CD8+ T cells from patients with narcolepsy and healthy controls recognize hypocretin neuron-specific antigens

Healthy and controlled immune response in COVID-19 is crucial for mild forms of the disease. Although CD8+ T cells play important role in this response, there is still a lack of studies showing the gene expression profiles in those cells at the beginning of the disease as potential predictors of more severe forms after the first week. We investigated a proportion of different subpopulations of CD8+ T cells and their gene expression patterns for cytotoxic proteins (perforin-1 (PRF1), granulysin (GNLY), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK)), cytokine interferon-γ (IFN-γ), and apoptotic protein Fas ligand (FASL) in CD8+ T cells from peripheral blood in first weeks of SARS-CoV-2 infection. Sixteen COVID-19 patients and nine healthy controls were included. The absolute counts of total lymphocytes (p = 0.007), CD3+ (p = 0.05), and CD8+ T cells (p = 0.01) in COVID-19 patients were significantly decreased compared to healthy controls. In COVID-19 patients in CD8+ T cell compartment, we observed lower frequency effector memory 1 (EM1) (p = 0.06) and effector memory 4 (EM4) (p < 0.001) CD8+ T cells. Higher mRNA expression of PRF1 (p = 0.05) and lower mRNA expression of FASL (p = 0.05) at the fifth day of the disease were found in COVID-19 patients compared to healthy controls. mRNA expression of PRF1 (p < 0.001) and IFN-γ (p < 0.001) was significantly downregulated in the first week of disease in COVID-19 patients who progressed to moderate and severe forms after the first week, compared to patients with mild symptoms during the entire disease course. GZMK (p < 0.01) and FASL (p < 0.01) mRNA expression was downregulated in all COVID-19 patients compared to healthy controls. Our results can lead to a better understanding of the inappropriate immune response of CD8+ T cells in SARS-CoV2 with the faster progression of the disease.

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NKG2D Polymorphism in Melanoma Patients from Southeastern Spain

Cancers ◽

10.3390/cancers11040438 ◽

2019 ◽

Vol 11 (4) ◽

pp. 438 ◽

Cited By ~ 1

Author(s):

Lourdes Gimeno ◽

Helios Martínez-Banaclocha ◽

M. Bernardo ◽

José Bolarin ◽

Luis Marín ◽

...

Keyword(s):

T Cells ◽

Natural Killer ◽

Cd8 T Cells ◽

Nk Cell ◽

Critical Role ◽

Healthy Controls ◽

Nucleotide Polymorphisms ◽

Southeastern Spain ◽

Melanoma Patients ◽

Risk Of Cancer

Background: Natural killer (NK) and CD8+ T cells are involved in the immune response against melanoma. C-Type lectin-like NK cell receptors are located in the Natural Killer Complex (NKC) region 12p13.2-p12.3 and play a critical role in regulating the activity of NK and CD8+ T cells. An association between polymorphisms in the NKC region, including the NKG2D gene and NKG2A promoter, and the risk of cancer has been previously described. The aim of this study was to analyze the association of polymorphisms in the NKC region with cutaneous melanoma in patients from southeastern Spain. Methods: Seven single-nucleotide polymorphisms (SNPs) in the NKG2D gene (NKC3,4,7,9,10,11,12), and one SNP in the NKG2A promoter (NKC17) were genotyped by a TaqMan 5′ Nuclease Assay in 233 melanoma patients and 200 matched healthy controls. Results: A linkage disequilibrium analysis of the SNPs performed in the NKC region revealed two blocks of haplotypes (Hb-1 and Hb-2) with 14 and seven different haplotype subtypes, respectively. The third most frequent haplotype from the block Hb-2—NK3 (CAT haplotype)—was significantly more frequent on melanoma patients than on healthy controls (p = 0.00009, Pc = 0.0006). No further associations were found when NKC SNPs were considered independently. Conclusions: Our results suggest an association between NKG2D polymorphisms and the risk of cutaneous malignant melanoma.

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Hypomethylation of STAT1 and HLA-DRB1 is associated with type-I interferon-dependent HLA-DRB1 expression in lupus CD8+ T cells

10.1101/398081 ◽

2018 ◽

Author(s):

Shaylynn Miller ◽

Patrick Coit ◽

Elizabeth Gensterblum-Miller ◽

Paul Renauer ◽

Nathan C Kilian ◽

...

Keyword(s):

Dna Methylation ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Type I Interferon ◽

Interferon Response ◽

Type I ◽

Mrna Levels ◽

Healthy Controls ◽

Genome Wide

AbstractObjectiveWe examined genome-wide DNA methylation changes in CD8+ T cells from lupus patients and controls, and investigated the functional relevance of some of these changes in lupus.MethodsGenome-wide DNA methylation of lupus and age, sex, and ethnicity-matched control CD8+ T cells was measured using the Infinium MethylationEPIC arrays. Measurement of relevant cell subsets was performed via flow cytometry. Gene expression was quantified by qPCR.ResultsLupus CD8+ T cells had 188 hypomethylated CpG sites compared to healthy matched controls. Among the most hypomethylated were sites associated with HLA-DRB1. Genes involved in the type-I interferon response, including STAT1, were also found to be hypomethylated. IFNα upregulated HLA-DRB1 expression on lupus but not control CD8+ T cells. Lupus and control CD8+ T cells significantly increased STAT1 mRNA levels after treatment with IFNα. The expression of CIITA, a key interferon/STAT1 dependent MHC-class II regulator, is induced by IFNα in lupus CD8+ T cells, but not healthy controls. Co-incubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to CD4+ T cell activation, determined by increased expression of CD69, in lupus patients but not in healthy controls. This can be blocked by neutralizing antibodies targeting HLA-DR.ConclusionsLupus CD8+ T cells are epigenetically primed to respond to type-I interferon. We describe an HLA-DRB1+ CD8+ T cell subset that can be induced by IFNα in lupus patients. A possible pathogenic role for CD8+ T cells in lupus that is dependent upon a high type-I interferon environment and epigenetic priming warrants further characterization.

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LAG3 and PD1 Regulate CD8+ T Cell in Diffuse Large B-cell Lymphoma Patients

Computational and Mathematical Methods in Medicine ◽

10.1155/2021/4468140 ◽

2021 ◽

Vol 2021 ◽

pp. 1-8

Author(s):

Ying Liu ◽

Xinhong Guo ◽

Lingbo Zhan ◽

Lei Wang ◽

Xinyou Wang ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Immune Checkpoints ◽

Healthy Controls ◽

Large B Cell Lymphoma ◽

Large B Cell

Background. Diffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous lymphoid malignancy. The unsatisfactory outcome for refractory patients has prompted efforts to explore new therapeutic approaches for DLBCL. However, the mechanisms involved in treatment associated with immune checkpoints remain unclear. This study is aimed at investigating the potential roles of programmed cell death protein 1 (PD1) and lymphocyte activation gene 3 (LAG3) in CD8+ T cells for treatment in DLBCL. Methods. Utilizing flow cytometry, we examined the content of T cells, the levels of cytokines, and the expression of PD1 and LAG3 in patients with DLBCL as well as in healthy controls. Levels of cytokines in CD8+ T cells from DLBCL patients before and after treatment were compared by blocking of PD1 and LAG3 in magnetic bead-sorted CD8+ T cells. Results. We found that the proportion of CD4+ T cells and CD8+ T cells was increased in DLBCL patients after treatment. The levels of cytokines trended toward those of healthy controls in treatment. PD1 (+), LAG3 (+), or PD1 (+) LAG3 (+) were all expressed in lower amounts in CD4+ T cells and CD8+ T cells after treatment than in untreated DLBCL patients. In addition, blockade of PD1 and LAG3 in sorted CD8+ T cells markedly inhibited cytokine production in response to treatment. Conclusion. PD1 and LAG3 in CD8+ T cells may be important targets of therapy and play therapeutic role in patients with DLBCL.

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Circulating cytokines and lymphocyte subsets in patients who have recovered from COVID-19

10.1101/2020.07.22.20160259 ◽

2020 ◽

Author(s):

Hasi Chaolu ◽

Xinri Zhang ◽

Xin Li ◽

Dongyan Li

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Lymphocyte Subsets ◽

Healthy Controls ◽

Tnf Α ◽

Ifn Γ ◽

Circulating Cytokines

To investigate the immune status of people who previously had COVID-19 infections, we recruited patients 2 weeks post-recovery and analyzed circulating cytokines and lymphocyte subsets. We measured levels of total lymphocytes, CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ NK cells, and the serum concentrations of interleukin (IL)-1, IL-4, IL-6, IL-8, IL-10, transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) by flow cytometry. We found that in most post-recovery patients, levels of total lymphocytes (66.67%), CD3+ T cells (54.55%), CD4+ T cells (54.55%), CD8 + T cells (81.82%), CD19+ B cells (69.70%), and CD56+ NK cells(51.52%) remained lower than normal, whereas most patients showed normal levels of IL-2 (100%), IL-4 (80.88%), IL-6 (79.41%), IL-10 (98.53%), TNF-α (89.71%), IFN-γ (100%) and IL-17 (97.06%). Compared to healthy controls, 2-week post-recovery patients had significantly lower absolute numbers of total lymphocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells, along with significantly higher levels of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17. Among post-recovery patients, T cells, particularly CD4+ T cells, were positively correlated with CD19+ B cell counts. Additionally, CD8+ T cells positively correlated with CD4+ T cells and IL-2 levels, and IL-6 positively correlated with TNF-α and IFN-γ. These correlations were not observed in healthy controls. By ROC curve analysis, post-recovery decreases in lymphocyte subsets and increases in cytokines were identified as independent predictors of rehabilitation efficacy. These findings indicate that the immune system has gradually recovered following COVID-19 infection; however, the sustained hyper-inflammatory response for more than 14 days suggests a need to continue medical observation following discharge from the hospital. Longitudinal studies of a larger cohort of recovered patients are needed to fully understand the consequences of the infection.

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Quantitative proteomic analyses of CD4+ and CD8+ T cells reveal differentially expressed proteins in multiple sclerosis patients and healthy controls

Clinical Proteomics ◽

10.1186/s12014-019-9241-5 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 9

Author(s):

Tone Berge ◽

Anna Eriksson ◽

Ina Skaara Brorson ◽

Einar August Høgestøl ◽

Pål Berg-Hansen ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

Cd8 T Cells ◽

Differentially Expressed ◽

Healthy Controls ◽

Differentially Expressed Proteins ◽

Proteomic Analyses ◽

Multiple Sclerosis Patients

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Dasatinib Has a Dual Mode of Action: Direct BCR-ABL1 Mediated Anti-Leukemic Effects Are Complemented by Promotion of Th1-Type and NK-Cell Mediated Cellular Immune Responses,

Blood ◽

10.1182/blood.v118.21.3760.3760 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3760-3760 ◽

Cited By ~ 1

Author(s):

Anna Kreutzman ◽

Jukka Vakkila ◽

Kimmo Porkka ◽

Satu Mustjoki

Keyword(s):

T Cells ◽

Immune Responses ◽

Nk Cells ◽

Cd8 T Cells ◽

K562 Cells ◽

Leukemic Cells ◽

Healthy Controls ◽

Median Percentage ◽

Blood Smears ◽

Cellular Immune

Abstract Abstract 3760 Background. Tyrosine kinase inhibitors (TKIs; imatinib, dasatinib, nilotinib) have dramatically improved outcome of CML. Besides inhibiting target kinases in leukemic cells, off-target kinases in immune effector cells are also affected. We have previously described that dasatinib therapy induces an oligoclonal expansion and mobilization of large granular lymphocytes (LGLs; CD8+ T-cells or NK-cells) in Ph+ leukemia patients. Importantly, LGL expansion is associated with improved therapy responses, but the actual mechanisms are unknown. In this study, we explored the function and anti-leukemic properties of LGLs. Methods. Peripheral blood samples from CML patients treated with dasatinib (n=10), imatinib (n=4), or nilotinib (n=7), or healthy controls (n=6) were used to analyze the activation and cytotoxicity of T- and NK-cells. Samples were collected before and after drug intake. The number of LGLs was determined from MGG stained blood smears and compared with granzyme B (GrB) positivity analyzed by flow cytometry. Th1-type cytokine (TNF-a, IFN-g) production was measured by flow cytometry after stimulation of mononuclear cells (MNCs) with a-CD3/CD28-antibodies. Unpurified and purified NK cells were cultured with K562 cells, and degranulation (CD107 analysis) and cytotoxicity were measured. Results. As GrB positivity correlated well (r=0.95, p<0.0001, n=17) with the number of LGLs counted from MGG stained blood smears, a GrB specific antibody was used to identify LGLs in further analyses. At diagnosis CML patients had more GrB+CD8+ T-cells than healthy controls (38 % vs. 11%, p=0.028). Also GrB+CD4+ T-cells were slightly increased, but did not differ significantly from healthy controls (3.6% vs. 0.8%, p=0.08). During dasatinib treatment the proportion of GrB+CD4+ (median at 6 months 28.1%, p=0.03) and GrB+CD8+ (70.9%, p=0.03) T cells increased significantly, whereas similar increase was not observed during imatinib (1.2% GrB+CD4+ and 30.0% GrB+CD8+ T-cells) or nilotinib (4.4% and 41.8%, respectively) therapies. In patients on dasatinib therapy, GrB+CD3+cells were more sensitive to CD3/CD28-antibody stimulation and a larger proportion of cells (13.7%) produced Th1-type cytokines (TNF-a+IFN-g) compared to imatinib (2.4%) or nilotinib patients (5.5%) or healthy controls (5%) under same conditions (p=0.015). As Th-1 cytokine-producing T cells are important in promoting cell-mediated immune responses, we next assessed whether dasatinib also enhances the cytolytic activity of NK cells. When MNC fraction was used as effector population (ratio 20:1), the median percentage of dead K562 cells was 18% in samples taken before dasatinib intake and 32% in samples taken 1h after dasatinib intake (p=0.004). Pre-dasatinib killing did not differ significantly from healthy volunteers (p=0.12). No increase in NK-cytotoxicity was observed after imatinib (11% vs. 8%) or nilotinib (10% vs. 10%) intake. Similar results were also obtained with purified NK-cells: the median percentage of dead K562 cells was 12% pre-dasatinib and 29% in post-dasatinib samples (p=0.06), whereas no differences were noticed with imatinib (30% vs. 28%) or nilotinib (14% vs. 15%) patients. The median percentage of dead K562 cells after incubation with pure NK-cells from healthy volunteers was 20%. Interestingly, the cytolytic ability of NK-cells differed significantly among dasatinib treated patients. When the patients were divided into two groups based on therapy response, patients who had achieved CMR within 12 months (n=4) had significantly higher cytotoxic capability compared to patients who had not (n=6): 46% vs. 28% of dead K562 cells in post-dasatinib samples (p=0.02). Conclusions. Dasatinib therapy resulted in increased numbers of GrB+ T-cells and generation of a Th1-type cellular immune response. In addition, 1h dasatinib exposure in vivo improved the cytotoxicity of NK-cells. These data support the dual mode of action of dasatinib: potent BCR-ABL1 inhibition in leukemic cells is accompanied by enhancement of cellular immunity, which likely have implications in be the long term control of Ph+ leukemia. Disclosures: Porkka: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria; Novartis: Honoraria.

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Developing Novel Approaches To Comprehensively Assess T Cell Repertoire Dynamics In The Early Post-Transplant Period

Blood ◽

10.1182/blood.v122.21.4618.4618 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4618-4618

Author(s):

Ute E. Burkhardt ◽

Joseph Kaplinsky ◽

Cindy Desmarais ◽

Kristen E. Stevenson ◽

Edwin P. Alyea ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Healthy Controls ◽

Transplant Recipients ◽

Hematopoietic Stem ◽

Post Transplant ◽

T Cell Reconstitution ◽

Tcr Repertoire ◽

Control Samples

Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a highly effective treatment modality for many hematologic malignancies, a major treatment-associated toxicity is the induction of a prolonged state of T cell immunodeficiency in the transplant recipient, which in turn contributes to critical clinical outcomes such as infectious complications, and the risk of relapse. Targeted deep sequencing of the T cell receptor beta-chain (TCRβ) has emerged as a promising technology for enabling the qualitative and quantitative monitoring of T cell recovery following transplant with unprecedented resolution. Major challenges remain, however, in the establishment of informative analysis tools for characterization of global TCRβ repertoire dynamics. In the current work, we developed and applied a novel analysis approach as a mean to gain detailed biological insight into T cell reconstitution following allo-HSCT. To this end, we isolated naïve and memory CD4+ and CD8+ T cells from peripheral blood mononuclear cells of 14 patients with advanced chronic lymphocytic leukemia who underwent allo-HSCT following reduced-intensity doses of fludarabine and busulfan. From these T cell subpopulations, genomic DNA was extracted at post-transplant day 30 (d30) and later time points informative for thymic-independent (4 month post-transplant; d120) and thymic-dependent (1 year post-transplant; d365) T cell immune recovery. Subsequently, a template library for sequencing on an Illumina GA2 system was generated through PCR amplification of the TCRβ CDR3 region using an established panel of 45 Vβ- and 13 Jβ-specific primers. We obtained a median of 394,872 (range 0-26,426,784) productive reads across our 168 samples. As a comparison group, we further studied repertoire data from naïve and memory CD4+ and CD8+ T cells collected from 9 healthy adult volunteers. To characterize how transplant perturbs the TCR repertoire, we first compared VDJ usage between the transplanted patients and the healthy controls. For each of the post-transplant and control samples, we tallied the number of clones from all sequenced compartments (CD4+ and CD8+, naïve and memory) that used each of the several thousand possible VDJ combinations. We performed pairwise comparisons of the resulting VDJ distributions for all 253 sample pairs at days 30, 120 and 365 by calculating the R2 and, separately, X2 statistics. Permutation analysis demonstrated that control samples were more similar to each other than either post-transplant day 30, 120 or 365 samples (P=2.5-5.0x10-5, 2.5-5.0x10-5 and ≤2.5x10-5 by X2; 2.5-5.0x10-5, 5.5-5.7x10-4 and 1.0-1.2x10-4 by R2, respectively). Of note, whereas control samples demonstrated a similar VDJ usage, such similarity was not observed among post-transplant samples at day 30, 120 or 365 (P=0.65, 0.53, and 0.60 by X2; P=0.014, 0.38, and 0.43 by R2, respectively). These results demonstrate that VDJ usage in transplant recipients remains more heterogeneous than in healthy controls throughout the entire first year of reconstitution. To understand whether this heterogeneity reflects equilibrium or dynamic changes of the TCR repertoire, we visualized the time course of reconstitution using principal component analysis of VDJ usage. We observed marked dynamism, in which most transplant recipients both experienced a greater degree of change than was represented by the controls, and explored regions of VDJ usage very different from that of controls. Preliminarily, we observed that several transplant recipients became more similar to controls over time, while others did not. Our results demonstrate that post-transplant T cell reconstitution follows both personal and highly dynamic trajectories across a range of clinical courses, and suggest that TCR sequencing in larger sample sizes is a promising avenue for future study. Ongoing analyses focus on investigating the correlates of this dynamism among the 14 transplant recipients through subgroup analysis based on their clinical course and sequence-level analysis. The results obtained through these novel computational and systems methods will be integrated with other experimental measures of immune reconstitution including immunophenotyping and TCR excision circle (TREC) analysis. Disclosures: No relevant conflicts of interest to declare.

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Increased Expression of Ganglioside GM1 in Peripheral CD4+ T Cells Correlates Soluble Form of CD30 in Systemic Lupus Erythematosus Patients

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/569053 ◽

2010 ◽

Vol 2010 ◽

pp. 1-8 ◽

Cited By ~ 13

Author(s):

Lingli Dong ◽

Shaoxian Hu ◽

Fang Chen ◽

Xiaomei Lei ◽

Wei Tu ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Memory T Cells ◽

Elevated Serum ◽

Soluble Form ◽

Membrane Microdomains ◽

Tnf Alpha ◽

Healthy Controls ◽

Soluble Cd30

Gangliosides GM1 is a good marker of membrane microdomains (lipid rafts) with important function in cellular activation processes. In this study we found that GM1 expression on CD4+ T cells and memory T cells (CD45RO/CD4) were dramatic increased after stimulation with phytohaemagglutinin in vitro. Next, we examined the GM1 expression on peripheral blood CD4+ T cells and CD8+ T cells from 44 patients with SLE and 28 healthy controls by flow cytometry. GM1 expression was further analyzed with serum soluble CD30 (sCD30), IL-10, TNF-alpha and clinical parameters. The mean fluorescence intensity of GM1 on CD4+ T cells from patients with SLE was significantly higher than those from healthy controls, but not on CD8+ T cells. Increased expression of GM1 was more marked on CD4+/CD45RO+ memory T cells from active SLE patients. Patients with SLE showed significantly elevated serum sCD30 and IL-10, but not TNF-alpha levels. In addition, we found that enhanced GM1 expression on CD4+ T cells from patients with SLE positively correlated with high serum levels of sCD30 and IgG as well as disease activity (SLEDAI scores). Our data suggested the potential role of aberrant lipid raft/GM1 on CD4+ T cells and sCD30 in the pathogenesis of SLE.

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