Downregulation of CD45 Signaling in COVID-19 Patients Is Reversed by C24D, a Novel CD45 Targeting Peptide

CD45, the predominant transmembrane tyrosine phosphatase in leukocytes, is required for the efficient induction of T cell receptor signaling and activation. We recently reported that the CD45-intracellular signals in peripheral blood mononuclear cells (PBMCs) of triple negative breast cancer (TNBC) patients are inhibited. We also reported that C24D, an immune modulating therapeutic peptide, binds to CD45 on immune-suppressed cells and resets the functionality of the immune system via the CD45 signaling pathway. Various studies have demonstrated that also viruses can interfere with the functions of CD45 and that patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are immune-suppressed. Given the similarity between the role of CD45 in viral immune suppression and our findings on TNBC, we hypothesized that the C24D peptide may have a similar “immune-resetting” effect on PBMCs from COVID-19 patients as it did on PBMCs from TNBC patients. We tested this hypothesis by comparing the CD45/TCR intracellular signaling in PBMCs from ten COVID-19 patients vs. PBMCs from ten healthy volunteers. Herein, we report our findings, demonstrating the immune reactivating effect of C24D via the phosphorylation of the tyrosine 505 and 394 in Lck, the tyrosine 493 in ZAP-70 and the tyrosine 172 in VAV-1 proteins in the CD45 signaling pathway. Despite the relatively small number of patients in this report, the results demonstrate that C24D rescued CD45 signaling. Given the central role played by CD45 in the immune system, we suggest CD45 as a potential therapeutic target.

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A novel MyD-1 (SIRP-1α) signaling pathway that inhibits LPS-induced TNFα production by monocytes

Blood ◽

10.1182/blood-2002-11-3596 ◽

2003 ◽

Vol 102 (7) ◽

pp. 2532-2540 ◽

Cited By ~ 52

Author(s):

Rosemary E. Smith ◽

Vanshree Patel ◽

Sandra D. Seatter ◽

Maureen R. Deehan ◽

Marion H. Brown ◽

...

Keyword(s):

Signaling Pathway ◽

Molecular Mechanisms ◽

Mononuclear Cells ◽

Kinase Inhibitor ◽

Tyrosine Phosphatase ◽

Peripheral Blood Mononuclear ◽

Necrosis Factor Alpha ◽

Purified Protein Derivative ◽

Sequential Activation ◽

Novel Target

Abstract MyD-1 (CD172) is a member of the family of signal regulatory phosphatase (SIRP) binding proteins, which is expressed on human CD14+ monocytes and dendritic cells. We now show a novel role for MyD-1 in the regulation of the innate immune system by pathogen products such as lipopolysaccharide (LPS), purified protein derivative (PPD), and Zymosan. Specifically, we demonstrate that ligation of MyD-1 on peripheral blood mononuclear cells (PBMCs) inhibits tumor necrosis factor alpha (TNFα) secretion but has no effect on other cytokines induced in response to each of these products. In an attempt to understand the molecular mechanisms underlying this surprisingly selective effect we investigated signal transduction pathways coupled to MyD-1. Ligation of the SIRP was found to recruit the tyrosine phosphatase SHP-2 and promote sequential activation of phosphatidylinositol (PI) 3-kinase, phospholipase D, and sphingosine kinase. Inhibition of LPS-induced TNFα secretion by MyD-1 appears to be mediated by this pathway, as the PI 3-kinase inhibitor wortmannin restores normal LPS-driven TNFα secretion. MyD-1-coupling to this PI 3-kinase-dependent signaling pathway may therefore present a novel target for the development of therapeutic strategies for combating TNFα production and consequent inflammatory disease. (Blood. 2003;102:2532-2540)

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Identification of Circulating Gene Expression Signatures of Intracranial Aneurysm in Peripheral Blood Mononuclear Cells

Diagnostics ◽

10.3390/diagnostics11061092 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1092

Author(s):

Vincent M. Tutino ◽

Haley R. Zebraski ◽

Hamidreza Rajabzadeh-Oghaz ◽

Muhammad Waqas ◽

James N. Jarvis ◽

...

Keyword(s):

Intracranial Aneurysm ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Intracellular Signaling ◽

Mononuclear Cells ◽

Differential Expression Analysis ◽

Validation Dataset ◽

Cytokine Signaling ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMCs) play an important role in the inflammation that accompanies intracranial aneurysm (IA) pathophysiology. We hypothesized that PBMCs have different transcriptional profiles in patients harboring IAs as compared to IA-free controls, which could be the basis for potential blood-based biomarkers for the disease. To test this, we isolated PBMC RNA from whole blood of 52 subjects (24 with IA, 28 without) and performed next-generation RNA sequencing to obtain their transcriptomes. In a randomly assigned discovery cohort of n = 39 patients, we performed differential expression analysis to define an IA-associated signature of 54 genes (q < 0.05 and an absolute fold-change ≥ 1.3). In the withheld validation dataset, these genes could delineate patients with IAs from controls, as the majority of them still had the same direction of expression difference. Bioinformatics analyses by gene ontology enrichment analysis and Ingenuity Pathway Analysis (IPA) demonstrated enrichment of structural regulation processes, intracellular signaling function, regulation of ion transport, and cell adhesion. IPA analysis showed that these processes were likely coordinated through NF-kB, cytokine signaling, growth factors, and TNF activity. Correlation analysis with aneurysm size and risk assessment metrics showed that 4/54 genes were associated with rupture risk. These findings highlight the potential to develop predictive biomarkers from PBMCs to identify patients harboring IAs.

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The analysis of the long-term impact of SARS-CoV-2 on the cellular immune system in individuals recovering from COVID-19 reveals a profound NKT cell impairment

10.1101/2020.08.21.20179358 ◽

2020 ◽

Cited By ~ 1

Author(s):

jia liu ◽

Xuecheng Yang ◽

Hua Wang ◽

Ziwei Li ◽

Hui Deng ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Annexin V ◽

Double Positive ◽

Peripheral Blood Mononuclear ◽

Cellular Immune System ◽

Cellular Immune

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects millions of people and killed hundred-thousands of individuals. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remained to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19 convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2 unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of Annexin V and 7-AAD double positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies, TIM-3 expression on CD4 and CD8 T cells, as well as PD-L1 expression on B cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by GzmB expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully capable to proliferate and produce effector cytokines upon TCR stimulation. Collectively, we provide the first comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease.

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A10 Presence and frequency of M184V mutation in the MOBIDIP trial

Virus Evolution ◽

10.1093/ve/vez002.009 ◽

2019 ◽

Vol 5 (Supplement_1) ◽

Author(s):

A Armero ◽

M L Chaix ◽

M L Nere ◽

E Delaporte ◽

M Peeters ◽

...

Keyword(s):

Mononuclear Cells ◽

Dual Therapy ◽

Significant Loss ◽

Resistance Mutations ◽

Hiv Reverse Transcriptase ◽

First Line ◽

Bioinformatics Pipeline ◽

Peripheral Blood Mononuclear ◽

Number Of Patients ◽

M184v Mutation

Abstract The MOBIDIP trial evaluated the simplification by protease (PI/r) monotherapy for HIV infection versus dual therapy and boosted protease inhibitor plus lamivudine (PI/r + 3TC) in controlled patients under second-line regimens. MOBIDIP was interrupted because of a significant number of patients with virological failure (VF) at week 48 (W48) in PI/r (33/133, ∼25%) versus in PI/r + 3TC (4/132, ∼3%). At the time of first-line VF, 96 per cent of patients harbored the M184V mutation. The presence of the M184V mutation was related to a protective effect against VF in the PI/r + 3TC arm. We developed a methodology that allows to determine the frequency of M184V/I mutations in the HIV reverse transcriptase (RT) gene in peripheral blood mononuclear cells (PBMC) obtained before MOBIDIP simplification. Paired-end sequences were obtained from 252 PBMC samples covering the first 855 bp of the RT gene (HXB2: 2485–3405) by MiSeq technology. These sequences were subjected to an in-house Bioinformatics pipeline. The results of our pipeline were compared to the output of PASeq (https://www.paseq.org), an open web-tool for the identification of drug resistance mutations. The M184V mutation was identified at a frequency greater than 1 per cent in 178 individuals (∼71%). The M184I mutation was observed in 34 patients (∼13%), always in the presence of stop codons, and is in agreement with expectations, as this mutation is a known APOBEC-targeted site. Sixty-seven patients (∼27%) had a frequency of the M184V mutation with values greater than 75 per cent. PASeq confirmed the presence of M184V mutation in 173 patients. The frequencies estimated by the PASeq tool and in-house pipeline were correlated up to 99.5 per cent. We found a significant loss of the M184V mutation archived in PBMC between the first-line regimen treatment failure and the beginning of the MOBIDIP trial. In patients under long-term antiretroviral therapy, as in our case, viral sub-populations could be lost, reducing the presence, and frequency of a mutation. In the next step, we will evaluate the association between the presence and frequency of M184V mutation and MOBIDIP results.

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The Lymphoproliferative Disease of Granular Lymphocytes: Updated Criteria for Diagnosis

Blood ◽

10.1182/blood.v89.1.256 ◽

1997 ◽

Vol 89 (1) ◽

pp. 256-260 ◽

Cited By ~ 137

Author(s):

Gianpietro Semenzato ◽

Renato Zambello ◽

Gordon Starkebaum ◽

Kazuo Oshimi ◽

Thomas P. Loughran

Keyword(s):

T Cell Receptor ◽

Mononuclear Cells ◽

Cell Receptor ◽

Absolute Number ◽

Lymphoproliferative Disease ◽

Peripheral Blood Mononuclear ◽

Criteria For Diagnosis ◽

Restricted Pattern ◽

Granular Lymphocytes

Abstract The lymphoproliferative disease of granular lymphocytes (LDGL), also referred to as LGL leukemia, is a heterogeneous disorder, but is clinically, morphologically, and immunologically distinct. Although LDGL has recently been included in the revised classification of lymphomas as an independent clinical entity, no consensus exists on the criteria to establish the diagnosis. The aim of this report was to refine the parameters needed to make the diagnosis of LDGL. We studied 11 patients with chronic granular lymphocytosis selected from among 195 cases observed by our institutions from three different geographic areas (North America, Europe, and Asia). These cases did not meet the current criteria for inclusion in LDGL, since all patients had less than 2,000 GL/μL. However, in each of these patients, we found evidence for expansion of a discrete GL population. Clonal rearrangement of the T-cell receptor (TCR) β gene was found in peripheral blood mononuclear cells (PBMC) of all nine patients with CD3+ LDGL. Using recently generated monoclonal antibodies (MoAbs) against the TCR Vβ gene regions, we identified a unique TCR Vβ on GL from each of three patients studied. In two patients with CD3− LDGL, we also identified a restricted pattern of reactivity, by staining with MoAbs against p58 antigen found on normal natural killer (NK) cells. The clinical features of these 11 patients with relatively low absolute number of GL were similar to those reported previously for patients with greater than 2,000 GL/μL. These data demonstrate that newer techniques such as MoAbs against Vβ gene regions and p58 molecules and molecular analyses are useful to identify expansions of discrete GL proliferations. Demonstration of an expansion of a restricted GL subset is evidence for the diagnosis of LDGL, even in patients with a relatively low GL count. Our results also contribute to distinguish between the end of normality and the beginning of pathology in the broad spectrum of GL lymphocytoses.

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Acquired DNA mutations associated with in vivo hydroxyurea exposure

Blood ◽

10.1182/blood.v95.11.3589 ◽

2000 ◽

Vol 95 (11) ◽

pp. 3589-3593 ◽

Cited By ~ 85

Author(s):

Valerie N. Hanft ◽

Steven R. Fruchtman ◽

Chrisley V. Pickens ◽

Wendell F. Rosse ◽

Thad A. Howard ◽

...

Keyword(s):

Mononuclear Cells ◽

Young Patients ◽

Myeloproliferative Disorders ◽

Cell Receptor ◽

Hypoxanthine Phosphoribosyl Transferase ◽

Peripheral Blood Mononuclear ◽

Dna Mutations ◽

Carcinogenic Potential ◽

Increased Risk

Abstract Hydroxyurea (HU) is an effective therapeutic agent for patients with myeloproliferative disorders (MPDs) or sickle cell disease (SCD). Short-term HU toxicities primarily include transient myelosuppression, but long-term HU risks have not been defined. The mutagenic and carcinogenic potential of HU is not established, although HU has been associated with an increased risk of leukemia in some patients with MPD. In this study, 2 assays were used to quantitate acquired somatic DNA mutations in peripheral blood mononuclear cells (PBMCs) after in vivo HU exposure. The HPRT assay measures hypoxanthine phosphoribosyl transferase (hprt) mutations, while the VDJ assay identifies “illegitimate” T-cell receptor Vγ-Jβ interlocus recombination events. PBMCs were analyzed from patients with MPD, adults and children with SCD, and normal controls. MPD patients with prolonged HU exposure had numbers of DNA mutations equivalent to patients with low HU exposure or controls. Similarly, adults with SCD had equivalent numbers of DNA mutations regardless of HU exposure. Children with SCD and 30-month HU exposure had equivalenthprt− mutations but significantly more VDJ mutations (1.82 ± 1.20 events per μg DNA) than children with 7-month HU exposure (1.58 ± 0.87 events) or no HU exposure (1.06 ± 0.45 events), P = .04 by analysis of variance. Taken together, these data suggest that the mutagenic and carcinogenic potential of in vivo HU therapy is low. Although increased numbers of illegitimate VDJ recombination events do not directly portend leukemia, young patients with SCD and HU exposure should be monitored serially for increases in DNA mutations.

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Extracellular ligation-dependent CD45RB enzymatic activity negatively regulates lipid raft signal transduction

Blood ◽

10.1182/blood-2008-04-150987 ◽

2009 ◽

Vol 113 (3) ◽

pp. 594-603 ◽

Cited By ~ 10

Author(s):

Kaushal Parikh ◽

Sibrand Poppema ◽

Maikel P. Peppelenbosch ◽

Lydia Visser

Keyword(s):

Signal Transduction ◽

Enzymatic Activity ◽

Lipid Raft ◽

Intracellular Signaling ◽

Tyrosine Phosphatase ◽

Cell Receptor ◽

Inhibitory Effect ◽

Tyrosine Phosphatases ◽

Peptide Arrays ◽

Substrate Peptide

Abstract CD45 is the most prominent membrane protein on lymphocytes. The function and regulation of this protein tyrosine phosphatase remain largely obscure, mainly because of the lack of a known ligand, and it still remains unknown whether such tyrosine phosphatases are subject to extracellular control at all. We report that an anti-CD45RB antibody (Ab) that prevents rejection and induces tolerance activates CD45RB tyrosine phosphatase enzymatic activity in T lymphocytes, allowing us to directly monitor the effects of increased CD45RB activity on signal transduction. Using both kinase substrate peptide arrays as well as conventional biochemistry, we also provide evidence of the various kinases involved in bringing about the inhibitory effect of this Ab on CD3-induced T-cell receptor signaling. Furthermore, we report that activated CD45RB translocates to lipid rafts and interferes with lipid raft localization and activation state of CD45 substrate Lck. Thus, these findings indeed prove that CD45 is subject to extracellular control and also define a novel mechanism by which receptor tyrosine phosphatases control lymphocyte biology and provide further insight into the intracellular signaling pathways effected by anti-CD45RB monoclonal Ab treatment.

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T-cell receptor V-gene usage in neoplasms of the central nervous system

Journal of Neurosurgery ◽

10.3171/jns.1993.78.4.0630 ◽

1993 ◽

Vol 78 (4) ◽

pp. 630-637 ◽

Cited By ~ 5

Author(s):

Adrian Merlo ◽

Luis Filgueira ◽

Markus Zuber ◽

Antonio Juretic ◽

Felix Harder ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Tumor Infiltrating Lymphocytes ◽

Cell Receptor ◽

Gene Products ◽

Peripheral Blood Mononuclear ◽

Infiltrating Lymphocytes

✓ The use of tumor-infiltrating lymphocytes in the treatment of central nervous system (CNS) neoplasms has met with serious obstacles due to difficulty of culture and poor characterization. Since in other tumors the therapeutic effects of tumor-infiltrating lymphocytes have been shown to rely on T-cell receptor engagement, the authors addressed the question as to whether expression of T-cell receptor variable (V) domains in cultured tumor-infiltrating lymphocytes from CNS is different from that of autologous cultured peripheral blood mononuclear cells. Infiltrating lymphocytes from CNS neoplasms, including primary malignancies, metastatic cancers, and meningiomas, were cultured in the presence of interleukin-2 and anti-CD3 monoclonal antibodies (MoAb's) in order to obtain optimum growth of T cells. Autologous peripheral blood mononuclear cells from the same patients were similarly cultured. After 4 to 5 weeks of culture, 97.3% ± 2.6% (mean ± standard deviation) of the resulting cell populations were CD3-positive lymphocytes. The expression of T-cell receptor V domains was then studied by using a panel of 12 MoAb recognizing gene products from T-cell receptor V-α 2, V-β 5, 6, 8, and 12, V-γ 4 and 9 families, and from two subfamilies of V-δ 2. Remarkably, in over 70% of all paired measurements, percentages of T cells expressing discrete T-cell receptor V-gene products were found to be virtually identical in tumor- and peripheral blood-derived cultured cell populations, with differences never exceeding 1%. In contrast, a different expression of individual V-gene products, concerning both α/β and γ/δ T-cell receptors, could be detected between cultured tumor-infiltrating lymphocytes and autologous peripheral blood-derived T lymphocytes in seven of 12 patients. In two cases, significant differences between the two populations were also observed in the proliferative responses obtained upon stimulation with staphylococcal enterotoxins that trigger defined V-β T-cell receptors. Altogether, these data suggest that the T-cell receptor repertoire of cultured tumor-infiltrating lymphocytes from CNS tumors, suitable for use in adoptive immunotherapies, differs from that of autologous cultured peripheral blood mononuclear cells.

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419. Rational design of bifunctional siRNAs that silence genes and recruit the innate immune system to treat ectopic pregnancies

Reproduction Fertility and Development ◽

10.1071/srb08abs419 ◽

2008 ◽

Vol 20 (9) ◽

pp. 99

Author(s):

S. Tong ◽

M. P. Gantier ◽

M. Belhke ◽

B. R. G. Williams

Keyword(s):

Immune System ◽

Gene Silencing ◽

Rational Design ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Micro Rna ◽

Peripheral Blood Mononuclear ◽

Sirna Sequence ◽

Sense Strand ◽

Ectopic Pregnancies

RNA interference (RNAi) is a new therapeutic approach, silencing genes to disrupt diseases. However, short interfering siRNAs (molecule used in RNAi) can have off-target effects, activating the immune system through RNA sensing toll-like receptors (TLR) 3, 7 and 8. We have previously proposed that in some diseases (cancers, ectopic pregnancies) it may be useful to enhance the immune response. A novel class of immunostimulatory siRNAs could be developed, silencing genes important to disease and recruiting the immune system to further aid disease clearance. We set out to develop a rational design strategy that enhances immunostimulatory properties to any siRNA sequence but maintains effective gene silencing. We screened a set of siRNAs targeting lamin. All were of the same sequence, except for different immunostimulatory motifs on the 3′ end of the sense strand. We also investigated a different design where we added a small micro-RNA like poly-uridine bulge (potentially immunostimulatory) on the sense strand. We used human peripheral blood mononuclear cells (PBMCs) to test for immunostimulation, and HEK 293-T-cells to test for lamin gene knockdown.Of all strategies tested, the poly-uridine bulge was best. It silenced the lamin gene as effectively as control, but caused a 2–3 fold increase of IFN-α and TNF-α. We verified this approach by adding the poly-uridine bulge onto an siRNA of low immunostimulatory potential targeting GFP. The bulge markedly enhanced immunostimulation in a dose response manner, and did not compromise gene knockdown. The addition of a poly-uridine bulge to siRNAs can increase immunostimulation without affecting gene silencing efficacy. Immunostimulatory siRNAs might be particularly efficacious to treat ectopic pregnancies where there are abundant immune cells, and functional TLR 7/8 in the trophoblast (unpublished observations). We now plan to test this immunostimulatory siRNA approach in an in vivo ectopic pregnancy model using JEG-3 cells xenographted in NOD-SCID mice.

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Superantigen immune stimulation activates epithelial STAT-1 and PI 3-K: PI 3-K regulation of permeability

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.279.5.g1094 ◽

2000 ◽

Vol 279 (5) ◽

pp. G1094-G1103 ◽

Cited By ~ 16

Author(s):

Derek M. McKay ◽

Fernando Botelho ◽

Peter J. M. Ceponis ◽

Carl D. Richards

Keyword(s):

Epithelial Cells ◽

Conditioned Medium ◽

Intracellular Signaling ◽

Mononuclear Cells ◽

Interferon Γ ◽

Transepithelial Resistance ◽

Mobility Shift ◽

Peripheral Blood Mononuclear ◽

Ussing Chambers ◽

Ifn Γ

Signal transducers and activators of transcription (STATs) are critical intracellular signaling molecules for many cytokines. We compared the ability of T84 epithelial cells to activate STATs in response to cytokines [interferon-γ (IFN-γ), interleukin (IL)-4, IL-10, and tumor necrosis factor-α (10 ng/ml)] and conditioned medium from superantigen [ Staphylococcus aureus enterotoxin B (SEB)]-activated peripheral blood mononuclear cells (PBMC) using electrophoretic mobility shift assays (EMSA). Of the cytokines tested, only IFN-γ caused a STAT-1 response. Exposure to SEB-PBMC-conditioned medium resulted in STAT-1 or STAT-1/3 activation, and inclusion of anti-IFN-γ antibodies in the conditioned medium abolished the STAT-1 signal. Cells treated with transcription factor decoys, DNA oligonucleotides bearing the STAT-1 recognition motif, and then SEB-PBMC-conditioned medium displayed a reduced STAT-1 signal on EMSA, yet this treatment did not prevent the drop in transepithelial resistance (measured in Ussing chambers) caused by SEB-PBMC-conditioned medium. In contrast, the phosphatidylinositol 3′-kinase (PI 3-K) inhibitor LY-294002 significantly reduced the drop in transepithelial resistance caused by SEB-PBMC-conditioned medium. Thus data are presented showing STAT-1 (±STAT-3) and PI 3-K activation in epithelial cells in response to immune mediators released by superantigen immune activation. Although the involvement of STAT-1/-3 in the control of barrier function remains a possibility, PI-3K has been identified as a regulator of T84 paracellular permeability.

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