Increased Expression Of HLA-DR Research Articles

Flow Cytometry as the Tool to Define Peripheral Blood Leukocyte Signatures in Acute EBV Infection.

Primary Epstein-Barr virus (EBV) infection which can manifest as infectious mononucleosis (IM) is commonly acquired during childhood. EBV primarily invades B cells leading to a lytic reaction; the control of the infection is handled by natural killer and T cells in immunocompetent individuals. The infection has a wide spectrum of clinical findings and can lead to serious complications in patients with certain underlying immunological dysfunctions. We retrospectively investigated peripheral white blood cell populations' surface marker characteristics in IM using a comprehensive flow cytometry marker panel. Twenty-one cases of IM and seventeen EBV-seropositive cases without IM serving as controls were included. We observed novel alterations in lymphocyte, neutrophil, and monocyte populations. In addition to increased activated cytotoxic T cells and low B cells, we demonstrated high T-large granular lymphocyte (T-LGL) populations in IM cases. Furthermore, despite T cells' increased HLA-DR expression, another activation marker, CD11b, was lower in T-LGL populations. Monocytes showed increased CD16 expression; CD64 was higher in neutrophils. Our findings point to monocyte and neutrophil activation which may account for acute clinical features and may contribute to the understanding of IM immunobiology. Furthermore, they may serve as a useful tool in investigating inherited and post-transplant conditions characterized by deficiencies in controlling EBV infection.

Inflammatory pathways confer resistance to chemoradiotherapy in anal squamous cell carcinoma

Anal squamous cell carcinoma (ASCC) is associated with immunosuppression and infection with human papillomavirus (HPV). Response to standard chemoradiotherapy (CRT) varies considerably. A comprehensive molecular characterization of CRT resistance is lacking, and little is known about the interplay between tumor immune contexture, host immunity, and immunosuppressive and/or immune activating effects of CRT. Patients with localized ASCC, treated with CRT at three different sites of the German Cancer Consortium (DKTK) were included. Patient cohorts for molecular analysis included baseline formalin fixed paraffin embedded biopsies for immunohistochemistry (n = 130), baseline RNA sequencing (n = 98), peripheral blood immune profiling (n = 47), and serum cytokine measurement (n = 35). Gene set enrichment analysis showed that pathways for IFNγ, IFNα, inflammatory response, TNFα signaling via NF-κB, and EMT were significantly enriched in poor responders (all p < 0.001). Expression of interferon-induced transmembrane protein 1 (IFITM1), both on mRNA and protein levels, was associated with reduced Freedom from locoregional failure (FFLF, p = 0.037) and freedom from distant metastasis (FFDM, p = 0.014). An increase of PD-L1 expression on CD4+ T-cells (p < 0.001) and an increase in HLA-DR expression on T-cells (p < 0.001) was observed in the peripheral blood after CRT. Elevated levels of regulatory T-cells and CXCL2 were associated with reduced FFLF (p = 0.0044 and p = 0.004, respectively). Inflammatory pathways in tissue in line with elevated levels of regulatory T-cells and CXCL2 in peripheral blood are associated with resistance to CRT. To counteract this resistance mechanism, the RADIANCE randomized phase-2 trial currently tests the addition of the immune checkpoint inhibitor durvalumab to standard CRT in locally advanced ASCC.

Proinflammatory role of monocytes in SARS-CoV-2 infection in chronic hemodialysis patients.

Fully mature monocytes that express CD14, but not CD16, undergo phagocytosis within tissues, whereas non-classical monocytes, CD14-low CD16+, represent <11% of peripheral monocytes and have primary pro-inflammatory functions. Inflammation plays a major role in Covid-19 disease and adds to the inflammation caused by chronic hemodialysis. The aim of our study was to monitor monocyte subsets in five patients with end-stage kidney disease (ESKD) over a 1-year period after a mild Covid-19 infection. Five ESKD patients with a mild Covid-19 infection were monitored using CD14, CD16, CD300e, HLA-DR, CD64, and CD45 panels using a BD FACS Canto flow cytometer. CD14-low CD16+ was dramatically (p=0,001) decreased in patients during Covid-19 infection, as previously described for patients without chronic renal failure. In addition, CD14-low CD16+ monocytes remained decreased for 10 months after recovery from Covid. Intermediate monocytes increased during Covid-19 infection and decreased 10 months after infection but this subtype of monocytes retained their inflammatory activity with a significant increase in HLA-DR expression after recovery from Covid infection. Our study shows that ESKD patients had a pro-inflammatory profile induced by Covid 19, but this status was prolonged significantly over a 10-month period. Thus, advanced renal failure treated by hemodialysis did not dramatically change the inflammatory response against to SARS Covid 2. It seems that monocytes retain their inflammatory status for many months in ESKD patients after a Covid-19 infection.

Role of complement activation in cellular activation by systemic lupus erythematosus (SLE) immune complexes (ICs)

Abstract Complement (C’) activation is a prominent feature of SLE ICs, but the mechanism by which C’ mediates cellular activation by ICs is not well understood. To investigate this question, we formed SLE ICs in situ in healthy donor (HD) whole blood (WB) by adding SLE plasma (n=1–3;10% v/v) or HD plasma (as a control) along with apoptotic bodies (generated from RAW 264.7 cells) or RNP-Sm antigen (Arotec Diagnostics, New Zealand) with and without C’ inhibitors (CI). For cellular binding studies, WB was incubated for 45 minutes at 37°C and deposition of C3c and human IgG (ICs) on blood cells was examined by flow cytometry. In these studies, we noted that C’ inhibition significantly reduced deposition of C3c and ICs on B cells {% reduction: C3c (97%) and IgG (97%)}, monocytes {% reduction: C3c (40%) and IgG (49%)} and neutrophils {% reduction: C3c (69%) and IgG (70%)}. To investigate cellular activation, WB was incubated for 22 hours and cellular activation was measured by HLA-DR expression on monocytes by flow cytometry and cytokine release (IL-8, IL-6 and TNF) by ELISA. Incubation of WB with SLE ICs, but not ICs made with HD plasma (control ICs), resulted in increased HLA-DR expression on monocytes (MFI control ICs: 1609 V SLE ICs: 11096), which was susceptible to C’ inhibition (MFI SLE ICs with CI: 4975). Similarly, inflammatory cytokine release by SLE ICs was also significantly inhibited with C’ inhibition {% inhibition: IL-8 (75 %), IL-6 (99%) and TNF (99 %)}. Taken together, our results show significant C’-dependent effects of SLE ICs on cellular binding and activation. Additional studies are underway to investigate the prevalence of C’ activating ICs in SLE patient and correlate their presence with clinical disease. Wendell F. Rosse Complement Research Award, Duke University Medical Center (SK) and Duke Scholars Award, Duke University Medical Center (GMA)

Pilot Trial of IFN-γ and Donor Lymphocyte Infusion to Treat Relapsed AML and MDS after Allogeneic Hematopoietic Stem Cell Transplantation

Background: Allogeneic stem cell transplantation (alloSCT) can cure high-risk myeloid malignancies. While non-relapse mortality has declined over the last several decades, more than half of alloSCT recipients die from recurrent leukemia, and little progress has been made in preventing or treating it. Only 10-20% of subjects with relapsed leukemia post-alloSCT become long-term survivors despite receiving donor lymphocyte infusions (DLI) or a second alloSCT. In mouse models, we identified that optimal CD4- and CD8- mediated graft-versus-leukemia (GVL) responses require interferon-gamma receptor (IFN-γR) expression on myeloblastic leukemias (Matte-Martone and Shlomchik, J Clin Invest. 2017). Other groups reported that IFN-γ restores HLA expression on myeloid blasts from patients who relapsed post-alloSCT. These data support the hypothesis that IFN-γ sensitizes myeloid leukemia cells to alloreactive T cell-mediated killing. We conducted a phase I trial to evaluate whether a standard dose of IFN-γ can safely be administered to patients with relapsed acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) post-alloSCT (NCT04628338). Methods: This investigator-initiated trial aimed to evaluate 1) the feasibility and safety of IFN-γ post alloSCT as monotherapy and combined with DLI; and 2) whether the standard dose IFN-γ is sufficient to upregulate IFN-γ-inducible molecules in myeloblasts in vivo. Major eligibility criteria included: 1) HLA-matched alloSCT recipient with relapsed AML/MDS; 2) measurable bone marrow (BM) myeloblasts (0.5-30%) by flow cytometry, 3) no increases in systemic immunosuppression for 4 weeks; and 4) no history of grade IV graft-versus-host disease (GVHD). The study had two stages: IFN-γ monotherapy (stage 1) with a fixed dose of IFN-γ (ACTIMMUNE®, Horizon Therapeutics) 100 mcg subcutaneously 3 times per week (outpatient self-injection) for 4 weeks and subsequent DLI combined with IFN-γ (stage 2) for 8 more weeks. Clinical responses were evaluated by BM pathology and donor chimerism at 4 weeks (pre-DLI), 12 weeks, and 6 months after the first dose of IFN-γ. BM, peripheral blood mononuclear cells, and plasma were collected before, 48-60 hours after IFN-γ, pre-DLI, and post-DLI. Phosphorylated STAT1 and HLA-ABC/DR, ICAM-1, and PDL-1 expression on BM leukemia blasts were measured by flow cytometry. Results: Four patients with post-transplant relapsed AML/MDS were enrolled (Table 1). All patients tolerated IFN-γ monotherapy well with anticipated injection site reactions, flu-like symptoms, and stable cytopenia. Three patients who received DLI achieved complete molecular and cytogenetics remissions and conversion to full donor chimerism coincident with steroid-sensitive GVHD. One of the three responders with donor T cell chimerism of >90% pre-treatment achieved a CR with IFN-γ monotherapy before DLI (Figure 1). All responders are currently alive and in complete remission without requiring additional anti-leukemia maintenance therapy (median follow-up 283 days; range 164-371). One patient who did not have available DLI and had poor donor T cell chimerism (8%) died from disease progression on day 139. BM blasts from pre-treatment phosphorylated pSTAT1 in response to in-vitro IFN-γ, demonstrating intact upstream IFN-γ R signaling. Blasts from all four patients increased HLA-DR expression 48-60 hours after IFN-γ therapy in vivo. We are performing single-cell RNAseq of BM cells to better understand IFN-γ -induced transcriptome changes across different maturation stages of leukemia blasts and donor-derived normal hematopoietic cells. Conclusion: IFN-g was safe in this pilot cohort of subjects with relapsed AML/MDS post-alloSCT. To our knowledge, this is the first time that IFN-γ has been tested on such subjects. In vivo, IFN-γ induced the predicted changes in myeloblasts. Most importantly, we observed durable CRs and conversion to 100% donor hematopoiesis with the IFN-γ/DLI combination, which is very promising for such poor-risk patients. These data provide a strong rationale for developing a phase II trial of IFN-γ/DLI for post-alloSCT relapse, and we are currently planning such a study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterization of peripheral T helper 17 (Th17) cells phenotype in postmenopausal women with estrogen insufficiency

Over the past few years, Th17 cells is considered a key player in osteoporosis pathogenesis. Although extensively studied in murine models, comprehensive Th17 cell characterization in osteoporotic women is elusive. We thus aimed to examine peripheral Th17 cells frequency and phenotypes in healthy and osteoporotic women. Our results demonstrated that Th17 cells were primarily CD4+CD45RA−CCR7−HALDR+CCR6lowT-cells. Compared to Pre-N, Post-L showed increased proportion of Th17 with concomitant decrease in Th1 cells. The Th17 cells frequency in effector memory CD4+ T cells was significantly elevated in Post-N with a decrease of Th1 cells in effector memory subsets compared to Pre-N and Post-L. Both Post-N and Post-L had decreased frequency of dual positive Th1-Th17 cells and increased HLA-DR expression on Th17 cells compared to Pre-N. Thus, our study demonstrates increased Th17 cells frequency and reduced Th1 cells frequency with effector memory phenotype in postmenopausal women with estrogen insufficiency and correlates with aging process.

Hyperoxia/Hypoxia Exposure Primes a Sustained Pro-Inflammatory Profile of Preterm Infant Macrophages Upon LPS Stimulation.

Preterm infants are highly susceptible to sustained lung inflammation, which may be triggered by exposure to multiple environmental cues such as supplemental oxygen (O2) and infections. We hypothesized that dysregulated macrophage (MФ) activation is a key feature leading to inflammation-mediated development of bronchopulmonary dysplasia (BPD) in preterm infants. Therefore, we aimed to determine age-dependent differences in immune responses of monocyte-derived MФ comparing cord blood samples derived from preterm (n=14) and term (n=19) infants as well as peripheral blood samples from healthy adults (n=17) after lipopolysaccharide (LPS) exposure. Compared to term and adult MФ, LPS-stimulated preterm MФ showed an enhanced and sustained pro-inflammatory immune response determined by transcriptome analysis, cytokine release inducing a RORC upregulation due to T cell polarization of neonatal T cells, and TLR4 surface expression. In addition, a double-hit model was developed to study pulmonary relevant exposure factors by priming MФ with hyperoxia (O2 = 65%) or hypoxia (O2 = 3%) followed by lipopolysaccharide (LPS, 100ng/ml). When primed by 65% O2, subsequent LPS stimulation in preterm MФ led to an exaggerated pro-inflammatory response (e.g. increased HLA-DR expression and cytokine release) compared to LPS stimulation alone. Both, exposure to 65% or 3% O2 together with subsequent LPS stimulation, resulted in an exaggerated pro-inflammatory response of preterm MФ determined by transcriptome analysis. Downregulation of two major transcriptional factors, early growth response gene (Egr)-2 and growth factor independence 1 (Gfi1), were identified to play a role in the exaggerated pro-inflammatory response of preterm MФ to LPS insult after priming with 65% or 3% O2. Preterm MФ responses to LPS and hyperoxia/hypoxia suggest their involvement in excessive inflammation due to age-dependent differences, potentially mediated by downregulation of Egr2 and Gfi1 in the developing lung.

Abstract CT141: A phase 1 trial of RTX-240, an allogeneic engineered red blood cell with cell -surface expression of 4-1BBL and trans-presented IL-15, in patients with advanced solid tumors

Abstract Short title: RTX-240 phase 1 in solid tumorsA phase 1 trial of RTX-240, an allogeneic engineered red blood cell with cell -surface expression of 4-1BBL and trans-presented IL-15, in patients with advanced solid tumorsBackground: T cell checkpoint inhibition has revolutionized the treatment of cancer, however the key challenge in cancer immunotherapy is the development of resistant disease. Immune agonists and cytokines are promising approaches, but have shown limited success in the clinic. RTX-240 is an allogeneic cellular therapy genetically engineered to express high-copy numbers of trimeric 4-1BBL and IL-15/IL-15R fusion proteins on the cell surface. RTX-240 is designed to activate and expand CD8+ memory T cells and NK cells, and is restricted to the normal biodistribution of red blood cells to mitigate toxicity. Safety, pharmacodynamic (PD) effects, pharmacokinetics and preliminary efficacy of RTX-240 were assessed in a phase 1 study in patients (pts) with solid tumors. Methods: Pts with relapsed/refractory solid tumors not eligible for standard therapy were treated in dose escalating cohorts with RTX-240 Q4 or Q6W until disease progression or unacceptable toxicity. An exploratory cohort of IV and intratumoral (IT) dosing was enrolled (QW IV and IT x3 in cycle 1 and Q4W IV in subsequent cycles). Results: As of 11 Dec 2020, 14 pts (median age 55) were treated across 4 dose levels (1x108 to 1x1010 cells) administered IV or IV/IT. Pts had received a median of 3.5 therapies (range, 1-10); 10 pts had received prior PD-1/PD-L1 inhibitor therapy. Common tumor types include colorectal or other GI cancers (n=5) and melanoma (n=5). No patients experienced DLTs and no related grade (gr) &amp;gt;3 AE were observed. The most common related AE (gr 1-2) were fatigue (4 pts); chills, decreased appetite, arthralgia (3 pts each); and fever, myalgia, dysgeusia, nausea and hyperhidrosis (2 pts each). Additional irAE include gr 2 pneumonitis (n=1) and gr 1 elevated ALT/AST (n=1); the majority of irAE were observed in cycle 2 and beyond. RTX-240 was detected at the end of infusion sample in a dose dependent manner. Five pts (Q4W IV dosing) were evaluable for response by RECIST v1.1. A confirmed partial response (PR) was observed in 1 pt with anal cancer. Disease control, including stable disease or PR, occurred in 4/5 pts. PD studies in peripheral blood from all pts indicated increased numbers of NK cells in 12/14 pts (change from baseline, range 1.1-3 fold) and memory CD8+ T cells in 10/14 pts (change from baseline, range 1.2-3.3 fold). Activation of both NK and memory CD8+ T cells was observed by increased HLA-DR expression in 11/14 and 14/14 pts, respectively. Optional on-treatment biopsies are collected and preliminary data in one pt suggests infiltration of activated NK and T cells into the tumor microenvironment following dosing with RTX-240. Conclusions: RTX-240 is tolerable and leads to activation, expansion and trafficking of memory CD8+ T cells and NK cells, with preliminary evidence of anti-tumor activity. Exploration of the dose and schedule are ongoing in this study (NCT04372706). Citation Format: Omid Hamid, Jason Luke, Alexander Spira, Geoffrey M. Kuesters, Iga Sienczylo, Gilad Gordon, Melissa L. Johnson. A phase 1 trial of RTX-240, an allogeneic engineered red blood cell with cell -surface expression of 4-1BBL and trans-presented IL-15, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT141.

Transformation of the T-lymphocyte profile in peripheral blood upon resection of tuberculous lung granuloma

It is known that subpopulations of peripheral blood T-lymphocytes in patients with tuberculous lung granuloma may be used for determining patient's condition and monitoring pathological process. The aim of this study was to monitor the number and functional state of peripheral blood T-lymphocytes upon resection of tuberculous granuloma of the lungs. Twenty-two patients with tuberculous lung granuloma were examined, including 12 men (54.5%) and 10 women (45.5%). The patients with immune-dependent diseases and HIV were not eligible for the study. All these patients underwent resection of the granuloma by minimally invasive method. The blood samples were examined twice: before and 5-7 days after surgery, i.e., total T-cell numbers, (CD45+CD3+), their subpopulations were also determined (CD3+CD4+, CD3+CD8+, CD3+CD4-CD8-, CD3+CD4+CD8+, CD3brightCD4-, CD3+CD16+CD56+, CD3+HLA-DR+, CD3+CD25+, CD3+CD25+HLA-DR+), as well as CD3+CD25+/CD3+HLA-DR+ ratio, performed by means of Coulter Epicx XL instrument (Beckman Coulter, USA). In addition, the changes in B- and NK-cell numbers were studied. Due to small size of the groups, distribution was considered abnormal, and the Wilcoxon Matched-Pair Test was used. The differences were considered significant at pw 0.05. The statistical programs Statistica v. 12.5.192.7 (StatSoft, USA) and GraphPad Prism v. 8.0.2 (GraphPad Software, USA) were used in the work. Resection of tuberculous granuloma of the lungs was followed by redistribution of T-, B-, and NK-cell numbers, as compared with preoperative level. The B-cell population increased by 35.8%, whereas the number of NK-cells decreased by 18.9% (pw 0.05). Despite such significant changes in two other populations, total number of T-lymphocytes decreased only slightly: the changes ranged within 3.2% for absolute counts, and 3.8% (pw 0.05) for relative values. The changes of T-cell subpopulation profile were different. During the postsurgical period, a 4.8% decrease in the CD3+CD4+-cell number was observed, whereas reduction in CD3+CD8+ subpopulation was more significant, and amounted to 26.2% (pw 0.05). The number of doublepositive cells increased by 13.6% (pw 0.05); γσT-cell counts decreased by 20.8% (pw 0.05), and absolute number of TNK-cells increased by 32.4%. A decreased number of γσT-cells in early postoperative period could be considered a favorable criterion for assessing the patient's condition, since some previous studies provide information about increased number of these cells in patients with tuberculous granuloma. Increased HLA-DR expression on T-lymphocytes may be another favorable factor, since the patients with tuberculous granuloma are known to have lesser amounts of such cells in peripheral blood than healthy persons. In general, a search for expression of activation molecules showed that there is a decrease of T-cells expressing CD25+, and an increased number of CD3+HLA-DR+-cells. The T-lymphocyte population co-expressing both CD25+ and HLA-DR+ shifts to the normal ranges after the surgery. The obtained data are supposed for monitoring results of treatment in the patients.

Mesothelin-Specific CAR T Cells Target Ovarian Cancer.

New therapeutic options for patients with ovarian cancer are urgently needed. Therefore, we evaluated the efficacy of two second-generation mesothelin (MSLN)-directed CAR T cells in orthotopic mouse models of ovarian cancer. Treatment with CAR T cells expressing an MSLN CAR construct including the CD28 domain (M28z) significantly prolonged survival, but no persistent tumor control was observed. Despite lower response rates, MSLN-4-1BB (MBBz) CAR T cells induced long-term remission in some SKOV3-bearing mice. Tumor-infiltrating M28z and MBBz CAR T cells upregulated PD-1 and LAG3 in an antigen-dependent manner while MSLN+ tumor cells expressed the corresponding ligands (PD-L1 and HLA-DR), demonstrating that coinhibitory pathways impede CAR T-cell persistence in the ovarian tumor microenvironment. Furthermore, profiling plasma soluble factors identified a cluster of M28z- and MBBz-treated mice characterized by elevated T-cell secreted factors that had increased survival, higher CD8+ T-cell tumor infiltration, less exhausted CAR T-cell phenotypes, and increased HLA-DR expression by tumor cells. Altogether, our study demonstrates the therapeutic potential of MSLN-CAR T cells to treat ovarian cancer. SIGNIFICANCE: These findings demonstrate that MSLN-directed CAR T cells can provide antitumor immunity against ovarian cancer.

Changes in the expression of HLA-DR on lymphocyte subpopulations of spouses having children with sporadic congenital heart defects without chromosomal diseases, under the influence of female’s autoserum

This study is aimed to investigate the effect of female autoserum on the HLA-DR expression in various subpopulations of lymphocytes obtained from spouses with children with sporadic congenital heart defects without chromosomal diseases. 78 married couples with children with congenital heart disease were included in the study group. The control group was formed from 35 married couples with healthy children. The immune response in a mixed culture of lymphocytes of spouses was evaluated by an increased HLA-DR expression in a mixed culture in relation to spontaneous cultures of lymphocytes. Primary staining of female and male lymphocytes by monoclonal antibodies to CD45 conjugated with various fluorescent dyes (PC-5 and PC-7) was performed to assess the immune response of female lymphocytes to male ones and vice versa. The activating effect of female autoserum on all subpopulations of female lymphocytes simultaneously occurred significantly less frequently in the study group compared to the control. The control group was characterized by the domination of the positive effect of female autoserum on HLA-DR expression for all subpopulations of female lymphocyte. For all female lymphocytes having HLA-DR molecule on its membrane, the blocking effect of female autoserum in the study group was significantly more expressed in relation to the control group. Thus, the effect of female autoserum is manifested in relation to the HLA-DR expression on its own lymphocytes, but not on the lymphocytes of the spouse.

DNAM1 and TIGIT balance the T cell response, with low T cell TIGIT expression corresponding to inflammation in psoriatic disease.

SummaryObjectivesSignals at the contact site of antigen-presenting cells (APCs) and T cells help orchestrate the adaptive immune response. CD155 on APCs can interact with the stimulatory receptor DNAM1 or inhibitory receptor TIGIT on T cells. The CD155/DNAM1/TIGIT axis is under extensive investigation as immunotherapy target in inflammatory diseases including cancer, chronic infection and autoimmune diseases. We investigated a possible role for CD155/DNAM1/TIGIT signaling in psoriatic disease.MethodsBy flow cytometry, we analyzed peripheral blood mononuclear cells of patients with psoriasis (n = 20) or psoriatic arthritis (n = 21), and healthy individuals (n = 7). We measured CD155, TIGIT, and DNAM1 expression on leukocyte subsets and compared activation-induced cytokine production between CD155-positive and CD155-negative APCs. We assessed the effects of TIGIT and DNAM1 blockade on T cell activation, and related the expression of CD155/DNAM1/TIGIT axis molecules to measures of disease activity.ResultsHigh CD155 expression associates with tumor necrosis factor (TNF) production in myeloid and plasmacytoid dendritic cells (DC). In CD1c+ myeloid DC, activation-induced CD155 expression associates with increased HLA-DR expression. CD8 T cells – but not CD4 T cells – express high levels of TIGIT. DNAM1 blockade decreases T cell pro-inflammatory cytokine production, while TIGIT blockade increased T cell proliferation. Finally, T cell TIGIT expression shows an inverse correlation with inflammation biomarkers in psoriatic disease.ConclusionCD155 is increased on pro-inflammatory APCs, while the receptors DNAM1 and TIGIT expressed on T cells balance the inflammatory response by T cells. In psoriatic disease, low TIGIT expression on T cells is associated with systemic inflammation.

Alterations in peripheral blood monocyte and dendritic cell subset homeostasis in relapsing-remitting multiple sclerosis patients

Antigen-presenting cells participate and are implicated in the pathogenesis of multiple sclerosis. In our study we assessed the frequency of plasmacytoid (pDC) and myeloid (mDC) dendritic cells and the classical, intermediate and non-classical monocytes subsets, as well as their phenotypic and functional profile. We evaluated peripheral blood from relapsing-remitting patients treated with IFN-β in remission and relapse phases and from healthy subjects. In remission, we observed a decrease of mDC/pDC ratio and a return to normal values in relapse. In both phases the frequency of non-classical monocytes decreases. Concerning the phenotypic characterization, an increased HLA-DR expression was observed in remission and a decrease in relapse, revealing alterations in monocytes and dendritic cells homeostasis.

CD56dim CD57- NKG2C+ NK cells retaining proliferative potential are possible precursors of CD57+ NKG2C+ memory-like NK cells.

Formation of the adaptive-like NK cell subset in response to HCMV infection is associated with epigenetic rearrangements, accompanied by multiple changes in the protein expression. This includes a decrease in the expression level of the adapter chain FcεRIγ, NKp30, and NKG2A receptors and an increase in the expression of NKG2C receptor, some KIR family receptors, and co-stimulating molecule CD2. Besides, adaptive-like NK cells are characterized by surface expression of CD57, a marker of highly differentiated cells. Here, it is shown that CD57-negative CD56dim NKG2C+ NK cells may undergo the same changes, as established by the similarity of the phenotypic expression pattern with that of the adaptive-like CD57+ NKG2C+ NK cells. Regardless of their differentiation stage, NKG2C-positive NK cells had increased HLA-DR expression indicating an activated state, both ex vivo and after cultivation in stimulating conditions. Additionally, CD57- NKG2C+ NK cells exhibited better proliferative activity compared to CD57+ NKG2C+ and NKG2C- NK cells, while retaining high level of natural cytotoxicity. Thus, CD57- NKG2C+ NK cells may represent a less differentiated, but readily expanding stage of the adaptive-like CD57+ NKG2C+ NK cells. Moreover, it is shown that NK cells have certain phenotypic plasticity and may both lose NKG2C expression and acquire it de novo during proliferation, induced by IL-2 and K562-mbIL21 feeder cells.

Antibiotics Differentially Modulate Lipoteichoic Acid-Mediated Host Immune Response

In Staphylococcus aureus bacteremia, our group has shown that a dysregulated balance of pro- and anti-inflammatory cytokine response biased towards an immunoparalysis phenotype is predictive of persistence and mortality, despite receipt of antibiotics. Certain antibiotics, as well as lipoteichoic acid (LTA) released from S. aureus, can modulate immune response ex vivo. Here, we evaluated the effects of three anti-staphylococcal antibiotics (vancomycin, tedizolid, and daptomycin) on the expression of cytokines and cell surface markers of immune activation (TNFα, HLA-DR) and immunoparalysis (IL-10, PD-L1) in human peripheral blood mononuclear cells (PBMC) exposed to high (10 μg) and low (1 μg) doses of LTA. Results suggested a dose-dependent relationship between LTA and induction of anti- and pro-inflammatory immune responses. Differential antibiotic effects were prominently observed at high but not low LTA condition. Vancomycin significantly induced IL-10 and TNFα expression, whereas daptomycin had no effects on cytokine response or expression of cell surface receptors. Tedizolid increased TNFα and modestly increased HLA-DR expression, suggesting a stimulatory effect. These findings suggest that anti-staphylococcal agents differentially alter LTA-mediated immune cell activation status and cytokine response, providing support for future clinical studies to better elucidate the complexities of host–microbial–antibiotic interaction that can help direct precision therapy for S. aureus bacteremia.

Interferon-gamma increases monocyte PD-L1 but does not diminish T-cell activation

Immune dysfunction can occur during sepsis or following major trauma. Decreased monocyte HLA-DR expression and cytokine responses are associated with mortality. Recent studies have shown that adaptive immune system defects can also occur in such patients, characterised by increased PD-L1 expression and associated T-cell anergy. The aim of this study was to determine the effects of an immune adjuvant, interferon-gamma, on monocyte PD-L1 expression and T-cell activation in an ex-vivo human whole blood model of infection. We found that with interferon-gamma treatment, monocytes had increased HLA-DR expression and augmented TNF-α production in response to LPS stimulation, with a decrease in IL-10 levels. Both LPS and interferon-gamma increased the level of monocyte PD-L1 expression, and that a combination of both agents synergistically stimulated a further increase in PD-L1 levels as measured by flow cytometry. However, despite elevated PD-L1 expression, both CD4 and CD8 T-cell activation was not diminished by the addition of interferon-gamma treatment. These findings suggest that PD-L1 may not be a reliable marker for T-cell anergy, and that interferon-gamma remains an adjuvant of interest that can improve the monocyte inflammatory response while preserving T-cell activation.

Age-related features of the pattern of lymphocyte subpopulations and functional activity of peripheral blood mononuclear cells in patients with chronic kidney disease before and after transplantation

Objective : to analyze the features of the pattern of lymphocyte subpopulations and the functional activity of peripheral blood mononuclear cells in older adult patients with chronic kidney disease. Materials and methods . The study featured 21 patients with chronic kidney disease (CKD), over 55 years of age, who underwent kidney transplantation (KT) from unrelated suboptimal donors. The average age was 61.4 ± 4.5 years (55 to 69). Comorbidity was assessed using the CIRS-G scale; the average number of points was 13.6 ± 5.09. The control group consisted of 21 volunteers, aged 55–70, without acute inflammatory diseases and signs of chronic kidney disease (CKD). The average age was 61.1 ± 4.4 years, the average CIRS-G score was 12.11 ± 6.04. In all patients, the pattern of lymphocyte subpopulations of peripheral blood was evaluated by flow cytometry. Vital computer laser cytomorphometry was used to assess the functional state of peripheral blood mononuclear cells. The Functional Activities Index (FAI) was evaluated to indirectly assess the degree of functional activity of cells. Results . In CKD patients before KT, there was a decrease in the proportion of CD4 cells (p = 0.009), an increase in the proportion of CD8 cells (p = 0.02), a decrease in the CD4/CD8 ratio (p = 0.017), an increase in the proportion of natural killers (p = 0.025) compared with healthy volunteers. Moreover, a decrease in the total proportion of CD3 cells, an increase in HLA-DR expression on CD3 cells, and an increase in the proportion of B cells were statistically insignificant: p = 0.137, p = 0.072 and p = 0.135, respectively. On the fifth day after KT, the proportion of CD3 cells increased (p = 0.017) mainly due to an increase in the proportion of CD4 cells (p = 0.002) compared to the pre- KT index. The proportion of natural killers (p = 0.002) and HLA-DR expression on CD3 cells (p &lt; 0.0001) also increased. An increase in the proportion of CD8 cells and in the CD4/CD8 ratio, and a decrease in the proportion of B cells were statistically insignificant: p = 0.439, p = 0.277, and p = 0.236, respectively. A decrease in FAI was noted in patients with CKD before KT in comparison with healthy volunteers (p = 0.0138). After ATP, this indicator significantly increased compared to the pre-KT value (p &lt; 0.0001) and exceeded the FAI value in healthy volunteers (p &lt; 0.0001). In healthy volunteers, there was no significant correlation between the functional activity of peripheral blood mononuclear cells and age (r = –0.263 [95% CI –0.6236; 0.1907], p = 0.264, r2 = 0.069). At the same time, significant negative correlation between FAI and age was noted in CKD patients: r = –0.52 [95% CI –0.7771; –0.1135], p = 0.0157, r2 = 0.27 before KT; r = –0.418 [95% CI –0.7559; –0.06256], p = 0.0272, r2 = 0.175 after KT. Conclusion . Older adult CKD patients before and after KT were likely to have significant changes in the morphofunctional state of peripheral blood mononuclear cells and pattern of lymphocyte subpopulations. Moreover, the severity of changes in the functional state of these cells had a strong correlation with age, which was not observed in the group of healthy volunteers. This should be considered when choosing immunosuppressive therapy in older kidney transplant recipients.

Increased HLA-DR expression and cortical demyelination in MS links with HLA-DR15.

ObjectiveTo investigate molecular changes in multiple sclerosis (MS) normal-appearing cortical gray matter (NAGM).MethodsWe performed a whole-genome gene expression microarray analysis of human brain autopsy tissues from 64 MS NAGM samples and 42 control gray matter samples. We further examined our cases by HLA genotyping and performed immunohistochemical and immunofluorescent analysis of all human brain tissues.ResultsHLA-DRB1 is the transcript with highest expression in MS NAGM with a bimodal distribution among the examined cases. Genotyping revealed that every case with the MS-associated HLA-DR15 haplotype also shows high HLA-DRB1 expression and also of the tightly linked HLA-DRB5 allele. Quantitative immunohistochemical analysis confirmed the higher expression of HLA-DRB1 in HLA-DRB1*15:01 cases at the protein level. Analysis of gray matter lesion size revealed a significant increase of cortical lesion size in cases with high HLA-DRB1 expression.ConclusionsOur data indicate that increased HLA-DRB1 and -DRB5 expression in the brain of patients with MS may be an important factor in how the HLA-DR15 haplotype contributes to MS pathomechanisms in the target organ.

Expansion of Human Mesenchymal Stromal/Stem Cells Using Standardized Xeno-Free, Serum-Free Culture Condition

Aims: mesenchymal stromal/stem cells (MSCs) is one of the most promising cell therapies to treat immune related diseases such as GvHD, Crohn's disease, osteoarthritis as well as to support function of heart, liver, and central nervous system. Many advanced clinical trials has been taking place to investigate the safety and efficacy of MSC therapy, however, it remains challenging to compare the results of these trials due to variations in culture conditions and tissue origins. These variations pose the urgent need of a standardized large-scale production and quality control of these cells become more urgent, as the use of MSCs are spreading around the globe and disease backgrounds. Numerous culture protocols for MSCs are available using FBS-containing, human platelet lysate/plasma supplementing, or serum-free xeno-free condition, either commercial or self-prepared media. In this study, we have established a standardized expansion protocol that can be used for MSCs derived from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC). Methods: we tested commercial xeno-free, serum-free media (MesenCult™-ACF Plus Medium/Stem Cell Technologies, MSC NutriStem® XF Medium/BI, Stempro® MSC SFM Medium/ThermoFisher, PowerStem MSC1 Medium/PAN, and StemMACS™ MSC Expansion Media/Miltenyi) in order to identify a common culture condition for all of these MSC sources. The cells were analysed for their population doubling time from P2 to P6, expression of surface markers including the positive markers CD73, CD90, and CD105, and the negative markers CD34, CD45, CD11b, CD19, and HLA-DR. Karyotype was conducted at early and later passage (P3 and P6, respectively). MSCs were tested for their differentiation capacity into osteoblast/osteocyte, adipocyte, and chondrocyte lineages. Bacterial and fungal sterility and mycoplasma test were performed and endotoxin concentration was determined before the cells were transfused into patients. Results: using UC for the primary screening, we identified two medium candidates that supported the isolation and culture of UC-MSCs. MSCs derived from BM and AD are capable of expansion in these media as well. However, one candidate induced an increased HLA-DR expression so that it failed to fulfill the minimal criteria of MSCs (Dominici et al., 2006). The last one showed the best results for MSCs from all tested tissues. Under our established protocol, MSCs grew exponentially until P6, which was the highest analyzed passage. The cells expressed CD73, CD90, CD105 in more than 95% cells and showed less than 2% of negative markers (CD34, CD45, CD11b, CD19, and HLA-DR). Karyotype analysis confirmed no chromosomal aberrations of the cultured cells. The cells were able to differentiate into osteoblast/osteocyte, adipocyte, and chondrocyte lineages. We are now conducting phase 1 clinical trial to address the safety and feasibility of UC-MSCs in the treatment of bronchopulmonary dysplasia and chronic obstructive pulmonary disease. Conclusion: we have established a standardized protocol for the xeno-free, serum-free culture of MSCs from different sources. BM-, AD-, and UC-derived MSCs expand in large-scale and maintain their cellular characteristics, karyotype integrity, and can be applied for cell therapy. Disclosures No relevant conflicts of interest to declare.

Salt increases monocyte CCR2 expression and inflammatory responses in humans.

Inflammation may play a role in the link between high salt intake and its deleterious consequences. However, it is unknown whether salt can induce proinflammatory priming of monocytes and macrophages in humans. We investigated the effects of salt on monocytes and macrophages in vitro and in vivo by performing a randomized crossover trial in which 11 healthy human subjects adhered to a 2-week low-salt and high-salt diet. We demonstrate that salt increases monocyte expression of CCR2, a chemokine receptor that mediates monocyte infiltration in inflammatory diseases. In line with this, we show a salt-induced increase of plasma MCP-1, transendothelial migration of monocytes, and skin macrophage density after high-salt diet. Macrophages demonstrate signs of an increased proinflammatory phenotype after salt exposure, as represented by boosted LPS-induced cytokine secretion of IL-6, TNF, and IL-10 in vitro, and by increased HLA-DR expression and decreased CD206 expression on skin macrophages after high-salt diet. Taken together, our data open up the possibility for inflammatory monocyte and macrophage responses as potential contributors to the deleterious effects of high salt intake.