001), interferon gamma (IFNγ) (p < 0.01), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (p < 0.05). The expression levels of CD33+ leukocytes and circulating IL-6 were higher (p < 0.05) among patients with arterial oxygen partial pressure-to-fractional inspired oxygen (PaO2/FiO2) ratios below 13.3 kPa compared to in the remaining patients. The expression levels of TNFα, IL-2, IL-4, IL-6, IL-8, and IL-10 were higher in patients treated with continuous renal replacement therapy (CRRT) (p < 0.05), and the levels of the maximum plasma creatinine and TNFα Spearman’s rank-order correlation coefficient (rho = 0.51, p < 0.05) and IL-8 (rho = 0.44, p < 0.05) correlated. Blood smears revealed neutrophil dysplasia with pseudo-Pelger forms being most common. Conclusion: These findings suggest that patients with severe COVID-19, in addition to augmented ILs, lymphopenia, and increased granulocytes, also had effects on the bone marrow.

HLA-G and CD152 Expression Levels Encourage the Use of Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells as an Alternative for Immunosuppressive Therapy.

Mesenchymal stromal cells (MSCs) have been used in immunosuppressive therapy due to their therapeutic effects, with the HLA-G molecule seeming to play a fundamental role. This work evaluated alternative MSC sources to bone marrow (BM), namely, umbilical cord tissue (UC), adipose tissue (AD) and dental pulp tissue (DP), and the influence of interferon-γ (IFN-γ) and hypoxia on the cultivation of these cells for use in immunosuppression therapies. Expression of costimulatory markers CD40, CD80 and CD86 and immunosuppressive molecules CD152 and HLA-G was analyzed. Lymphocyte inhibition assays were also performed. Sequencing of the HLA-G gene from exons 1 to 5 was performed using next-generation sequencing to determine the presence of alleles. UC-derived MSCs (UCMSCs) expressed higher CD152 and HLA-G1 under standard cultivation. UCMSCs and DP-derived MSCs (DPSCs) secreted similar levels of HLA-G5. All MSC sources inhibited the proliferation of peripheral blood mononuclear cells (PBMCs); growth under regular versus hypoxic conditions resulted in similar levels of inhibition. When IFN-γ was added, PBMC growth was inhibited to a lesser extent by UCMSCs. The HLA-G*01:04:01:01 allele appears to generate a more efficient MSC response in inhibiting lymphocyte proliferation. However, the strength of this conclusion was limited by the small sample size. UCMSCs are an excellent alternative to BM in immunosuppressive therapy: they express high concentrations of inhibitory molecules and can be cultivated without stimuli, which minimizes cost.

Key markers for diagnosis of minimal residual disease in multiple myeloma

Introduction. Therapeutic advances in recent years, the appearance on the market each time of new drugs that allow patients to achieve complete remission, a long period without progression dictate the need to control treatment by monitoring residual disease in multiple myeloma. Monitoring of multiple myeloma is the detection of a small number of tumor cells after therapy in a patient, which may be the cause of recurrence of the disease – control of minimal residual disease (MRD). This article discusses the key diagnostic markers (CD45, CD56 and CD19) of minimal residual multiple myeloma disease at initial diagnosis and after induction therapy. There are various diagnostic methods of research that can reliably assess the response to therapy and predict the occurrence of relapse. The main methods for detecting MRD are allele-specific polymerase chain reaction, next-generation sequencing and multicolor flow cytometry. The diagnosis of MRD by flow cytometry is the most widely used quite fast, quantitative, sensitive and affordable method, it is used for the primary diagnosis of multiple myeloma, as well as for monitoring MRD. It is important to identify the aberrant immunophenotype during the initial diagnosis for the correct subsequent assessment of MRD. MRD of multiple myeloma is considered an important component in the course of patient therapy before hematopoietic stem cell autotransplantation to assess the effectiveness of therapy, control and prognosis of the disease.The aim of the work MRD-study with the key diagnostic markers in multiple myeloma (CD45, CD56 and CD19).Materials and methods. The study was carried out in 59 patients with multiple myeloma. For all patients, in addition to standard methods of diagnosis and staging of the disease, bone marrow morphology (myelogram) and immunophenotype were examined. Eight-color flow cytometry with a panel of monoclonal antibodies for the diagnosis of plasma cell tumors Euro-Flow 2012 was used to diagnose MRD.Results. MRD was assessed by markers CD45, CD56 and CD19 in patients with multiple myeloma after induction therapy. The incidence of MRD-positivity for CD45 was 87.5 %, for CD56 – 97.5 %, for CD19 – 95.5 %. The incidence of MRD-negative status was for CD45 was 12.5 %, for CD56 – 2.5 % and for CD19 – 4,5 %.Conclusion. The use of a complex of these markers allows the most accurate determination of the MRD-negative status, taking into account the primary immunophenotype of malignant plasma cells.

ORAL WALDENSTROM MACROGLOBULINEMIA

<h3>Objective</h3> Waldenstrom macroglobulinemia (WM) is a rare B-cell lymphoproliferative disorder that is characterized by bone marrow infiltration with lymphoplasmacytic cells and immunoglobulin M monoclonal gammopathy. The extramedullary infiltration of WM is uncommon but predicts a worse prognosis. To date, there has been only 1 reported case of biopsy-proven oral manifestation of WM. <h3>Case Summary</h3> A 72-year-old man with a medical history significant for hypertension and WM, which was in good clinical and laboratory control on ibrutinib, a Bruton tyrosine kinase inhibitor, for the past 4 years, was referred by his dentist for evaluation of an asymptomatic palatal mass. The patient was known to have medication-related osteonecrosis of the jaw stage 1 of left mandible. This was secondary to Zometa prescribed to address skeletal involvement of WM. Intraoral examination demonstrated a firm, nontender, 3 × 3 cm sessile mass with surface ulcerations involving the right hard and soft palate. A panoramic radiograph did not reveal any right maxillary sinus pathology, any central lesion, or any periapical radiolucency associated with the right maxillary teeth. A biopsy of the lesion was obtained. The pathology report indicated diffuse infiltration of small to intermediate-sized lymphoplasmacytic cells within the lamina propria that were positive for B-cell markers CD19, CD20, and CD22 (on immunoperoxidase studies) with kappa light chain restriction (with in situ hybridization) confirming the diagnosis of WM. This extramedullary manifestation of WM held great importance because it indicated disease progression in the context of his current management with ibrutinib. He underwent a bone marrow biopsy that demonstrated a previously undetected Bruton tyrosine kinase mutation. He was enrolled in a Phase 1 trial of dasatinib, a tyrosine kinase inhibitor. There was almost complete resolution of the palatal mass after 6-month systemic treatment with dasatinib. However, despite noted improvement in oral WM, the patient died 7 months later. <h3>Conclusions</h3> Although rare, oral health care providers should be cognizant of potential oral manifestation of WM and should communicate the findings to the oncology team because it can be of vital importance in the overall treatment plan. As in this case, the diagnosis of oral WM signified the progression of underlying malignancy.

Bacillus Calmette-Guerin (BCG) induces superior anti-tumour responses by Vδ2+ T cells compared with the aminobisphosphonate drug zoledronic acid.

Vδ2+ T cells can recognize malignantly transformed cells as well as those infected with mycobacteria. This cross-reactivity supports the idea of using mycobacteria to manipulate Vδ2+ T cells in cancer immunotherapy. To date, therapeutic interventions using Vδ2+ T cells in cancer have involved expanding these cells in or ex vivo using zoledronic acid (ZA). Here, we show that the mycobacterium Bacillus Calmette–Guérin (BCG) also causes Vδ2+ T-cell expansion in vitro and that resulting Vδ2+ cell populations are cytotoxic toward tumour cell lines. We show that both ZA and BCG-expanded Vδ2+ cells effectively killed both Daudi and THP-1 cells. THP-1 cell killing by both ZA and BCG-expanded Vδ2+ cells was enhanced by treatment of targets cells with ZA. Although no difference in cytotoxic activity between ZA- and BCG-expanded Vδ2+ cells was observed, BCG-expanded cells degranulated more and produced a more diverse range of cytokines upon tumour cell recognition compared to ZA-expanded cells. ZA-expanded Vδ2+ cells were shown to upregulate exhaustion marker CD57 to a greater extent than BCG-expanded Vδ2+ cells. Furthermore, ZA expansion was associated with upregulation of inhibitory markers PD-1 and TIM3 in a dose-dependent manner whereas PD-1 expression was not increased following expansion using BCG. Intradermal BCG vaccination of rhesus macaques caused in vivo expansion of Vδ2+ cells. In combination with the aforementioned in vitro data, this finding suggests that BCG treatment could induce expansion of Vδ2+ T cells with enhanced anti-tumour potential compared to ZA treatment and that either ZA or BCG could be used intratumourally as a means to potentiate stronger anti-tumour Vδ2+ T-cell responses.