Key Immune Cell Subsets Are Dysregulated in Patients with Myelofibrosis

Abstract Introduction: Myelofibrosis (MF) is a clonal disorder associated mainly with JAK2V617F and MPL mutations. Recently, a new mutation in the gene encoding calreticulin (CALR) was discovered in the majority of JAK2/MPL negative patients. MF is burdened by a high rate of potentially life-threatening infections. The issue of recurrent and opportunistic infections is increased after the introduction in clinical practice of JAK inhibitors with immunosuppressive activity. However, the role of crucial immune cell subsets is still poorly characterized. Here, we investigated the phenotype/function of selected immune cells in MF. Specifically, we focused on circulating regulatory (Tregs) and IL-17-producing T cells (Th17 cells), monocytes and dendritic cells (DCs). Monocyte-derived DCs were also characterized. Methods: We characterized circulating Th17 cells, Tregs, monocytes and DCs of 17 untreated MF patients and 8 healthy controls (HC) by flow cytometry. Th17 cells were identified as CD4+ CD161+ CD196+ cells while Tregs were enumerated as CD4+ CD25high CD127low T cells. We also tested the in vitro suppressive activity of circulating CD4+ CD25+ Tregs with a mixed leukocyte reaction assay. Two subpopulations of circulating DCs, myeloid CD11c+ and plasmacytoid CD123+cells, were enumerated as well. In addition, after immunomagnetic selection, we tested both phenotype of circulating monocytes and their capacity to differentiate into CD14-derived immature and mature DCs, using a specific cytokines cocktail. JAK2V617F and MPL mutations were detected with RT-PCR while the presence of CALR mutations were tested with Exon 9 Next Generation Sequencing assay. Results: JAK2V617F (11 cases), MPL (3 cases), and CARL (3 cases) mutations were detected. We found that circulating CD4+CD25highCD127low Tregs were reduced in MF patients as compared with healthy controls (p=0.043), although their suppressive ability was maintained. We also found a lower number of circulating Th17 cells (p=0.0026) in MF patients. This finding was particularly evident in JAK2V617F+(p=0.008) and CARL+(p=0.03) patients. Despite their number was in the normal range, circulating monocytes from MF patients showed reduced expression of the CD86 co-stimulatory molecule. Moreover, as compared with the normal counterparts, immature monocytes-derived DCs from patients maintained low CD14 expression without upregulating the CD80 co-stimulatory molecule expression (p=0.0063). Interestingly, at variance with plasmacytoid DCs, a reduced number of circulating myeloid DCs was observed in MF patients as compared with that of HC (p=0.01). Conclusions: Here we demonstrated that specific crucial subsets of immune cells show quantitative and/or qualitative abnormalities in MF patients. These findings may be useful to better understand the increased susceptibility of these patients to infections, since Th17 cells play a role in bacterial and fungal infections while myeloid DCs regulate Th1 activity. Of note, DCs inhibition might result in increased propensity to infections and compromised immune response to cancer.In addition, since monocytes are DC precursors, alterations in their differentiation pathway may contribute to develop defective immune responses. Disclosures Martinelli: NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

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Generation of IL-8 and IL-9 Producing CD4+T Cells Is Affected by Th17 Polarizing Conditions and AHR Ligands

Mediators of Inflammation ◽

10.1155/2014/182549 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 13

Author(s):

Michaela Gasch ◽

Tina Goroll ◽

Mario Bauer ◽

Denise Hinz ◽

Nicole Schütze ◽

...

Keyword(s):

T Cells ◽

T Helper Cells ◽

Th17 Cells ◽

Immune Cell ◽

T Helper Cell ◽

Helper Cell ◽

T Helper ◽

Helper Cells ◽

Aryl Hydrocarbon

The T helper cell subsets Th1, Th2, Th17, and Treg play an important role in immune cell homeostasis, in host defense, and in immunological disorders. Recently, much attention has been paid to Th17 cells which seem to play an important role in the early phase of the adoptive immune response and autoimmune disease. When generating Th17 cells underin vitroconditions the amount of IL-17A producing cells hardly exceeds 20% while the nature of the remaining T cells is poorly characterized. As engagement of the aryl hydrocarbon receptor (AHR) has also been postulated to modulate the differentiation of T helper cells into Th17 cells with regard to the IL-17A expression we ask how far do Th17 polarizing conditions in combination with ligand induced AHR activation have an effect on the production of other T helper cell cytokines. We found that a high proportion of T helper cells cultured under Th17 polarizing conditions are IL-8 and IL-9 single producing cells and that AHR activation results in an upregulation of IL-8 and a downregulation of IL-9 production. Thus, we have identified IL-8 and IL-9 producing T helper cells which are subject to regulation by the engagement of the AHR.

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Immune cell composition in normal human kidneys

Scientific Reports ◽

10.1038/s41598-020-72821-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Jun-Gyu Park ◽

Myeongsu Na ◽

Min-Gang Kim ◽

Su Hwan Park ◽

Hack June Lee ◽

...

Keyword(s):

T Cells ◽

Immune Cells ◽

Immune Cell ◽

Kidney Diseases ◽

Myeloid Cells ◽

Human Kidney ◽

Effector Memory ◽

Immune Cell Subsets ◽

Immunological Mechanisms ◽

Normal Human

Abstract An understanding of immunological mechanisms in kidney diseases has advanced using mouse kidneys. However, the profiling of immune cell subsets in human kidneys remains undetermined, particularly compared with mouse kidneys. Normal human kidneys were obtained from radically nephrectomised patients with urogenital malignancy (n = 15). Subsequently, human kidney immune cell subsets were analysed using multicolor flow cytometry and compared with subsets from C57BL/6 or BALB/c mice under specific pathogen-free conditions. Twenty kidney sections from healthy kidney donors or subjects without specific renal lesions were additionally analysed by immunohistochemistry. In human kidneys, 47% ± 12% (maximum 63%) of immune cells were CD3+ T cells. Kidney CD4+ and CD8+ T cells comprised 44% and 56% of total T cells. Of these, 47% ± 15% of T cells displayed an effector memory phenotype (CCR7− CD45RA− CD69−), and 48% ± 19% were kidney-resident cells (CCR7− CD45RA− CD69+). However, the proportions of human CD14+ and CD16+ myeloid cells were approximately 10% of total immune cells. A predominance of CD3+ T cells and a low proportion of CD14+ or CD68+ myeloid cells were also identified in healthy human kidney sections. In mouse kidneys, kidney-resident macrophages (CD11blow F4/80high) were the most predominant subset (up to 50%) but the proportion of CD3+ T cells was less than 20%. These results will be of use in studies in which mouse results are translated into human cases under homeostatic conditions or with disease.

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Global immune characterization of HBV/HCV-related hepatocellular carcinoma identifies macrophage and T-cell subsets associated with disease progression

Cell Discovery ◽

10.1038/s41421-020-00214-5 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Guohe Song ◽

Yang Shi ◽

Meiying Zhang ◽

Shyamal Goswami ◽

Saifullah Afridi ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

T Cell ◽

Single Cell ◽

Immune Cells ◽

Immune Cell ◽

T Cell Subsets ◽

M2 Macrophage ◽

Advanced Hcc ◽

Immune Cell Subsets

AbstractDiverse immune cells in the tumor microenvironment form a complex ecosystem, but our knowledge of their heterogeneity and dynamics within hepatocellular carcinoma (HCC) still remains limited. To assess the plasticity and phenotypes of immune cells within HBV/HCV-related HCC microenvironment at single-cell level, we performed single-cell RNA sequencing on 41,698 immune cells from seven pairs of HBV/HCV-related HCC tumors and non-tumor liver tissues. We combined bio-informatic analyses, flow cytometry, and multiplex immunohistochemistry to assess the heterogeneity of different immune cell subsets in functional characteristics, transcriptional regulation, phenotypic switching, and interactions. We identified 29 immune cell subsets of myeloid cells, NK cells, and lymphocytes with unique transcriptomic profiles in HCC. A highly complex immunological network was shaped by diverse immune cell subsets that can transit among different states and mutually interact. Notably, we identified a subset of M2 macrophage with high expression of CCL18 and transcription factor CREM that was enriched in advanced HCC patients, and potentially participated in tumor progression. We also detected a new subset of activated CD8+ T cells highly expressing XCL1 that correlated with better patient survival rates. Meanwhile, distinct transcriptomic signatures, cytotoxic phenotypes, and evolution trajectory of effector CD8+ T cells from early-stage to advanced HCC were also identified. Our study provides insight into the immune microenvironment in HBV/HCV-related HCC and highlights novel macrophage and T-cell subsets that could be further exploited in future immunotherapy.

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Overexpansion of Th17 and Th1/17 Cells in Patients with Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v112.11.2698.2698 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2698-2698

Author(s):

Elena E. Solomou ◽

A. Tsanaktsi ◽

V. Fertakis ◽

K. Dallas ◽

S. Karambina ◽

...

Keyword(s):

T Cells ◽

Transcription Factor ◽

Cd4 T Cells ◽

Th17 Cells ◽

Mononuclear Cells ◽

Mapk Pathway ◽

Immunosuppressive Treatment ◽

Healthy Controls ◽

Orphan Nuclear Receptor

Abstract IL17-producing T cells have been recently described as a distinct T cell helper population (Th17 cells) characterized by expression of membrane CD4 and IL23R and intracellular expression of the orphan nuclear receptor RORgt. In Th17 cells the transcription factor RORgt induces the transcription of IL17 gene, whereas in Th1 cells the transcription factor Tbet is responsible for the transcription of IFNg gene. Th1 along with Th17 cells are thought to contribute to the pathogenesis of autoimmune diseases. In murine models Th17 cells are fully polarized. In humans a proportion of Th17 cells are also positive for interferon gamma (IFN-g); they are named Th1/17 cells and their function is yet unclear. In patients with colitis and seronegative arthritis Th17 cells are increased. The induction of Th17 and Th1/17 in patients with MDS has not been previously evaluated. To examine the expression of Th17 and Th1/17 cells in this disease, peripheral blood mononuclear cells (PBMC) from patients with MDS were cultured in vitro for 6 days in RPMI-1640, 15% FBS supplemented with PHA (0.1 μg/mL) and IL-2 (10 ng/mL). Percentages of CD4+IL23R+IL-17+ T cells (Th17) and CD4+IL23R+IL17+IFN-g+ T cells (Th1/17) in patients with MDS were determined by flow cytometry: Th17 cells were markedly increased in patients (n=30) compared to healthy controls (n=15), (17.5% ± 3.4 vs 2.5% ± 0.4, p=0.008). Th1/Th17 cells were also significantly increased in MDS patients compared to controls (15.17% ± 2.80 vs 2.56% ± 0.80, p=0.008). None of the patients had been on immunosuppressive treatment or transfused before sampling. In multi-transfused patients with no underlying hematologic disease examined (n=3) the Th17 and Th1/17 populations were comparable to those of healthy donors. In patients with MDS the majority of the Th17 cells expressed also IFNg (90.07% ± 2.87) whereas in healthy controls only 59.7% ± 5.5 of the Th17 cells were also positive for IFNg (p<0.0001). There were no differences between different subtypes of MDS (RA, RARS, and RAEB). Using confocal microscopy, purified CD4+ T cells from PBMC cultures from patients (n=5) showed increased Tbet and RORgt expression at the single-cell level compared to controls (n=3),(T-bet: 22.03 ± 1.20 vs 11.60 ± 0.35 arbitrary units respectively, p<0.0001 and RORãt: 28.90 ± 0.35 vs 21.03 ± 1.20 arbitrary units, p=0.0008. For each sample 100 cells were analyzed). We next asked whether kinases involved in the induction of Tbet are also involved in the induction of RORgt. We analyzed the effects of rottlerin, a PKC-theta inhibitor, SB203580, a p38 MAPK pathway inhibitor, and PD98059, an ERK pathway inhibitor, on Th17 and Th1/17 cell induction in patients (n=7) and controls (n=4). Rottlerin decreased the Th17 content in patients and controls by 45.0%, and the Th1/17 content by 64.8%. SB203580 showed a 17% and 18% decrease on Th17 and on Th1/17 content, respectively, in patients and controls. PD98059 showed no effect on Th17 and Th1/17 populations in patients and controls. By immunoblots, in normal CD4+T cells rottlerin decreased both T-bet and RORgt protein levels by 50% and 20%, respectively. SB203580, decreased RORgt levels by 25%, and PD98059 did not obviously decrease Tbet but decreased RORgt levels by 20%. CD4+IL23R+IL-17+ T cells and CD4+IL23R+IL17+IFN-g+ T cells are increased in most patients with MDS. T cells have recently been implicated in MDS pathogenesis. Although more studies are needed in order to define the role of Th17 and Th1/17 cells in the pathogenesis of MDS, our in vitro data with the kinase inhibitors may suggest a probable therapeutic target for patients with MDS.

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Authentic Patient-Derived Hepatitis C Virus Infects and Productively Replicates in Primary CD4+and CD8+T LymphocytesIn Vitro

Journal of Virology ◽

10.1128/jvi.01790-17 ◽

2017 ◽

Vol 92 (3) ◽

Cited By ~ 4

Author(s):

Georgia Skardasi ◽

Annie Y. Chen ◽

Tomasz I. Michalak

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Lymphocytes ◽

Immune Cells ◽

Immune Cell ◽

Cell Types ◽

Hcv Rna ◽

Biophysical Properties

ABSTRACTAccumulated evidence indicates that immune cells can support the replication of hepatitis C virus (HCV) in infected patients and in culture. However, there is a scarcity of data on the degree to which individual immune cell types support HCV propagation and on characteristics of virus assembly. We investigated the ability of authentic, patient-derived HCV to infectin vitrotwo closely related but functionally distinct immune cell types, CD4+and CD8+T lymphocytes, and assessed the properties of the virus produced by these cells. The HCV replication system in intermittently mitogen-stimulated T cells was adapted to infect primary human CD4+or CD8+T lymphocytes. HCV replicated in both cell types although at significantly higher levels in CD4+than in CD8+T cells. Thus, the HCV RNA replicative (negative) strand was detected in CD4+and CD8+cells at estimated mean levels ± standard errors of the means of 6.7 × 102± 3.8 × 102and 1.2 × 102± 0.8 × 102copies/μg RNA, respectively (P< 0.0001). Intracellular HCV NS5a and/or core proteins were identified in 0.9% of CD4+and in 1.2% of CD8+T cells. Double staining for NS5a and T cell type-specific markers confirmed that transcriptionally competent virus replicated in both cell types. Furthermore, an HCV-specific protease inhibitor, telaprevir, inhibited infection in both CD4+and CD8+cells. The emergence of unique HCV variants and the release of HCV RNA-reactive particles with biophysical properties different from those of virions in plasma inocula suggested that distinct viral particles were assembled, and therefore, they may contribute to the pool of circulating virus in infected patients.IMPORTANCEAlthough the liver is the main site of HCV replication, infection of the immune system is an intrinsic characteristic of this virus independent of whether infection is symptomatic or clinically silent. Many fundamental aspects of HCV lymphotropism remain uncertain, including the degree to which different immune cells support infection and contribute to virus diversity. We show that authentic, patient-derived HCV productively replicatesin vitroin two closely related but functionally distinct types of T lymphocytes, CD4+and CD8+cells. The display of viral proteins and unique variants, the production of virions with biophysical properties distinct from those in plasma serving as inocula, and inhibition of replication by an antiviral agent led us to ascertain that both T cell subtypes supported virus propagation. Infection of CD4+and CD8+T cells, which are central to adaptive antiviral immune responses, can directly affect HCV clearance, favor virus persistence, and decisively influence the development and progression of hepatitis C.

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Targeting the RhoA-ROCK pathway to reverse T-cell dysfunction in SLE

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2016-209850 ◽

2016 ◽

Vol 76 (4) ◽

pp. 740-747 ◽

Cited By ~ 33

Author(s):

Cristina Rozo ◽

Yurii Chinenov ◽

Reena Khianey Maharaj ◽

Sanjay Gupta ◽

Laura Leuenberger ◽

...

Keyword(s):

T Cells ◽

Lupus Erythematosus ◽

Th17 Cells ◽

Mononuclear Cells ◽

Activity Levels ◽

Healthy Controls ◽

Rock Inhibitor ◽

Peripheral Blood Mononuclear ◽

Rock Activity

ObjectivesDeregulated production of interleukin (IL)-17 and IL-21 contributes to the pathogenesis of autoimmune disorders such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Production of IL-17 and IL-21 can be regulated by ROCK2, one of the two Rho kinases. Increased ROCK activation was previously observed in an SLE cohort. Here, we evaluated ROCK activity in a new SLE cohort, and an RA cohort, and assessed the ability of distinct inhibitors of the ROCK pathway to suppress production of IL-17 and IL-21 by SLE T cells or human Th17 cells.MethodsROCK activity in peripheral blood mononuclear cells (PBMCs) from 29 patients with SLE, 31 patients with RA and 28 healthy controls was determined by ELISA. SLE T cells or in vitro-differentiated Th17 cells were treated with Y27632 (a pan-ROCK inhibitor), KD025 (a selective ROCK2 inhibitor) or simvastatin (which inhibits RhoA, a major ROCK activator). ROCK activity and IL-17 and IL-21 production were assessed. The transcriptional profile altered by ROCK inhibitors was evaluated by NanoString technology.ResultsROCK activity levels were significantly higher in patients with SLE and RA than healthy controls. Th17 cells exhibited high ROCK activity that was inhibited by Y27632, KD025 or simvastatin; each also decreased IL-17 and IL-21 production by purified SLE T cells or Th17 cells. Immune profiling revealed both overlapping and distinct effects of the different ROCK inhibitors.ConclusionsROCK activity is elevated in PBMCs from patients with SLE and RA. Production of IL-17 and IL-21 by SLE T cells or Th17 cells can furthermore be inhibited by targeting the RhoA-ROCK pathway via both non-selective and selective approaches.

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884 Engineered toxin body mediated depletion of TIGIT expressing immune cells for cancer immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.884 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A926-A926

Author(s):

Elizabeth Saputra ◽

Garrett Cornelison ◽

Jennifer Mitchell ◽

Karia Williams ◽

Andrea Mendiola ◽

...

Keyword(s):

T Cells ◽

Immune Cells ◽

Immune Cell ◽

Single Agent ◽

Antibody Fragment ◽

Cell Surface Receptor ◽

Mouse Tumor ◽

List Type ◽

Proof Of Concept

BackgroundTIGIT (T cell immunoreceptor with Ig and ITIM domains) is an exciting novel target for immuno-oncology which functions as an immune checkpoint on multiple immune cell types including memory CD8+, CD4+ Treg, and memory CD4+ cells. TIGIT upregulation on tumor infiltrating lymphocytes (TILs) has been observed in multiple cancer types and contributes to an immunosuppressive tumor microenvironment (TME). Interestingly, TIGIT is commonly co-expressed with PD-1 on Tregs in the TME, tumor antigen specific CD8+ T cells and CD8+ TILs, leading to weakened anti-tumor immune responses.1–2 To date, TIGIT inhibiting monoclonal antibodies (mAb) have shown little activity as a monotherapy in clinical and preclinical studies. 3–4 Therefore, current clinical trials are now focused on combining TIGIT mAbs with known commercial PD-1 or PD-L1 mAbs. A TIGIT-specific engineered toxin body (ETB) represents a wholly new approach to targeting TIGIT expressing cells including those co-expressing TIGIT and PD-1.MethodsETBs targeting TIGIT were designed to deplete TIGIT-expressing TILs, including Tregs, directly in the TME. ETBs are proteins that consist of an antibody fragment genetically fused to a proprietary de-immunized (DI) form of the Shiga-like toxin A subunit (SLTA). These proteins are specific for a cell surface receptor, and function through triggering rapid internalization upon binding, followed by an enzymatic and irreversible termination of ribosomal protein synthesis resulting in cellular apoptosis. Here we provide proof of concept for ETBs as a novel modality for the depletion of TIGIT-expressing immune cells.ResultsTIGIT-targeting ETBs exhibit potent in vitro cytotoxicity of TIGIT over-expressing cell lines (IC50<1nM). These ETBs also lead to apoptotic depletion of ex vivo TIGIT-expressing regulatory T cells (Tregs) from healthy donors. In mixed culture assays, TIGIT ETBs increase the proliferation of TIGIT negative T cells by depleting TIGIT-expressing T cells.ConclusionsStudies to assess pharmacodynamics and efficacy of TIGIT targeting ETBs using a double knock-in (TIGIT and PD-1) mouse tumor model are ongoing, but these early proof of concept in vitro data support the hypothesis that ETBs can deplete TIGIT positive immune cell populations including those co-expressing PD-1. It is possible that targeted TIGIT inhibition through ETB-induced cell death could tip the balance towards tumor regression by eliminating this novel checkpoint (and TIGIT/PD-1 co-expression) at the level of the TME.ReferencesJinhua X, Ji W, Shouliang C, Liangfeng Z. Expression of immune checkpoints in T cells of esophageal cancer patients. Oncotarget 2016;7(39):1–10.Blessin NC, Simon R, Kluth M, Fischer K, et al. Patterns of TIGIT expression in lymphatic tissue, inflammation and cancer. Dis Markers 2019;2019:1–13.Johnston RJ, Comps-Agrar L, Hackney J, Yu X, et al. The immunoreceptor TIGIT regulates anti-tumor and antiviral CD8(+) T effector function. Cancer Cell 2014;26(6):923–927.Bendell JC, Bedrad P, Bang Y-J, LoRusso P, et al. Phase Ia/Ib dose-escalation study of the anti-TIGIT antibody Tiragolumab as a single agent and in combination with atezolizumab in patients with advanced solid tumors. Proceedings: AACR Annual Meeting 2020; April 27–28, 2020 and June 22–24, 2020; Philadelphia, PA.

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Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus

10.1101/2020.05.15.098335 ◽

2020 ◽

Cited By ~ 1

Author(s):

Craig M. Rive ◽

Eric Yung ◽

Lisa Dreolini ◽

Daniel J. Woodsworth ◽

Robert A. Holt

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Immune Cell ◽

Wild Type ◽

Immune Cell Subsets ◽

Car T

AbstractAnti-CD19 CAR-T therapy for B cell malignancies has shown clinical success, but a major limitation is the logistical complexity and high cost of manufacturing autologous cell products. Direct infusion of viral gene transfer vectors to initiate in vivo CAR-T transduction, expansion and anti-tumor activity could provide an alternative, universal approach for CAR-T and related immune effector cell therapies that circumvents ex vivo cell manufacturing. To explore the potential of this approach we first evaluated human and murine CD8+ T cells transduced with VSV-G pseudotyped lentivectors carrying an anti-CD19CAR-2A-GFP transgene comprising either an FMC63 (human) or 1D3 (murine) anti-CD19 binding domain. To evaluate CD19 antigen-driven CAR-T proliferation in vitro we co-cultured transduced murine T cells with an excess of irradiated splenocytes and observed robust expansion over a 9 week period relative to control T cells transduced with a GFP transgene (mean fold expansion +/- SD: ID3-CD19CAR-GFP modified T cells, 12.2 +/- 0.09 (p < 0.001); FMC63-CD19CAR-GFP modified T cells 8.8 +/- 0.03 (p < 0.001). CAR-T cells isolated at the end of the expansion period showed potent B cell directed cytolytic activity in vitro. Next, we administered approximately 20 million replication-incompetent lentiviral particles carrying either ID3-CD19CAR-GFP, FMC63-CD19CAR-GFP, or GFP-only transgene to to wild-type C57BL/6 mice by tail vein infusion and monitored the dynamics of immune cell subsets isolated from peripheral blood at weekly intervals. We saw emergence of a persistent CAR-transduced CD3+ T cell population beginning week 3-4 that reaching a maximum of 13.5 +/- 0.58 % (mean +/- SD) and 7.8 +/- 0.76% of the peripheral blood CD3+ T cell population in mice infused with ID3-CD19CAR-GFP lentivector or FMC63-CD19CAR-GFP lentivector, respectively, followed by a rapid decline, in each case of, the B cell content of peripheral blood. Complete B cell aplasia was apparent by week 5 and was sustained until the end of the protocol (week 8). None of these changes were observed in mice infused with GFP-only control lentivector, and significant CAR positive populations were not observed within other immune cell subsets, including macrophage, natural killer, or B cells. Within the T cell compartment, CD8+ effector memory cells were the predominant CAR-positive subset. Modest weight loss of 5.5 +/- 2.97 % (mean +/- SD) observed in some animals receiving an anti-CD19CAR-GFP transgene during the protocol. These results indicate that direct IV infusion of lentiviral particles carrying an anti-CD19 CAR transgene can transduce T cells that then fully ablate endogenous B cells in wild type mice. Based on these results it may be useful to further explore, using currently available vectors, the feasibility of systemic gene therapy as a modality for CAR-T intervention.

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Metabolic stress and disease-stage specific basigin expression of peripheral blood immune cell subsets in COVID-19 patients

10.1101/2020.09.18.20194175 ◽

2020 ◽

Author(s):

Peter J. Siska ◽

Katrin Singer ◽

Jana Klitzke ◽

Nathalie Kauer ◽

Sonja-Maria Decking ◽

...

Keyword(s):

T Cells ◽

T Cell Activation ◽

Cell Activation ◽

Immune Cell ◽

Metabolic Stress ◽

Recovery Phase ◽

Disease Stage ◽

Immune Cell Subsets ◽

Ros Accumulation

Coronavirus disease 2019 (COVID-19) is driven by dysregulated immune responses yet the role of immunometabolism in COVID-19 pathogenesis remains unclear. By investigating 47 patients with confirmed SARS-CoV-2 infection and 16 uninfected controls, we found an immunometabolic dysregulation specific for patients with progressed disease that was reversible in the recovery phase. Specifically, T cells and monocytes exhibited increased mitochondrial mass, accumulated intracellular ROS and these changes were accompanied by disrupted mitochondrial architecture. Basigin (CD147), but not established markers of T cell activation, was up-regulated on T cells from progressed COVID-19 patients and correlated with ROS accumulation, reflected in the transcriptome. During recovery, basigin and ROS decreased to match the uninfected controls. In vitro analyses confirmed the correlation and showed a down-regulation of ROS by dexamethasone treatment. Our findings provide evidence of a basigin-related and reversible immunometabolic dysregulation in COVID-19.

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MO695THE ASSOCIATION OF CIRCULATING CD14++CD16+ MONOCYTES, NATURAL KILLER CELLS AND REGULATORY T CELLS SUBPOPULATIONS WITH CARDIOVASCULAR DISEASE IN A COHORT OF PERITONEAL DIALYSIS PATIENTS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab101.0017 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Anila Duni ◽

Olga Balafa ◽

George Vartholomatos ◽

Margarita Oikonomou ◽

Paraskevi Tseke ◽

...

Keyword(s):

Cardiovascular Disease ◽

T Cells ◽

Peritoneal Dialysis ◽

Nk Cells ◽

Immune Cells ◽

Immune Cell ◽

Normal Range ◽

Immune Cell Subsets ◽

Cell Subpopulations

Abstract Background and Aims Advanced chronic kidney disease (CKD) is characterized by elevated expression of the proinflammatory and pro-atherogenic CD14++CD16+ monocytes subset. The role of lymphocyte subpopulations including natural killer (NK) cells and CD4+CD25+ regulatory T cells (Tregs) in the modulation of inflammation and immunity and subsequent cardiovascular implications have received increasing attention. The role of immune cell subpopulations remains to be determined in peritoneal dialysis (PD) patients. The aim of this pilot study was to investigate potential correlations between blood levels of CD14++CD16+ monocytes, NK cells and Tregs with phenotypes of established cardiovascular disease (CVD), including coronary artery disease (CAD) and heart failure (HF) in a cohort of PD patients. Method 29 stable PD patients (mean age 66.96 years ±14.5, 62% males) were enrolled. Exclusion criteria were a history of malignancy, autoimmune disease, active or chronic infections and a recent (< 3 months) cardiovascular event. Demographic, laboratory and bioimpedance measurements data (overhydration, extracellular and total body water and their ratios) were collected. The analysis of peripheral blood immune cell subsets was performed using flow cytometry (FC). Additionally, in 7 PD patients the distribution of the immune cells was evaluated by FC at two time points: T0 (before initiation of PD - CKD stage G5) and T1 (after PD start). Results The median dialysis vintage was 34.5 (range 3.2-141) months. Overall, PD patients had 527 ± 199 monocytes and 1731 ± 489 lymphocytes while mean percentage of CD14++CD16+ monocytes was 9.3 ±6.36% (normal range 2-8%), NK cells 16.6±10.3% (normal range 5-15%) and Tregs 2.1±1.76% (normal range 1-3%). There was no correlation of either of these cell subpopulations with age, PD vintage, inflammation markers (CRP, fibrinogen, albumin, hsTroponin-I), overhydration markers or comorbidities. Only increased NK cells were associated with the presence of HF in PD (24.87 vs 14.92%, p 0.047). In multiple regression analysis, NK cells levels were strongly associated with the presence of edema (beta coef=13.7, p<0.001) and CAD (beta coef=7.1, p=0.046). At T0 mean percentage of CD14++CD16+ monocytes, NK cells and Tregs were 9.7 ±4.5%, 17.1 ±3.84% and 2.38± 1.26% respectively whereas at T1 mean percentage of CD14++CD16+ monocytes was 13.3% ±8.4, NK cells 19.8±6.47% and Tregs 1.5±0.6%. Paired t-test of cell subpopulations (T0 vs T1) showed that only the Tregs were significantly decreased (p =0.018), while the other subpopulations did not differ and remained increased. Conclusion Our study is the first to evaluate the potential association between specific immune cell subsets and cardiovascular disease in long-term PD patients. Increased NK cells levels directly correlate both with the presence of HF and CAD in PD patients. Longitudinal results suggest that CD14++CD16+ and NK cells remain increased after PD start, while Tregs decrease further. The state of pro-inflammation and immune deregulation appear to persist after initiating PD. Future research is required to evaluate the role of immune cells subsets as potential tools to identify patients who are at the highest risk for complications and to guide interventions that may improve clinical outcomes.

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