Activation Markers and Regulatory T cells in Children with Chronic Pyelonephritis Associated with Bacterial Uropathogens

Abstract The development of inhibitory antibodies (inhibitors) against FVIII is the most critical complication in the treatment of hemophilia A patients as hemostasis can no longer be reestablished by FVIII replacement therapy. Immune tolerance induction by frequent FVIII infusions is demanding, costly and not successful in all treated patients leading to an urgent need for the development of new therapeutic approaches for the prevention or treatment of FVIII inhibitors. Regulatory T cells (Tregs) are important for the maintenance of tolerance and have a high therapeutic potential in the context of autoimmune or inflammatory immune disorders. As Tregs are polyclonal, treatment with Treg pools comprises the risk of a general immunosuppression. Thus, the establishment of antigen-specific Tregs could be of great benefit for a broad range of patients, including inhibitor positive hemophilia A patients. To create such specific Tregs, a FVIII-specific scFv isolated out of a synthetic phage display library was used to generate a second generation chimeric antigen receptor (CAR). To verify the specificity of the CAR for FVIII and the functionality of the recombined cytoplasmic domain (CD28 and CD3zeta), naïve CD4 T cells were retrovirally transduced with the generated CAR construct and a proliferation assay was conducted in the presence of plate-bound or soluble FVIII, as well as soluble FVIII presented by autologous irradiated PBMCs. Proliferation of transduced cells was more effective when FVIII was presented plate-bound or by PBMCs. In a therapeutically-relevant setting, this would be very promising, as transduced T cells should not be activated by soluble FVIII in the bloodstream but rather by FVIII presented on antigen-presenting cells in lymphatic organs. Next, functionality of the CAR construct in Tregs was addressed. Transduced Tregs showed extracellular expression of the scFv and could be stimulated MHC-independently with FVIII. Such stimulated cells showed increased expression of Treg activation markers LAP and GARP. Thus, by using the generated CAR for transduction of Tregs, it was possible to create FVIII-specific Tregs that can be stimulated MHC-independently, opening new possibilities for therapeutic approaches in hemophilia A patients with FVIII inhibitors. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Rabbit ATG but not horse ATG promotes expansion of functional CD4+CD25highFOXP3+ regulatory T cells in vitro

Blood ◽

10.1182/blood-2008-01-130146 ◽

2008 ◽

Vol 111 (7) ◽

pp. 3675-3683 ◽

Cited By ~ 154

Author(s):

Xingmin Feng ◽

Sachiko Kajigaya ◽

Elena E. Solomou ◽

Keyvan Keyvanfar ◽

Xiuli Xu ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Expression Patterns ◽

Cell Number ◽

Therapeutic Effects ◽

Peripheral Blood Mononuclear ◽

Activation Markers

Abstract Regulatory T cells (Treg) play important roles in suppressing immune responses and maintaining tolerance. Rabbit antithymocyte globulin (rATG) and horse ATG (hATG) are widely used in the treatment of immune-mediated syndromes, but their effects on Treg are unknown. We show here that in vitro culture of normal human peripheral blood mononuclear cells (PBMCs) with a low-dose rATG resulted in marked expansion of functional Treg by converting CD4+CD25− T cells to CD4+CD25+ T cells. hATG did not expand but rather decreased Treg. Immuno-blot showed increased expression of FOXP3 and NFAT1 in CD4+CD25− and CD4+CD25+ T cells exposed to rATG. PBMCs treated with rATG displayed increased interleukin-10 in culture supernatants than those treated with hATG. Furthermore, rATG and hATG showed differences in their potential to stimulate CD4+ T cells as examined using different activation markers. Microarray revealed that rATG induced markedly different gene-expression patterns in PBMCs, compared with hATG-treated or untreated PBMCs. Our findings indicate that rATG expanded Treg, probably through transcriptional regulation by enhanced NFAT1 expression, in turn conferring CD4+CD25− T cell FOXP3 expression and regulatory activity. The therapeutic effects of rATG may occur not only because of lymphocyte depletion but also enhanced Treg cell number and function.

Download Full-text

In Vitro-Generated Antigen-Specific CD4+ CD25+ Foxp3+ Regulatory T Cells Control the Severity of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions

Journal of Virology ◽

10.1128/jvi.00697-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 6838-6851 ◽

Cited By ~ 50

Author(s):

Sharvan Sehrawat ◽

Susmit Suvas ◽

Pranita P. Sarangi ◽

Amol Suryawanshi ◽

Barry T. Rouse

Keyword(s):

T Cells ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Regulatory T Cells ◽

Inflammatory Disease ◽

Activation Markers ◽

Control Virus ◽

Simplex Virus

ABSTRACT Generating and using regulatory T cells (Tregs) to modulate inflammatory disease represents a valuable approach to therapy but has not yet been applied as a means to control virus-induced immunopathological reactions. In this report, we developed a simplified technique that used unfractionated splenocytes as a precursor population and showed that stimulation under optimized conditions for 5 days with solid-phase anti-CD3 monoclonal antibody in the presence of transforming growth factor β (TGF-β) and interleukin-2 could induce up to 90% of CD4+ T cells to become Foxp3+ and able to mediate suppression in vitro. CD11c+ dendritic cells were intricately involved in the conversion process and, once modified in the presence of TGF-β, could convert Foxp3− CD4+ cells into Foxp3+ CD4+cells by producing TGF-β. The converted cells had undergone cell division, and the majority of them expressed activation markers along with surface molecules that would facilitate their migration into tissue sites. The primary reason for our study was to determine if such in vitro-converted Tregs could be used in vivo to influence the outcome of a virus-induced immunoinflammatory lesion in the eye caused by herpes simplex virus infection. We could show in three separate models of herpetic stromal keratitis that adoptive transfers of in vitro-converted Tregs effectively diminished lesion severity, especially when given in the initial phases of infection. The suppression effect in vivo appeared to be polyspecific. The protocol we have developed could provide a useful additional approach to control virus-induced inflammatory disease.

Download Full-text

Revealing the specificity of regulatory T cells in murine autoimmune diabetes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715590115 ◽

2018 ◽

Vol 115 (20) ◽

pp. 5265-5270 ◽

Cited By ~ 20

Author(s):

Allyson Spence ◽

Whitney Purtha ◽

Janice Tam ◽

Shen Dong ◽

Youmin Kim ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Autoimmune Diabetes ◽

Nod Mice ◽

Cell Receptor ◽

Antigen Specificity ◽

Activation Markers ◽

Nonobese Diabetic ◽

Organ Specific ◽

Control Organ

Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Compared with Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαβ pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9–23 and proinsulin. Consistently, insulin B:9–23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28−/− recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes.

Download Full-text

HLA-G and CD8+ regulatory T cells in the inflammatory environment of pre-eclampsia

Reproduction ◽

10.1530/rep-15-0608 ◽

2016 ◽

Vol 152 (6) ◽

pp. 741-751 ◽

Cited By ~ 12

Author(s):

Priscila Vianna ◽

Andressa G Mondadori ◽

Moisés E Bauer ◽

Dinara Dornfeld ◽

José A B Chies

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Pregnant Women ◽

Gene Polymorphisms ◽

Cell Activation ◽

Low Frequency ◽

Activation Markers ◽

Maternal Immune System ◽

Tnf Α ◽

Th17 Cytokines

During pregnancy, the maternal immune system is tolerant to foetal antigens via the engagement of immune regulatory mechanisms. Failure in regulating the maternal immunity to foetal antigens may lead to pre-eclampsia (PE). We addressed the role ofHLA-Ggene polymorphisms and protein expression as well as regulatory T cells and Th1/Th2/Th17 cytokines in healthy and pathological pregnancies. Blood samples from 26 pregnant women with PE, 25 non-PE and 7 strictly healthy pregnant women were assessed. PBMCs were phenotyped for early activation markers (CD25 and CD69), regulatory T-cell markers (CD8+CD28−and CD4+CD25highFoxp3+), ILT-2 (HLA-G receptor) and HLA-G. Lymphocyte proliferation was estimated and levels of IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α and IL-17 were measured. HLA-G polymorphisms (rs66554220 and rs1063320) were genotyped by PCR. PE women exhibited low levels of HLA-G in PBMCs and low frequency of regulatory CD8+CD28−T cells. High amounts of the pro-inflammatory cytokines IL-17, IL-2 and TNF-α as well as IL-4 and IL-10 and an increased proliferative cell activation profile were observed in PE. The allelic and genotypic frequencies of theHLA-Ggene polymorphisms and the frequency of CD4+CD25highFoxp3+T cells did not vary among the groups. Our data suggest that the cytokine imbalance presented in PE is associated with a deficient immune regulatory profile, contributing to an impaired immune tolerance between mother and foetus.

Download Full-text

Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001749 ◽

2021 ◽

Vol 9 (2) ◽

pp. e001749

Author(s):

Helena Van Damme ◽

Bruno Dombrecht ◽

Máté Kiss ◽

Heleen Roose ◽

Elizabeth Allen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Tumor Growth ◽

Fusion Proteins ◽

Antitumor Immunity ◽

Flow Cytometric Analysis ◽

Activation Markers ◽

Cell Lung Carcinoma ◽

Treg Population

BackgroundModulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker.MethodsWe employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models.ResultsWe were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs.ConclusionsCollectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy.

Download Full-text

An imbalance of esophageal effector and regulatory T cell subsets in experimental eosinophilic esophagitis in mice

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00148.2009 ◽

2009 ◽

Vol 297 (3) ◽

pp. G550-G558 ◽

Cited By ~ 22

Author(s):

Xiang Zhu ◽

Meiqin Wang ◽

Caleb H. Crump ◽

Anil Mishra

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Eosinophilic Esophagitis ◽

Critical Role ◽

T Cell Subsets ◽

Activation Markers ◽

Activated T Cell ◽

Anti Inflammatory Cytokine

We recently reported a critical role for T cells in the induction of eosinophilic esophagitis (EE) in mice; however, the role of specific T cell subsets in disease pathogenesis is not yet understood. In the current study, we tested the hypothesis that allergen-induced EE develops in response to the disproportion of functionally different effector and regulatory T cells in the esophagus. Fluorescence-activated cell sorter analysis was performed to examine activated T cell subsets using the cell surface activation markers CD25 and CD69. A significant increase in activated CD4+ and CD4− T cells was observed in the total esophageal cells isolated from the mouse model of EE. Furthermore, an imbalance in the effector and regulatory T cells was observed in the esophagus. The esophageal CD4+CD45RBhigh effector T cells in allergen-challenged mice increased compared with saline-challenged mice (65.4 ± 3.6 × 103 to 44.8 ± 4.2 × 103), whereas CD4+CD45RBlow mostly regulatory T cells decreased in allergen-challenged mice compared with saline-challenged mice (5.8 ± 0.9 × 103 from 10.2 ± 1.7 × 103). The functional characteristics were examined by analysis of the pro- and anti-inflammatory cytokine profile of purified low and high CD4+CD45RB subsets from the spleen. Additionally, a significantly reduced interleukin (IL)-2 production by CD4+CD45RBlow cells in allergen-challenged mice compared with saline-challenged mice was observed. The reduced IL-2 in the CD4+CD45RBlow subset may be associated with reduction of CD4+CD45RBlow subset. In conclusion, our results suggest that local regulatory interaction of CD45RBhigh and CD45RBlow CD4+ T cells may be required for protective and pathogenic immunity in EE.

Download Full-text

Activation of CD4 and CD8 T cell receptors and regulatory T cells in response to human proteins

PeerJ ◽

10.7717/peerj.4462 ◽

2018 ◽

Vol 6 ◽

pp. e4462 ◽

Cited By ~ 2

Author(s):

Borros M. Arneth

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Regulatory T Cells ◽

Serum Proteins ◽

Human Albumin ◽

System Response ◽

Activation Markers ◽

Cytoplasmic Proteins ◽

Intracellular Cytokines ◽

Human Proteins

This study assessed in detail the influence of four different human proteins on the activation of CD4+ and CD8+ T lymphocytes and on the formation of regulatory T cells. Human whole-blood samples were incubated with four different human proteins. The effects of these proteins on the downstream immune-system response, on the expression of extracellular activation markers on and intracellular cytokines in T lymphocytes, and on the number of regulatory T cells (T-reg cells) were investigated via flow cytometry. Incubation with β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which are cytoplasmic proteins, increased the expression of both extracellular activation markers (CD69 and HLA-DR) and intracellular cytokines but did not significantly affect the number of T-reg cells. In contrast, incubation with human albumin or insulin, which are serum proteins, reduced both extracellular activation markers and intracellular cytokine expression and subsequently increased the number of T-reg cells. These findings may help to explain the etiological basis of autoimmune diseases.

Download Full-text

Generation of Human Regulatory T Cells Using NOD/SCID/γ CNULL Mice Model.

Blood ◽

10.1182/blood.v104.11.2787.2787 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2787-2787

Author(s):

Hidefumi Hiramatsu ◽

Hisanori Fujino ◽

Toshio Heike ◽

Mamoru Ito ◽

Tatsutoshi Nakahata

Keyword(s):

Stem Cells ◽

T Cells ◽

Regulatory T Cells ◽

Hematopoietic Stem Cells ◽

Mice Model ◽

Activation Markers ◽

Hematopoietic Stem ◽

Inhibition Of Proliferation ◽

Human T Cells ◽

Nog Mice

Abstract We have reported a NOD/SCID/γcnull (NOG) mice model, which enable efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells. Using this model, we investigated whether various subpopulations of T cells were generated in this unique murine microenvironment. Freshly collected cord blood was depleted of phagocytes with Silica ® followed by CD34 positive selection using auto MACS ®. The purity of CD34 positive cells always exceeded 98%. These cells were transplanted into irradiated NOG mice intravenously. About 3 months after the transplantation, human T cells in peripheral blood, bone marrow and spleen were analyzed by flow cytometry. As we have reported previously, more than half of the human cells seen in the spleen were human CD3+ T cells and as many as 30% of them expressed CD4 and CD25 without activation markers such as CD69. To examine if these CD4+ CD25+ cells have regulatory activity, CD4+ CD25− cells were stimulated with anti-human CD3 antibody along with irradiated autologous antigen presenting cells in the presence of limiting dose of CD4+ CD25+ cells. The inhibition of proliferation by CD4+ CD25+ cells was analyzed by 3H-thymidine uptake. CD4+ CD25+ cells successfully suppressed the CD4+ CD25− T cell proliferation and RT-PCR analysis revealed the expression of Foxp3, a marker for regulatory T cells, specifically in the CD4+ CD25+ cell population. These results suggest that regulatory T cells can develop from hematopoietic stem cells in our NOG mice model. As human T cells appear first in the thymus of NOG mice, these regulatory T cells are considered to arise in the murine thymus. Our model provides a new and versatile tool to investigate development and function of human regulatory T cells, which are often difficult to study because of complicated history of infection or genetic differences among individuals.

Download Full-text