Co-expression of Foxp3 and Helios facilitates the identification of human T regulatory cells in health and disease.

Foxp3 is regarded as the major transcription factor for T regulatory (Treg) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4+ T conventional (Tconv) cells activated in vitro by T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a Treg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3+ Treg cells and can potentially serve as a marker of human Treg. Here, we confirm that Foxp3 expression is readily upregulated by Tconv upon TCR stimulation in vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hTconv in a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4+ T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon Tconv cell activation in vivo and that Foxp3 expression alone can be used to quantitate Treg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of Treg in humans.

Control of regulatory T cell homeostasis.

CD4+ Foxp3+ T Regulatory (Treg) cells play a critical role in the homeostasis and maintenance of the immune system. The understanding of different aspects of Treg cells biology remains an intensively investigated subject as altering their generation, stability, or function by drugs or biologics may have therapeutic value in the treatment of autoimmune and inflammatory diseases as well as cancers. This review will focus on recent studies on the role of cytokines, T Cell Receptor (TCR) and co-stimulatory/co-inhibitory molecules signaling, location and metabolism on the homeostasis and stability of Treg cells. The potential for therapeutic manipulation of each of these factors will be discussed.

Cutting Edge: Inhibition of the Interaction of NK Inhibitory Receptors with MHC Class I Augments Antiviral and Antitumor Immunity.

NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I-specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti-MHC-I mAb that blocks NK cell inhibitory receptor binding at a site distinct from the TCR binding site. Treatment of unmanipulated mice with this mAb disrupted immune homeostasis, markedly activated NK and memory phenotype T cells, enhanced immune responses against transplanted tumors, and augmented responses to acute and chronic viral infection. mAbs of this type represent novel checkpoint inhibitors in tumor immunity, potent tools for the eradication of chronic infection, and may function as adjuvants for the augmentation of the immune response to weak vaccines.

T Follicular Regulatory Cell Suppression of T Follicular Helper Cell Function Is Context-Dependent in vitro.

The production of antibody-secreting plasma cells and memory B cells requires the interaction of T follicular helper (Tfh) cells with B cells in the follicle and is modulated by T follicular regulatory (Tfr) cells. We compare the effects of Tfr cells in an in vitro model of bystander Tfh function in the absence of BCR engagement and in a model in which mimics cognate T-B interactions in which the BCR is engaged. In the absence of Tfr cells, Tfh cells from primed mice induce naive B cell differentiation into GC B cells and class switch recombination (CSR) in the presence of anti-CD3 alone or anti-CD3/IgM in a contact-dependent manner. Addition of primed Tfr cells efficiently suppressed GC B cell proliferation, differentiation and CSR in the anti-CD3 alone cultures, but only moderately suppressed BCR-stimulated B cells. When stimulated with anti-CD3 alone, IL-4 is critical for the induction of GC B cells and CSR. IL-21 plays a minimal role in GC B cell differentiation, but a greater role in switching. When the BCR is engaged, IL-4 is primarily required for switching and IL-21 only modestly affects switching. CD40L expression was critical for Tfh-mediated B cell proliferation/differentiation in the absence of B cell engagement. When the BCR was engaged, proliferation of CD40 deficient B cells was partially restored, but was susceptible to suppression by Tfr. These studies suggest that in vitro Tfr suppressor function is complex and is modulated by BCR signaling and CD40-CD40L interactions.

The Effects of Endurance Exercise and Diet on Atherosclerosis in Young and Aged ApoE-/- and Wild-Type Mice.

BACKGROUND: Atherosclerosis is the leading cause of death worldwide. The disease development is by and large driven by old age and lifestyle factors, such as diet, physical activity, and smoking. In the present study, we have investigated the effect of exercise and diet on the development of atherosclerosis in young and aged mice. OBJECTIVE: This study aimed at comparing multiple age-dependent factors that may influence atherosclerosis in a transgenic mouse model. METHODS: Young (14 weeks) and aged (49-52 weeks) C57BL/6 wild-type (WT) and atherosclerosis-prone ApoE-/- mice were subjected to physical endurance exercise on a treadmill, with or without a high-fat diet. Five weeks later, the frequencies of regulatory T cells (TREGs) in lymph nodes were assessed by flow cytometry, plasmatic cytokines (interleukin [IL]-1ß, IL-6, IL-10, IL-17, interferon-γ, tumor necrosis factor-α, and transforming growth factor [TGF]-ß1) levels were determined by Luminex assay. Lipids (cholesterol and triglycerides) and anti-heat shock protein 60 (HSP60) autoantibodies were measured by ELISA. Aortic lesion sizes were assessed by en face imaging. Microarray analysis and qPCR of skeletal muscle gene expression were also performed. RESULTS: Exercise leads to a reduction of aortic lesions in young ApoE-/- and aged WT mice independent of diet. In most groups, this reduction was followed by an increased proportion of TREGs and TGF-ß1 levels. Moreover, gene expression analysis showed that exercise seems to affect the AMPK signaling pathway. In particular, PGC-1α1 mRNA was induced in aged WT mice, whereas it was reduced in young ApoE-/- mice. In addition, GSEA analysis showed a marked reduction in the insulin signaling pathway in aged ApoE-/- mice. CONCLUSION: Practicing endurance exercise seems to be enough for reducing early aortic lesion formation, independent of diet. However, this was only true in mice with smaller aortic lesions, since mice with large, advanced, complicated atherosclerotic plaques did not show any reduction in lesion size with exercise training.

Elevated sodium leads to the increased expression of HSP60 and induces apoptosis in HUVECs.

Atherosclerosis is the leading cause of death in the world. We have previously shown that expression of heat shock protein 60 (HSP60) on the surface of endothelial cells is the main cause of initiating the disease as it acts as a T cell auto-antigen and can be triggered by classical atherosclerosis risk factors, such as infection (e.g. Chlamydia pneumoniae), chemical stress (smoking, oxygen radicals, drugs), physical insult (heat, shear blood flow) and inflammation (inflammatory cytokines, lipopolysaccharide, oxidized low density lipoprotein, advanced glycation end products). In the present study, we show that increasing levels of sodium chloride can also induce an increase in intracellular and surface expression of HSP60 protein in human umbilical vein endothelial cells. In addition, we found that elevated sodium induces apoptosis.

In vitro immunoregulatory effects of thymoglobulin on human immune cell subpopulations.

Thymoglobulin (ATG) is a polyclonal rabbit antibody against human thymocytes used as a T cell-depleting agent in organ transplantation. Its polyclonal character suggests that its effect may go far beyond just T cell depletion. The aim of this study was to further elucidate possible mechanisms underlying the suppressive activity of ATG. For in vitro studies, human peripheral blood mononuclear cells (PBMC) were incubated with ATG or control Ig for various time points. Foxp3+ regulatory cells (Tregs) and monocytes were phenotypically analyzed by flow cytometry and functionally tested by in vitro suppression assays. Cytokine levels were determined by quantitative RT- PCR, Multiplex or ELISA techniques. In vitro, the frequencies of Foxp3+ Tregs increased when human PBMC were stimulated with ATG as compared with stimulation by rabbit Ig or without stimulation. ATG-treated cells suppressed proliferation of autologous PBMC stimulated with anti-CD3 and anti-CD28 monoclonal antibodies and this suppression could be reversed by exogenous IL-2. The Foxp3+ expression dropped down on day 10, which suggests that it is transient. Monocytes and natural killer cells stimulated with ATG down-modulated CD16. Monocytes suppressed the proliferation of autologous PBMC. However, there were not statistically significant differences in IL-10, TNF-α and TGF-ß1 secretion by monocytes stimulated with ATG or control rabbit Ig. These findings suggest that ATG has immunomodulatory effects that go beyond T cell depletion and induction of Foxp3+ Tregs. The induction of immunosuppressive monocytes might have a protective role in delaying transplant rejection.

Differential depletion of total T cells and regulatory T cells and prolonged allotransplant survival in CD3Ɛ humanized mice treated with polyclonal anti human thymocyte globulin.

Thymoglobulin (ATG) is a polyclonal rabbit antibody against human thymocytes used as a T cell-depleting agent to prevent or treat allotransplant rejection. The aim of the present study was to investigate the effect of low dose ATG treatment exclusively on T cells using a humanized BALB/c human CD3Ɛ transgenic mouse model expressing both human and murine T cell receptors (TCR). Mice received a single intravenous (i.v.) injection of ATG. Blood and peripheral lymphoid organs were obtained after different time points. We found a significant T cell depletion in this mouse model. In addition, regulatory T cells (Tregs) proved to be less sensitive to depletion than the rest of T cells and the Treg:non-Treg ratio was therefore increased. Finally, we also investigated the effect of ATG in a heterotopic allogenic murine model of heart transplantation. Survival and transplant function were significantly prolonged in ATG-treated mice. In conclusion, we showed (a) an immunosuppressive effect of ATG in this humanized mouse model which is exclusively mediated by reactivity against human CD3Ɛ; (b) provided evidence for a relative resistance of Tregs against this regimen; and

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis: Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65 peptides.

OBJECTIVE: The aim of this study was to identify atherogenic and atheroprotective peptides of bacterial HSP60 [taking mycobacterial HSP65 (mbHSP65) as a potent paradigmatic representative] that could be used as candidates for an orally applied tolerizing vaccine against atherosclerosis. METHODS: ApoE(-/-) mice were immunized with mbHSP65 protein or peptides, given mbHSP65 orally and then kept either on chow or high cholesterol diet. Atherosclerosis was assessed by en face and immunohistological analysis. Anti-HSP autoantibodies were detected by ELISA. The number and in vitro suppressive function of splenic and lymph node regulatory T cells (Tregs) were analyzed by flow cytometry. Specific T cell reactivity against mbHSP65 protein or peptides was assessed by proliferation assay. RESULTS: Decreased lesion size was accompanied by (a) increased splenic Treg numbers; (b) increased interleukin (IL)-10 mRNA levels in the aorta; (c) increased levels of anti-mbHSP65 and anti-mouse HSP60 antibodies pointing to pro-eukaryotic HSP60 humoral crossreaction, not curtailed by oral tolerization; (d) most importantly, we identified and functionally characterized novel atherogenic and atheroprotective mbHSP65 epitopes. CONCLUSION: Atheroprotective mbHSP65 peptides may be considered as potential candidates for the development of a tolerizing vaccine to prevent and treat atherosclerosis, while keeping protective immunity to non-atherogenic domains of mbHSP65 intact.

The role of heat shock proteins in atherosclerosis.

Atherosclerosis is a chronic, multifactorial disease that starts in youth, manifests clinically later in life, and can lead to myocardial infarction, stroke, claudication, and death. Although inflammatory processes have long been known to be involved in atherogenesis, interest in this subject has grown in the past 30-40 years. Animal experiments and human analyses of early atherosclerotic lesions have shown that the first pathogenic event in atherogenesis is the intimal infiltration of T cells at arterial branching points. These T cells recognize heat shock protein (HSP)60, which is expressed together with adhesion molecules by endothelial cells in response to classic risk factors for atherosclerosis. Although these HSP60-reactive T cells initiate atherosclerosis, antibodies to HSP60 accelerate and perpetuate the disease. All healthy humans develop cellular and humoral immunity against microbial HSP60 by infection or vaccination. Given that prokaryotic (bacterial) and eukaryotic (for instance, human) HSP60 display substantial sequence homology, atherosclerosis might be the price we pay for this protective immunity, if risk factors stress the vascular endothelial cells beyond physiological conditions.

Optimized protocol for the isolation of spleen-resident murine neutrophils.

Neutrophils are an important cellular component of the innate immune system that provides immediate protection to the host from infection. Neutrophil infiltration into inflamed peripheral tissues during infection is beneficial for immunity through phagocytosis of microbes, the release of antimicrobial factors, and secretion of proinflammatory cytokines. Recent reports further suggest that spleen-infiltrating neutrophils play a role in the adaptive immune response by providing survival signals to B cells. However, neutrophils may have detrimental effects on immunity in inflammatory diseases where their recruitment to lymphoid tissues and activation occur abnormally. To determine the contribution of neutrophils that reside in secondary lymphoid tissues to adaptive immunity, direct evaluation of the functional properties of tissue-resident neutrophils is required. We have developed a modified magnetic bead isolation approach for purifying neutrophils from inflamed spleens of autoimmune-prone mice by negative selection. Using this approach, we yielded neutrophils with greater than 90% purity without compromising cell viability. Equally important, the isolation procedure had little effect on the activation of neutrophils and did not impair phagocytic function. Thus, isolation of spleen-resident neutrophils by this optimized approach could be useful for interrogating the functional role of murine neutrophils in normal and abnormal immune responses.