PHENOMENON OF BIMODAL EXPRESSION OF WASP IN PATIENTS WITH WISKOTT–ALDRICH SYNDROME ASSESSED BY FLOW CYTOMETRY

Compensatory or revertant somatic mutations (RSM) is a well-known phenomenon in patients with PIs. RSM can lead to the restoration of functional protein expression in some cell populations and thus influence the severity of clinical manifestations in patients with PI. The WAS gene, a mutation that leads to the development of Wiskott–Aldrich syndrome (WAS), is associated with high levels of RSM. In our study, we aimed to describe in detail the RSM phenomenon in patients with WAS, and also to try to study its effect on the severity of the clinical phenotype. Materials and methods of research: a single-center, prospective, open, continuous, non-randomized, comparative study was conducted. The study included 39 male patients with WAS from 2 months and up to 18 years old with mutation in WAS gene, who underwent assessment of WASP expression using flow cytometry. In 2 out of 9 patients, use of magnetic selection made it possible to isolate a population of lymphocytes expressing WASP. Germinal and somatic mutations were identified using Sanger sequencing. To assess severity of clinical manifestations of WAS, the original detailed scoring classification was used. Results: 9 patients had bimodal expression of WASP, which equaled to 23% of all examined. The median age of these patients was 3 years [7 months. – 15 years]. In two patients a presence of RSM was confirmed in WASP-expressing populations. Linear assessment of partially restored WASP expression revealed different patterns of lymphocyte subpopulation involvement in all 9 patients. The effect of RSM on the severity of clinical manifestations of WAS in the studied group of patients was statistically insignificant (p=0,39). Conclusion: according to the data obtained, patients with WAS, demonstrating a bimodal pattern of WASP expression, represent a very heterogeneous group in terms of the severity of clinical manifestations, involvement of cell populations, and the nature of RSM. This work is a methodological prerequisite for further comprehensive study of RSM phenomenon in patients with various PIs.

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Clonal B-cell populations in patients with idiopathic thrombocytopenic purpura

Blood ◽

10.1182/blood.v76.11.2321.bloodjournal76112321 ◽

1990 ◽

Vol 76 (11) ◽

pp. 2321-2326 ◽

Cited By ~ 39

Author(s):

D van der Harst ◽

D de Jong ◽

J Limpens ◽

PM Kluin ◽

Y Rozier ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Autoimmune Diseases ◽

B Cell ◽

Malignant Lymphoma ◽

Idiopathic Thrombocytopenic Purpura ◽

Clinical Manifestations ◽

Cell Populations ◽

Dna Rearrangement ◽

Thrombocytopenic Purpura

Idiopathic thrombocytopenic purpura (ITP) may be associated with other autoimmune diseases and the development of lymphoproliferative malignancies. In Sjogren's disease, Graves' disease, and essential mixed cryoglobulinemia, which are also associated with the development of B-cell neoplasia, clonal B-cell expansions have been detected. Eleven patients with ITP were investigated for the presence of a clonal excess (CE) using kappa-lambda flow cytometry and DNA analysis for rearrangement of immunoglobulin heavy and light chain genes in blood and/or spleen lymphocytes. In 10 of 11 patients, clonal B-cell populations were found by one or both tests. In three of these patients, oligoclonal B-cell populations were suggested by the combined findings. In all four patients with a small paraproteinemia, the isotype was confirmed by either flow cytometry or DNA rearrangement analysis. Our data suggest that the oligoclonal expansions are not restricted to CD5+ B cells, as in the majority of patients this subset was below the detection level of flow cytometry or DNA rearrangement analysis. None of the patients developed clinical manifestations of malignant lymphoma during a follow-up period of 10 to 44 months after sampling. We conclude that clonal excess populations of B cells are not a unique feature of malignant lymphoma, but may occur in autoimmune diseases, suggesting a benign (oligo)clonal B-cell proliferation.

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Recent advances in understanding the pathophysiology of Wiskott-Aldrich syndrome

Blood ◽

10.1182/blood-2008-12-115253 ◽

2009 ◽

Vol 113 (25) ◽

pp. 6288-6295 ◽

Cited By ~ 157

Author(s):

Marita Bosticardo ◽

Francesco Marangoni ◽

Alessandro Aiuti ◽

Anna Villa ◽

Maria Grazia Roncarolo

Keyword(s):

Bone Marrow ◽

Wide Spectrum ◽

Clinical Manifestations ◽

Cellular Mechanisms ◽

Gene Encoding ◽

Hematopoietic Stem ◽

Wiskott Aldrich Syndrome ◽

Alternative Therapeutic Approach ◽

Unrelated Donors ◽

Was Gene

Abstract Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency caused by mutations in the gene encoding for WASP, a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in WASP result in a wide spectrum of clinical manifestations ranging from the relatively mild X-linked thrombocytopenia to the classic full-blown WAS phenotype characterized by thrombocytopenia, immunodeficiency, eczema, and high susceptibility to developing tumors and autoimmune manifestations. The life expectancy of patients affected by severe WAS is reduced, unless they are successfully cured by bone marrow transplantation from related identical or matched unrelated donors. Because many patients lack a compatible bone marrow donor, the administration of WAS gene–corrected autologous hematopoietic stem cells could represent an alternative therapeutic approach. In the present review, we focus on recent progress in understanding the molecular and cellular mechanisms contributing to the pathophysiology of WAS. Although molecular and cellular studies have extensively analyzed the mechanisms leading to defects in T, B, and dendritic cells, the basis of autoimmunity and thrombocytopenia still remains poorly understood. A full understanding of these mechanisms is still needed to further implement new therapeutic strategies for this peculiar immunodeficiency.

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Development of flow cytometry assay for Wiskott–Aldrich syndrome diagnosis by WASP protein evaluation

Pediatric Hematology/Oncology and Immunopathology ◽

10.24287/1726-1708-2020-19-2-141-151 ◽

2020 ◽

Vol 19 (2) ◽

pp. 141-151

Author(s):

D. E. Pershin ◽

O. B. Lodoeva ◽

M. S. Fadeeva ◽

I. V. Mersiyanova ◽

A. L. Khoreva ◽

...

Keyword(s):

Flow Cytometry ◽

Threshold Value ◽

Clinical Severity ◽

Malignant Neoplasms ◽

Flow Cytometry Assay ◽

Old Patients ◽

Wiskott Aldrich Syndrome ◽

Increased Risk ◽

Short Period ◽

Was Gene

Wiskott–Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by microplatelet thrombocytopenia, eczema, frequent infections and an increased risk of autoimmune disorders and malignant neoplasms. Mutation detection in WAS gene is the gold standard for diagnosis of this disorder. This gene encodes a WASP protein, which works as regulator of cell cytoskeleton and is involved in the transmission of many intracellular signals. Nowadays there is no rapid and reliable method that allows to confirm WAS in a short period of time. Early detection of WAS in patients enables initiation of a donor search and preparation for the HSCT procedure. It also helps to avoid the development of severe and life-threatening conditions during waiting for genetic confirmation of the diagnosis by using pathogenetic therapy. Currently ﬂow cytometry is one of the leading laboratory methods that permits to get the information about the expression of a protein in several hours. The study below describes rapid and reliable based on flow cytometry assay for WAS diagnosis. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. The study included 46 patients with suspected WAS from 2 months to 17 years old. Patients were examined from January 2018 to January 2020. WAS gene defect was confirmed in 35 patients. It was calculated that normal threshold value for WASP expression is 7.07 with sensitivity and specificity 100% and 93.1% respectively. Besides negative correlation between WASP expression index and WAS clinical severity was shown (r = –0.63). This flow cytometry assay can be used for chimerism detection in WAS patients after HSCT. The flow cytometry assay for WASP protein evaluation is rapid, highly sensitive and highly specific. It allows to speed up diagnosis of this disorder.

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Clonal B-cell populations in patients with idiopathic thrombocytopenic purpura

Blood ◽

10.1182/blood.v76.11.2321.2321 ◽

1990 ◽

Vol 76 (11) ◽

pp. 2321-2326 ◽

Cited By ~ 2

Author(s):

D van der Harst ◽

D de Jong ◽

J Limpens ◽

PM Kluin ◽

Y Rozier ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Autoimmune Diseases ◽

B Cell ◽

Malignant Lymphoma ◽

Idiopathic Thrombocytopenic Purpura ◽

Clinical Manifestations ◽

Cell Populations ◽

Dna Rearrangement ◽

Thrombocytopenic Purpura

Abstract Idiopathic thrombocytopenic purpura (ITP) may be associated with other autoimmune diseases and the development of lymphoproliferative malignancies. In Sjogren's disease, Graves' disease, and essential mixed cryoglobulinemia, which are also associated with the development of B-cell neoplasia, clonal B-cell expansions have been detected. Eleven patients with ITP were investigated for the presence of a clonal excess (CE) using kappa-lambda flow cytometry and DNA analysis for rearrangement of immunoglobulin heavy and light chain genes in blood and/or spleen lymphocytes. In 10 of 11 patients, clonal B-cell populations were found by one or both tests. In three of these patients, oligoclonal B-cell populations were suggested by the combined findings. In all four patients with a small paraproteinemia, the isotype was confirmed by either flow cytometry or DNA rearrangement analysis. Our data suggest that the oligoclonal expansions are not restricted to CD5+ B cells, as in the majority of patients this subset was below the detection level of flow cytometry or DNA rearrangement analysis. None of the patients developed clinical manifestations of malignant lymphoma during a follow-up period of 10 to 44 months after sampling. We conclude that clonal excess populations of B cells are not a unique feature of malignant lymphoma, but may occur in autoimmune diseases, suggesting a benign (oligo)clonal B-cell proliferation.

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POS0369 ELEVATED EXPRESSION OF TIM-3 ON NEUTROPHILS CORRELATES WITH DISEASE ACTIVITY AND SEVERITY OF ANKYLOSING SPONDYLITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.3516 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 414.2-415

Author(s):

X. Huang ◽

T. W. Li ◽

J. Chen ◽

Z. Huang ◽

S. Chen ◽

...

Keyword(s):

Flow Cytometry ◽

Ankylosing Spondylitis ◽

Disease Activity ◽

Immune Cells ◽

Clinical Manifestations ◽

Innate Immune ◽

Innate Immune Cells ◽

Bacterial Killing ◽

Markers Of Inflammation ◽

Significant Difference

Background:Ankylosing spondylitis (AS) is a type of common, chronic inflammatory disease that compromises the axial skeleton and sacroiliac joints, causing inflammatory low back pain and progressive spinal stiffness, over time some patients develop spinal immobility and ankylosis which can lead to a decrease in quality of life. The last few decades, evidence has clearly indicated that neutrophil also plays key roles in the progression of AS. However, the immunomodulatory roles and mechanisms of neutrophils in AS are poorly understood. T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) has been reported as an important regulatory molecule, expressed and regulated on different innate immune cells, plays a pivotal role in several autoimmunity diseases. Recent study indicates that Tim3 is also expressed on neutrophils. However, the frequency and roles of Tim3-expressing neutrophils in AS was not clear.Objectives:In this study, we investigated the expression of Tim3 on neutrophils in AS patients and explored the correlation between the level of Tim3-expressing neutrophils and the disease activity and severity of AS.Methods:Patients with AS were recruited from Guangdong Second Provincial General Hospital (n=62). Age/sex-matched volunteers as Healthy controls (HC) (n=39). The medical history, clinical manifestations, physical examination, laboratory measurements were recorded. The expression of costimulatory molecules including programmed death 1 (PD-1), Tim-3 on neutrophils were determined by flow cytometry. The mRNA expression of PD-1 and Tim-3 was determined by real-time PCR. The levels of Tim3-expressing neutrophils in AS patients were further analyzed for their correlation with the markers of inflammation such as ESR,CRP,WBC and neutrophil count(NE), as well as disease activity and severity of AS. The expression of Tim3 on neutrophils was monitored during the course of treatment (4 weeks).Results:The expression of Tim3 on neutrophils in patients with AS was increased compared to the HC (Figure 1A). However, significant difference was observed in the frequency of PD-1-expressing neutrophils between AS patients and HC (Figure 1B). The expression analysis of Tim-3 mRNA, but not PD-1, confirmed the results obtained from flow cytometry (Figure 1C). The level of Tim3-expressing neutrophils in patients with AS showed an positive correlation with ESR, CRP and ASAS-endorsed disease activity score (ASDAS) (Figure 1D). Moreover, the frequency of Tim3-expressing neutrophils in active patients(ASDAS≥1.3) was increased as compare with the inactive patients (ASDAS<1.3) (Figure 1E). As shown in Figure 1F, the frequency of Tim3-expressing neutrophils decreased after the treatment.Conclusion:Increased Tim-3 expression on neutrophils may be a novel indicator to assess disease activity and severity in AS, which may serves as a negative feedback mechanism preventing potential tissue damage caused by excessive inflammatory responses in AS patients.References:[1]Han, G., Chen, G., Shen, B. & Li, Y., Tim-3: an activation marker and activation limiter of innate immune cells. FRONT IMMUNOL 4 449 (2013).[2]Vega-Carrascal, I. et al., Galectin-9 signaling through TIM-3 is involved in neutrophil-mediated Gram-negative bacterial killing: an effect abrogated within the cystic fibrosis lung. J IMMUNOL 192 2418 (2014).Figure 1.(A,B)The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by flow cytometry.(C) The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by RT-PCR.(D)The correction between Tim3-expressing neutrophils and ESR,CRP,ASDAS.(E) The expression of Tim3 on neutrophils in active and inactive patients.(F) Influence of treatment on the frequency of Tim3-expressing neutrophils.Disclosure of Interests:None declared

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Comparison of Lethal and Nonlethal Mouse Models of Orientia tsutsugamushi Infection Reveals T-Cell Population-Associated Cytokine Signatures Correlated with Lethality and Protection

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed6030121 ◽

2021 ◽

Vol 6 (3) ◽

pp. 121

Author(s):

Alison Luce-Fedrow ◽

Suchismita Chattopadhyay ◽

Teik-Chye Chan ◽

Gregory Pearson ◽

John B. Patton ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Host Response ◽

Cd8 T Cell ◽

Cd4 T Cell ◽

Cell Populations ◽

Murine Models ◽

Orientia Tsutsugamushi ◽

Multicolor Flow Cytometry ◽

Interstrain Difference

The antigenic diversity of Orientia tsutsugamushi as well as the interstrain difference(s) associated with virulence in mice impose the necessity to dissect the host immune response. In this study we compared the host response in lethal and non-lethal murine models of O. tsutsugamushi infection using the two strains, Karp (New Guinea) and Woods (Australia). The models included the lethal model: Karp intraperitoneal (IP) challenge; and the nonlethal models: Karp intradermal (ID), Woods IP, and Woods ID challenges. We monitored bacterial trafficking to the liver, lung, spleen, kidney, heart, and blood, and seroconversion during the 21-day challenge. Bacterial trafficking to all organs was observed in both the lethal and nonlethal models of infection, with significant increases in average bacterial loads observed in the livers and hearts of the lethal model. Multicolor flow cytometry was utilized to analyze the CD4+ and CD8+ T cell populations and their intracellular production of the cytokines IFNγ, TNF, and IL2 (single, double, and triple combinations) associated with both the lethal and nonlethal murine models of infection. The lethal model was defined by a cytokine signature of double- (IFNγ-IL2) and triple-producing (IL2-TNF-IFNγ) CD4+ T-cell populations; no multifunctional signature was identified in the CD8+ T-cell populations associated with the lethal model. In the nonlethal model, the cytokine signature was predominated by CD4+ and CD8+ T-cell populations associated with single (IL2) and/or double (IL2-TNF) populations of producers. The cytokine signatures associated with our lethal model will become depletion targets in future experiments; those signatures associated with our nonlethal model are hypothesized to be related to the protective nature of the nonlethal challenges.

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OP0083 INFECTIOUS AND AUTOINFLAMMATORY MODIC TYPE 1 CHANGES HAVE DIFFERENT PATHOMECHANISMS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.825 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 45.2-45

Author(s):

I. Heggli ◽

R. Schüpbach ◽

N. Herger ◽

T. A. Schweizer ◽

A. Juengel ◽

...

Keyword(s):

Flow Cytometry ◽

Immune Response ◽

T Cell ◽

Cell Populations ◽

Gene Set ◽

Go Enrichment ◽

Modic Type ◽

And Control ◽

Infectious Etiology

Background:Modic type 1 changes (MC1) are vertebral bone marrow (BM) edema that associate with non-specific low back pain (LBP). Two etiologies have been described. In the infectious etiology the anaerobic aerotolerant Cutibacterium acnes (C. acnes) invades damaged intervertebral discs (IVDs) resulting in disc infection and endplate damage, which leads to the evocation of an immune response. In the autoinflammatory etiology disc and endplate damage lead to the exposure of immune privileged disc cells and matrix to leukocytes, thereby evoking an immune response in the BM. Different etiologies require different treatment strategies. However, it is unknown if etiology-specific pathological mechanisms exist.Objectives:The aim of this study was to identify etiology-specific dysregulated pathways of MC1 and to perform in-depth analysis of immune cell populations of the autoinflammatory etiology.Methods:BM aspirates and biopsies were obtained from LBP patients with MC1 undergoing spinal fusion. Aspirates/biopsies were taken prior screw insertion through the pedicle screw trajectory. From each patient, a MC1 and an intra-patient control aspiration/biopsy from the adjacent vertebral level was taken. If C. acnes in IVDs adjacent to MC1 were detected by anaerobic bacterial culture, patients were assigned to the infectious, otherwise to the autoinflammatory etiology.Total RNA was isolated from aspirates and sequenced (Novaseq) (infectious n=3 + 3, autoinflammatory n=5 + 5). Genes were considered as differentially expressed (DEG) if p-value < 0.01 and log2fc > ± 0.5. Gene ontology (GO) enrichment was performed in R (GOseq), gene set enrichment analysis (GSEA) with GSEA software.Changes in cell populations of the autoinflammatory etiology were analyzed with single cell RNA sequencing (scRNAseq): Control and MC1 biopsies (n=1 + 1) were digested, CD45+CD66b- mononuclear cells isolated with fluorescence activated cell sorting (FACS), and 10000 cells were sequenced (10x Genomics). Seurat R toolkit was used for quality-control, clustering, and differential expression analysis.Transcriptomic changes (n=5 + 5) of CD45+CD66b+ neutrophils isolated with flow cytometry from aspirates were analyzed as for total bulk RNAseq. Neutrophil activation (n=3 + 3) was measured as CD66b+ expression with flow cytometry. CD66bhigh and CD66blow fractions in MC1 and control neutrophils were compared with paired t-test.Results:Comparing MC1 to control in total bulk RNAseq, 204 DEG in the autoinflammatory and 444 DEG in the infectious etiology were identified with only 67 shared genes (Fig. 1a). GO enrichment revealed “T-cell activation” (p = 2.50E-03) in the autoinflammatory and “complement activation, classical pathway” (p=1.1E-25) in the infectious etiology as top enriched upregulated biological processes (BP) (Fig 1b). ScRNAseq of autoinflammatory MC1 showed an overrepresentation of T-cells (p= 1.00E-34, OR=1.54) and myelocytes (neutrophil progenitor cells) (p=4.00E-05, OR=2.27) indicating an increased demand of these cells (Fig. 1c). Bulk RNAseq analysis of neutrophils from the autoinflammatory etiology revealed an activated, pro-inflammatory phenotype (Fig 1d), which was confirmed with more CD66bhigh neutrophils in MC1 (+11.13 ± 2.71%, p=0.02) (Fig. 1e).Figure 1.(a) Venn diagram of DEG from total bulk RNAseq (b) Top enriched upregulated BP of autoinflammatory (left) and infectious (right) etiology (c) Cell clustering of autoinflammatory MC1 BM (d) Enrichment of “inflammatory response” gene set in autoinflammatory MC1 neutrophils (e) Representative histogram of CD66b+ expression in MC1 and control neutrophils.Conclusion:Autoinflammatory and infectious etiologies of MC1 have different pathological mechanisms. T-cell and neutrophil activation seem to be important in the autoinflammatory etiology. This has clinical implication as it could be explored for diagnostic approaches to distinguish the two MC1 etiologies and supports developing targeted treatments for both etiologies.Disclosure of Interests:None declared

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Herpes zoster recurrence within 1 month: A case report

European Journal of Inflammation ◽

10.1177/20587392211021214 ◽

2021 ◽

Vol 19 ◽

pp. 205873922110212

Author(s):

Nan Zhao ◽

Yulan Geng ◽

Yexian Li ◽

Lijuan Liu ◽

Yanjia Li ◽

...

Keyword(s):

Flow Cytometry ◽

Case Report ◽

Herpes Zoster ◽

B Cells ◽

Varicella Zoster Virus ◽

Lower Limit ◽

Skin Disease ◽

Clinical Manifestations ◽

Varicella Zoster ◽

Humoral Immune

Herpes zoster (HZ), caused by the varicella-zoster virus, is an infectious skin disease that rarely recurs after initial presentation. The mechanism underlying HZ recurrence is currently under investigation. In this article, we report a case of HZ relapse within 1 month. Analysis of patient’s clinical manifestations, histopathological features, and flow cytometry results indicated that the absolute and percentage values of B cells were below the lower limit. We hypothesized that the patient had abnormal humoral immune function, which may be one reason leading to the HZ relapse within 1 month. The findings of this case will serve as useful reference for HZ recurrence for clinicians. This case was impactful and added to the literature on HZ recurrence.

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