Tregs

Regulatory T cells (Treg cells) are a small subset of immune cells that are dedicated to curbing excessive immune activation and maintaining immune homeostasis. Accordingly, deficiencies in Treg cell development or function result in uncontrolled immune responses and tissue destruction and can lead to inflammatory disorders such as graft-versus-host disease, transplant rejection and autoimmune diseases. As Treg cells deploy more than a dozen molecular mechanisms to suppress immune responses, they have potential as multifaceted adaptable smart therapeutics for treating inflammatory disorders. Indeed, early-phase clinical trials of Treg cell therapy have shown feasibility, tolerability and potential efficacy in these disease settings. In the meantime, progress in the development of chimeric antigen receptors and in genome editing (including the application of CRISPR-Cas9) over the past two decades has facilitated the genetic optimization of primary T cell therapy for cancer. These technologies are now being used to enhance the specificity and functionality of Treg cells. In this Review, we describe the key advances and prospects in designing and implementing Treg cell-based therapy in autoimmunity and transplantation.

The infection with Trypanosoma cruzi leads to a vigorous and apparently uncontrolled inflammatory response in the heart. Although the parasites trigger specific immune response, the infection is not completely cleared out, a phenomenon that in other parasitic infections has been attributed to CD4+CD25+ T cells (Tregs). Then, we examined the role of natural Tregs and its signaling through CD25 and GITR in the resistance against infection with T. cruzi. Mice were treated with mAb against CD25 and GITR and the parasitemia, mortality and heart pathology analyzed. First, we demonstrated that CD4+CD25+GITR+Foxp3+ T cells migrate to the heart of infected mice. The treatment with anti-CD25 or anti-GITR resulted in increased mortality of these infected animals. Moreover, the treatment with anti-GITR enhanced the myocarditis, with increased migration of CD4+, CD8+, and CCR5+ leukocytes, TNF-alpha production, and tissue parasitism, although it did not change the systemic nitric oxide synthesis. These data showed a limited role for CD25 signaling in controlling the inflammatory response during this protozoan infection. Also, the data suggested that signaling through GITR is determinant to control of the heart inflammation, parasite replication, and host resistance against the infection.

CD4 effector T cells, also called helper T (Th) cells, are the functional cells for executing immune functions. Balanced immune responses can only be achieved by proper regulation of the differentiation and function of Th cells. Dysregulated Th cell function often leads to inefficient clearance of pathogens and causes inflammatory diseases and autoimmunity. Since the establishment of the Th1–Th2 dogma in the 1980s, different lineages of effector T cells have been identified that not only promote but also suppress immune responses. Through years of collective efforts, much information was gained on the function and regulation of different subsets of Th cells. In this review, we attempt to sample the essence of what has been learnt in this field over the past two decades. We will discuss the classification and immunological functions of effector T cells, the determinants for effector T cell differentiation, as well as the relationship between different lineages of effector T cells.

Regulatory T cells (Tregs) represent a low number of T‐cell population under normal conditions, and they play key roles for maintaining immune system in homeostasis. The number of these cells is extensively increased in nearly all cancers, which is for dampening responses from immune system against cancer cells, metastasis, tumor recurrence, and treatment resistance. The interesting point is that apoptotic Tregs are stronger than their live counterparts for suppressing responses from immune system. Tregs within the tumor microenvironment have extensive positive cross‐talks with other immunosuppressive cells including cancer‐associated fibroblasts, cancer cells, macrophage type 2 cells, and myeloid‐derived suppressor cells, and they have negative interactions with immunostimulatory cells including cytotoxic T lymphocytes (CTL) and natural killer cells. A wide variety of markers are expressed in Tregs, among them forkhead box P3 (FOXP3) is the most specific marker and the master regulator of these cells. Multiple signals are activated by Tregs including transforming growth factor‐β, signal transducer and activator of transcription, and mTORC1. Treg reprogramming from an immunosuppressive to immunostimulatory proinflammatory phenotype is critical for increasing the efficacy of immunotherapy. This would be applicable through selective suppression of tumor‐bearing receptors in Tregs, including FOXP3, programmed death‐1, T‐cell immunoglobulin mucin‐3, and CTL‐associated antigen‐4, among others. Intratumoral Tregs can also be targeted by increasing the ratio for CTL/Treg.

Aims:  HIV  infection  is  a  secondary  immunodeficiency  disorder
associated  with  loss  ofCD4+Tcells  and  immune  suppression.
Tregs  are  immune  suppressive  cells  and  involved  in  peripheral
tolerance.  Tregs  expressCD4
+
CD25
+
on  the  surface  and  Foxp3
+
internally.  Relative  frequency  of  Tregs  during  HIV/AIDS  is  not
fully known. CD4
+
CD25
+
Foxp3
+
Tcells (Tregs) play a vital role in
peripheral  tolerance  but  its  role  during  HIV/AIDS  is  not  fully
known.  Hence  this  study  was  conducted  with  the  aim  to
characterize the Tregs frequency among HIV patientsand healthy
controls.
Study design: Cross sectional study
Place  and  Duration  of  study: Department  of  Microbiology,
University  of  Madras,  chennai  and  Government  Hospital  Of
Thoracic Medicine, Chennai between October 2012 to  December
2013.
Methodology: 108  individuals  comprising  of  71  HIV  positive
patients (44 patients undergoing HAART and 27 without HAART)
and 37 were HIV negative healthy controls were enrolled in this
study.  PBMCs  were  isolated  and  stained  with  CD4/FITC,
CD25/APC, FoxP3/PE antibodies (BD Pharmingen) and analyzed
for Tregs frequency by flow cytometry (BD FACSCalibur).
Results:  Our  study  showed  that  healthy  controls  had  a  Tregs
frequency  of  4.1%.  When  Tregs  frequency  analysed  among
HIV/AIDS  patients  who  are  yet  to  receive  HAART  we  found  a
significant reduction in Tregs population i.e. 1.2%. Encouragingly
this deep plunge in Tregs population was found to be restored and
surged upon HAART upto 12.6%. There was no difference when
compared between gender or age or absolute CD4 count.
Conclusion:HAART is known to decrease viral load and improve
the  patient’s  CD4  cells  count.  Here  we  report  that  HAART  has
improved  not  only  the  CD4  cells  but  also  the  Tregs  frequency. This  study  add  one  more  dimention
to  Tregs  in  infectious  diseases
especially HIV/AIDS.

To analyze immune cell populations accurately, a large number of Peripheral Blood Mononuclear Cells (PBMCs) must be obtained from blood samples. Traditional manual isolation and SepMate TM isolation of PBMCs consistently yield bloodstained plasma layers and overall low numbers of CD4+ and CD8+ cells. Here, we describe an optimized protocol, using PBS with EDTA to increase PBMC yield from pregnant patients. This protocol enables analysis of CD4+, CD8+, and Regulatory T Cells and is potentially applicable to any immune cell population.

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-z signaling domain. The CDRgrafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR + TCR deficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR + TCR deficient Tregs did not impair the function of these HLA-A2 + islets, whereas similarly engineered A2-CAR + TCR deficient CD4 + conventional T cells rejected the islets in less than 2 weeks. A2-CAR + TCR deficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas' disease.

Infertility in women could be a result of an excessive production of antisperm antibody (ASA). Paternal lymphocyte immunization (PLI) could decrease the ASA levels, but the mechanism is still unclear. The aim of this study was to analyse the impact of a PLI-induced ASA decline on regulatory T-cell populations and serum interleukin-10 (IL-10) levels in women with unexplained infertility. Samples were obtained from patients who came to Sayyidah Mother and Child Hospital in Jakarta from July 2018 to April 2019 with infertility problems. The inclusion criterion for this study was unexplained infertility. Each patient was examined for ASA titres using husband's sperm auto-agglutination test (HSAaT) method, and patients with ASA titres >1:128 were given PLI subcutaneously every 3 weeks. ASA titres were evaluated again 2 weeks after PLI with HSAaT. A total of 12 samples were analysed. Regulatory T-cell populations were evaluated using flow cytometry and human forkhead box P3 FoxP3 s...

Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases;
however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss
of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors
expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m.
injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained
for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT
expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR
Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after
vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population
in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle
were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of
rAAV type 1–AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and
indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.

Schistosoma mansoni soluble egg antigens (SEA) profoundly regulate the infected host's immune system. We previously showed that SEA prevents type 1 diabetes in NOD mice and that splenocytes from SEA-treated mice have reduced ability to transfer diabetes to NOD.scid recipients. To further characterize the mechanism of diabetes prevention we examined the cell types involved and showed that CD25(+) T-cell depletion of splenocytes from SEA-treated donors restored their ability to transfer diabetes. Furthermore, SEA treatment increased the number and proportional representation of Foxp3(+) T cells in the pancreas of NOD mice. We have used in vitro systems to analyze the effect of SEA on the development of NOD Foxp3(+) T cells. We find that SEA can induce Foxp3 expression in naïve T cells in a TGF-beta-dependent manner. Foxp3 induction requires the presence of DC, which we also show are modified by SEA to upregulate C-type lectins, IL-10 and IL-2. Our studies show that SEA can have a direct effect on CD4(+) T cells increasing expression of TGF-beta, integrin beta8 and galectins. These effects of SEA on DC and T cells may act in synergy to induce Foxp3(+) Treg in the NOD mouse.

OBJECTIVE Infertility in women could be a result of an excessive production of antisperm antibody (ASA). Paternal lymphocyte immunization (PLI) could decrease the ASA levels, but the mechanism is still unclear. The aim of this study was to analyse the impact of a PLI-induced ASA decline on regulatory T-cell populations and serum interleukin-10 (IL-10) levels in women with unexplained infertility. Methods Samples were obtained from patients who came to Sayyidah Mother and Child Hospital in Jakarta from July 2018 to April 2019 with infertility problems. The inclusion criterion for this study was unexplained infertility. Each patient was examined for ASA titres using husband's sperm auto-agglutination test (HSAaT) method, and patients with ASA titres >1:128 were given PLI subcutaneously every 3 weeks. ASA titres were evaluated again 2 weeks after PLI with HSAaT. A total of 12 samples were analysed. Regulatory T-cell populations were evaluated using flow cytometry and human forkh...

Infertility in women could be a result of an excessive production of antisperm antibody (ASA). Paternal lymphocyte immunization (PLI) could decrease the ASA levels, but the mechanism is still unclear. The aim of this study was to analyse the impact of a PLI-induced ASA decline on regulatory T-cell populations and serum interleukin-10 (IL-10) levels in women with unexplained infertility. Samples were obtained from patients who came to Sayyidah Mother and Child Hospital in Jakarta from July 2018 to April 2019 with infertility problems. The inclusion criterion for this study was unexplained infertility. Each patient was examined for ASA titres using husband's sperm auto-agglutination test (HSAaT) method, and patients with ASA titres >1:128 were given PLI subcutaneously every 3 weeks. ASA titres were evaluated again 2 weeks after PLI with HSAaT. A total of 12 samples were analysed. Regulatory T-cell populations were evaluated using flow cytometry and human forkhead box P3 FoxP3 s...

T-regulatory (Treg) cells are important to immune homeostasis, and Treg cell deficiency or dysfunction leads to autoimmune disease. A histone/protein acetyltransferase (HAT), p300, was recently found to be important for Treg function and stability, but further insights into the mechanisms by which p300 or other HATs affect Treg biology are needed. Here we show that CBP, a p300 paralog, is also important in controlling Treg function and stability. Thus, while mice with Treg-specific deletion of CBP or p300 developed minimal autoimmune disease, the combined deletion of CBP and p300 led to fatal autoimmunity by 3 to 4 weeks of age. The effects of CBP and p300 deletion on Treg development are dose dependent and involve multiple mechanisms. CBP and p300 cooperate with several key Treg transcription factors that act on the Foxp3 promoter to promote Foxp3 production. CBP and p300 also act on the Foxp3 conserved noncoding sequence 2 (CNS2) region to maintain Treg stability in inflammatory e...

CCAAT enhancer binding protein-delta (C/EBPδ) is a transcription factor that regulates inflammatory processes mediating bystander neuronal injury and CNS autoimmune inflammatory disease. The mechanism of the involvement of C/EBPδ in these processes remains to be determined. Here, we examined the cellular source(s) and mechanisms by which C/EBPδ may be involved in an animal model of multiple sclerosis. Mice deficient in C/EBPδ expression exhibited less severe clinical disease than wild-type littermates in response to induction of experimental autoimmune encephalomyelitis (EAE) by vaccination with a myelin oligodendrocyte glycoprotein (MOG) fragment. This reduction in EAE severity was associated with a significant alteration in the complement of major CNS T-helper (Th) cell subtypes throughout disease, manifest as reduced ratios of Th17 cells to regulatory T-cells (Tregs). Studies in bone marrow chimeric mice indicated that C/EBPδ expression by peripherally derived immune cells mediat...

Graphical Abstract Highlights d APC controls microtubule organization and NFAT-driven cytokine gene expression d APC silencing impairs NFAT nuclear localization and activation d NFAT associates with microtubules that control its localization and its activation d Apc Min/+ Tregs have intrinsically reduced differentiation and IL-10 production