The Involvement of Regulatory T Cells in Immunopathophysiology of Autoimmune Neutropenia in Infancy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3835-3835
Author(s):  
Kazuhiro Nakamura ◽  
Mizuka Miki ◽  
Hiroshi Kawaguchi ◽  
Takashi Sato ◽  
Emi Kurita ◽  
...  
Keyword(s):  
T Cells ◽  
Autoimmune Disease ◽  
Regulatory T Cells ◽  
Matched Control ◽  
Age Groups ◽  
Foxp3 Expression ◽  
Autoimmune Neutropenia ◽  
Spontaneous Disappearance ◽  
Standard Value ◽  
Infancy And Early Childhood

Abstract CD4+25+ regulatory T cells (Treg) have been recognized to play a significant role in the immunopathogenesis of autoimmune disease, tumors, and organ transplantation. Several human autoimmune diseases in adulthood have been reported to associate with alternations in Treg. To date little is known about the standard value of Treg in infancy and childhood and its involvement in pediatric autoimmune disease. Autoimmune neutropenia (AIN) in infancy is a common disorder characterized by chronic neutropenia, the detection of antineutrophil antibodies in sera, and spontaneous resolution of neutropenia within several months to a few years. In this study, to elucidate the involvement of Treg in AIN known as a representative autoimmune disease in infancy and early childhood, we examined the frequencies of Treg in various normal age groups and AIN patient. Blood samples from normal neonates (n=22), infants (1 month through 3 years in age, n=21), adults (n=12) and AIN patients (1 month through 3 year in age, n=22) were obtained after informed consent. The frequency of Treg was assessed by the expression of CD4, CD25, and FOXP3 using flow cytometry. As shown in Table, the frequency of CD4+25high Treg known as pure Treg fraction was highest in neonates and decreased by aging. Notably CD4+25high Treg in AIN patients was fewer than age-matched control infants (P<0.01). Furthermore, the ratio of intracytosolic FOXP3 expression of each Treg fraction in AIN patients was fewer than age-matched control infants (P<0.05). The spontaneous disappearance of antineutrophil antibody and the restoration of the frequency of CD4+25high cells that preceded the normalization of the neutrophil count were found in two patients with AIN. These results suggest that Treg could contribute to the immunopathophysiology of AIN in infancy. Percentage of CD25+, CD25high, and CD25low in CD4+ cells Neonates Infants Adults Patients with AIN Date represent mean±SD. CD25+ 11.9±2.6 10.1±2.0 18.5±3.9 9.5±1.9 CD25high 8.3±2.7 5.6±1.9 4.2±1.1 4.0±1.6 CD25low 3.6±1.2 4.5±1.9 14.3±3.6 5.5±1.5

The Decrease of Regulatory T Cells through the Low Levels of FOXP3 Expression Mediated by NFAT1 in Infancy with Autoimmune Neutropenia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 666-666
Author(s):  
Kazuhiro Nakamura ◽  
Mizuka Miki ◽  
Syuhei Karakawa ◽  
Rie Onodera ◽  
Emi Kurita ◽  
...  
Keyword(s):  
T Cells ◽  
Autoimmune Disease ◽  
Regulatory T Cells ◽  
Age Groups ◽  
Foxp3 Expression ◽  
Autoimmune Neutropenia ◽  
Foxp3 Mrna ◽  
Healthy Infants ◽  
Standard Value ◽  
Infancy And Early Childhood

Abstract CD4+ /CD25+ regulatory T cells have been recognized to regulate the maintenance of self-tolerance, and several human autoimmune diseases in adulthood. To date, little is known about the standard value of regulatory T cells in infancy and childhood and about its involvement in pediatric autoimmune disease. Autoimmune neutropenia (AIN) in infancy is a common disorder characterized by chronic neutropenia, the detection of antineutrophil antibodies in sera, and spontaneous resolution of neutropenia within several months to a few years. In this study we examined the frequencies of regulatory T cells in various normal age groups and AIN patients to elucidate the involvement of regulatory T cells in AIN known as a representative autoimmune disease in infancy and early childhood. Blood samples from healthy neonates (n = 22), infants (1 month through 3 years in age, n = 21), adults (n = 12) and AIN patients (1 month through 3 years in age, n = 22) were obtained after informed consent. The frequencies of regulatory T cells were assessed by the expression of CD4, CD25, and FOXP3 using flow cytometry. The mRNA expressions of FOXP3 and its transcription factor, NFAT in CD4+ /CD25+ regulatory T cells were determined by quantitative real-time PCR. The frequency of CD4+ /CD25high regulatory T cells known as pure regulatory T cells fraction was highest in neonates and decreased by aging. Notably, the number of CD4+ /CD25high regulatory T cells in AIN patients significantly decreased, compared with that of age-matched healthy infants (P < 0.01) and the ratio of intracytosolic FOXP3 expression of each regulatory T cell fraction in AIN patients was lower than that of age-matched healthy infants (P < 0.05). Next, we purified CD4+ /CD25+ T cells by using FACS-Aria (more than 95% of purity) and the mRNA expressions in CD4+ /CD25+ T cells were quantified. The expressions of FOXP3 and NFAT1 mRNA in AIN patients were significantly lower than those of healthy infants (P < 0.05). The low level of FOXP3 mRNA in patients well correlated with the expression level of NFAT1 mRNA, implying that the expression of FOXP3 was mediated by NFAT1. Furthermore, the decreased frequency of CD4+ /CD25high regulatory T cells and FOXP3-positive cells in AIN patients were restored to normal levels when the neutrophil count was spontaneously recovered. These results strongly suggest that regulatory T cells play an important role in the immunopathophysiology of AIN in childhood.

scholarly journals Conversion of T-Effector Cells to Immunosuppressive T-Regulatory-like Cells By CRISPR/Cas9-Mediated Integration of a FOXP3 Transgene

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3490-3490 ◽  
Author(s):  
Yuchi Honaker ◽  
Yufei Xiang ◽  
Logan Fisher ◽  
Karen Sommer ◽  
Troy R. Torgerson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autoimmune Disease ◽  
Regulatory T Cells ◽  
Peripheral Blood ◽  
Gene Editing ◽  
Ex Vivo ◽  
Foxp3 Expression ◽  
Self Tolerance ◽  
Equity Ownership

Abstract Regulatory T cells (Treg) are distinct among T cell subtypes, having the primary role of suppressing adaptive immune responses. The importance of these cells in immune self-tolerance is underscored by the genetically inherited syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), which is caused by an inactivating mutation in FOXP3. FOXP3 is a transcription factor that is a determinant of regulatory T cell function. Patients with IPEX syndrome suffer from the rapid and severe onset of multi-organ autoimmunity, including severe enteropathy, Type I diabetes, thyroiditis, skin inflammation and other features. In mouse models of IPEX, neonatal transplantation of wild-type Tregs is sufficient to prevent the development of disease. Less-severe Treg defects have also been implicated in the etiology of a variety of prevalent autoimmune diseases. It is possible that the pivotal role for Tregs in self-tolerance could be exploited clinically to improve therapies for autoimmunity and other diseases of tolerance. However, the use of autologous ex vivo expanded Treg as a clinical cell therapy is problematic: Tregs are present in low numbers in the peripheral blood, they expand slowly in culture ex vivo, and they may lack antigen specificities necessary for efficient suppression in specialized tissues. They may also down-regulate FOXP3 expression and lose functional activity in vivo in the setting of chronic inflammation. Additionally, autologous Tregs from patients with autoimmune disease may exhibit cell intrinsic dysfunction, while IPEX patients do not even have Tregs. To overcome these issues, we developed a gene editing approach to enforce stable expression of FOXP3 in primary human CD4+ peripheral blood T cells. CRISPR/Cas9 ribonucleoprotein and an AAV6-delivered donor template were developed to target a MND promoter-FOXP3 cDNA expression cassette (linked to a cell surface LNGFR tag by a 2A ribosome skip peptide) to the FOXP3 locus by homology directed repair (HDR). Highly efficient HDR rates were achieved across multiple donors (~34%; 5 donors in 9 experiments). For therapy of IPEX caused by FOXP3 missense mutations, integration of the functional coding sequence simultaneously abolishes endogenous FOXP3 expression. Following gene editing, expression of FOXP3 was sufficient to drive Treg-like phenotypic changes, including the up-regulation of CD25 and inhibitory receptors and down-regulation of CD127 and inflammatory cytokines. Further, consistent with the translatability of this approach into clinical manufacturing, FOXP3+ cells could be enriched to >90% purity by a simple LNGFR antibody column and expanded 20-fold within one week. Importantly, transfer of these edited Treg-like cells (edTreg) to NOD-scid-IL2Rγ-/- mice prevented xeno-graft vs. host disease (xeno-GvHD) mediated by co-transferred autologous effector T cells; xeno-GvHD protection correlated with long-term survival of the edTregs, and a marked reduction in effector T cell expansion and tissue infiltration. These data support the development of edited regulatory T cells for the treatment of IPEX and other autoimmune disease. Disclosures Scharenberg: Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership.

scholarly journals CRISPR Screen in Regulatory T Cells Reveals Ubiquitination Modulators of Foxp3

2020 ◽  
Author(s):  
Jessica T. Cortez ◽  
Elena Montauti ◽  
Eric Shifrut ◽  
Yusi Zhang ◽  
Oren Shaked ◽  
...  
Keyword(s):  
T Cells ◽  
Autoimmune Disease ◽  
Regulatory T Cells ◽  
Tumor Immunity ◽  
Screening Method ◽  
Foxp3 Expression ◽  
Negative Regulator ◽  
Multiple Cancer ◽  
Primary Mouse ◽  
Gene Regulatory

AbstractRegulatory T cells (Tregs) are required to control immune responses and maintain homeostasis but are a significant barrier to anti-tumor immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of pro-inflammatory properties2, can promote autoimmunity and/or facilitate more effective tumor immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. Despite improved functional genetic tools that now allow for systematic interrogation, dissection of the gene regulatory programs that modulate Foxp3 expression has not yet been reported. In this study, we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Tregs and applied this technology to perform a targeted loss-of-function screen of ∼490 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We discovered several novel modulators including ubiquitin-specific peptidase 22 (Usp22), Ataxin 7 like 3 (Atxn7l3) and ring finger protein 20 (Rnf20). Members of the deubiquitination module of the SAGA chromatin modifying complex, Usp22 and Atxn7l3, were discovered to be positive regulators that stabilized Foxp3 expression; whereas the screen suggested Rnf20, an E3 ubiquitin ligase, is a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein and created defects in their suppressive function that led to spontaneous autoimmunity but protected against tumor growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Tregs could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Tregs. These results reveal novel modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

scholarly journals Secretome screening reveals immunomodulating functions of IFNα-7, PAP and GDF-7 on regulatory T-cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mei Ding ◽  
Rajneesh Malhotra ◽  
Tomas Ottosson ◽  
Magnus Lundqvist ◽  
Aman Mebrahtu ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cell Function ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Secreted Proteins ◽  
External Stimulation ◽  
Marker Proteins ◽  
Positive Regulators ◽  
Conformational Model

AbstractRegulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.

scholarly journals FOXP3 Defines Regulatory T Cells in Human Tumor and Autoimmune Disease

2009 ◽  
Vol 69 (9) ◽  
pp. 3995-4000 ◽  
Author(s):  
Ilona Kryczek ◽  
Rebecca Liu ◽  
Guobin Wang ◽  
Ke Wu ◽  
Xiaogong Shu ◽  
...  
Keyword(s):  
T Cells ◽  
Autoimmune Disease ◽  
Regulatory T Cells ◽  
Human Tumor

scholarly journals A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease

2018 ◽  
Vol 95 ◽  
pp. 1-14 ◽  
Author(s):  
Laurence B. Peterson ◽  
Charles J.M. Bell ◽  
Sarah K. Howlett ◽  
Marcin L. Pekalski ◽  
Kevin Brady ◽  
...  
Keyword(s):  
T Cells ◽  
Autoimmune Disease ◽  
Regulatory T Cells

scholarly journals CD38 Correlates with an Immunosuppressive Treg Phenotype in Lupus-Prone Mice

2021 ◽  
Vol 22 (21) ◽  
pp. 11977
Author(s):  
Jocelyn C. Pérez-Lara ◽  
Enrique Espinosa ◽  
Leopoldo Santos-Argumedo ◽  
Héctor Romero-Ramírez ◽  
Gabriela López-Herrera ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Immune Activation ◽  
Lupus Erythematosus ◽  
Clinical Signs ◽  
Foxp3 Expression ◽  
Cell Receptor ◽  
Treg Cells ◽  
Transmembrane Glycoprotein ◽  
Ifn Γ

CD38 is a transmembrane glycoprotein expressed by T-cells. It has been reported that patients with systemic lupus erythematosus (SLE) showed increased CD38+CD25+ T-cells correlating with immune activation and clinical signs. Contrariwise, CD38 deficiency in murine models has shown enhanced autoimmunity development. Recent studies have suggested that CD38+ regulatory T-cells are more suppressive than CD38− regulatory T-cells. Thus, we have suggested that CD38 overexpression in SLE patients could play a role in regulating immune activation cells instead of enhancing it. This study found a correlation between CD38 with FoxP3 expression and immunosuppressive molecules (CD69, IL-10, CTLA-4, and PD-1) in T-cells from lupus-prone mice (B6.MRL-Faslpr/J). Additionally, B6.MRL-Faslpr/J mice showed a decreased proportion of CD38+ Treg cells regarding wild-type mice (WT). Furthermore, Regulatory T-Cells (Treg cells) from CD38-/- mice showed impairment in expressing immunosuppressive molecules and proliferation after stimulation through the T-cell receptor (TCR). Finally, we demonstrated an increased ratio of IFN-γ/IL-10 secretion in CD38-/- splenocytes stimulated with anti-CD3 compared with the WT. Altogether, our data suggest that CD38 represents an element in maintaining activated and proliferative Treg cells. Consequently, CD38 could have a crucial role in immune tolerance, preventing SLE development through Treg cells.

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