Large-scale expansion of rat CD4+ CD25+ T(reg) cells in the absence of T-cell receptor stimulation

T-cell receptor (TCR) stimulation is both central to homeostatic maintenance of CD4(+) CD25(+) regulatory T cells (T(reg) cells) in vivo and a prerequisite for the initiation of suppression by T(reg) cells, both in vivo and in vitro. However, TCR-independent stimulation of T(reg) cells, e.g. with superagonistic CD28-specific monoclonal antibodies (CD28-SA), not only expands these cells in vivo but, as we show here, also mediates large-scale expansion of rat T(reg) cells in vitro. Interestingly, CD28-SA stimulation plus interleukin (IL)-2 was even superior to conventional costimulation plus IL-2 in promoting T(reg) cell growth in vitro. Despite their highly activated phenotype suppression by T(reg) cells expanded in the absence of TCR stimulation remained fully dependent on TCR-triggering for initiation and cell contact was required to exert suppression. With regard to the regulation of suppression by CD28 stimulation we observed that neither the presence of a conventional anti-CD28 monoclonal antibody nor a CD28-SA generally rendered conventional T cells resistant to suppression by preactivated T(reg) cells. Taken together, we provide a novel protocol for long-term propagation of T(reg) cells in vitro and our data are the first to reveal a difference in the signals required for activation and expansion of T(reg) cells and those, involving the TCR, necessary for the initiation of suppression.     

CD5 plays an inhibitory role in the suppressive function of murine CD4(+) CD25(+) T(reg) cells

A subset of CD4(+) T cells, the CD4(+) CD25(+) regulatory T (T(reg)) cells in the lymphoid organs and peripheral blood are known to possess suppressive function. Previous in vitro and in vivo studies have indicated that T cell receptor (TCR) signal is required for development of such 'natural regulatory (T(reg)) cells' and for activation of the effector function of CD4(+) CD25(+) regulatory T cells. CD5 is a cell surface molecule present on all T cells and a subtype of B lymphocytes, the B-1 cells, primarily localized to coelomic cavities, Peyer's patches, tonsils and spleen. CD5 acts as a negative regulator of T cell and B cell signaling via recruitment of SHP-1. Here, we demonstrate that T(reg) cells obtained from CD5(-/-) mice are more potent than those from wild type mice in suppressing the in vitro cell proliferation of anti-CD3 stimulated CD4(+) CD25(-) responder T cells. This phenomenon was cell contact and GITR dependent. Lack of CD5 expression on T(reg) cells (from spleen, lymph node and thymus) did not affect the intracellular levels of Foxp3. However, CD5(-/-) T(reg) thymocytes were able to elicit a higher Ca(2+) response to TCR + co-stimulatory signals than the wild type cells. CD5(-/-) mice expressed more Foxp3 mRNA in the colon than wild type mice, and additionally, the severity of the dextran sulfate sodium (DSS)-induced colitis in CD5(-/-) mice was less than the wild type strain. We suggest that manipulation of CD5 expression or the downstream signaling components of CD4(+) CD25(+) T(reg) cells as a potential strategy for therapeutic intervention in cases of auto-immune disorders.     

Onchocerca volvulus-specific antibody and cytokine responses in onchocerciasis patients after 16 years of repeated ivermectin therapy

The recommended control option against onchocerciasis is repeated ivermectin treatment, which will need to be implemented for decades, and it remains unknown how repeated ivermectin therapy might affect immunity against Onchocerca volvulus in the long term. O. volvulus-specific antibody reactivity and cellular cytokine production were investigated in onchocerciasis patients receiving ivermectin (150 microg/kg) annually for 16 years. In treated patients, the T helper type 2 (Th2) cytokine interleukin (IL)-5 and T regulatory IL-10 in response to O. volvulus antigen (OvAg) and bacteria-derived Streptolysin O (SL-O) diminished to levels found in infection-free endemic controls; also, cellular release of Th1-type interferon (IFN)-gamma at 16 years post initial ivermectin treatment (p.i.t.) approached control levels. In ivermectin-treated onchocerciasis patients, IL-5 production in responses to the mitogen phytohaemagglutinin (PHA) decreased, but IL-10 in response PHA increased, and neither attained the cytokine production levels of endemic controls. At 16 years p.i.t., O. volvulus-specific IgG1 and IgG4 subclass reactivity still persisted at higher levels in onchocerciasis patients than in O. volvulus exposed but microfilariae-free endemic controls. In addition, cytokine responses remained depressed in onchocerciasis patients infected concurrently with Mansonella perstans and Necator americanus or Entamoeba histolytica/dispar. Thus, long-term ivermectin therapy of onchocerciasis may not suffice to re-establish fully a balanced Th1 and Th2 immune responsiveness in O. volvulus microfilariae-negative individuals. Such deficient reconstitution of immune competence may be due to an as yet continuing and uncontrolled reinfection with O. volvulus, but parasite co-infections can also bias and may prevent the development of such immunity.     

Tolerogen-induced interferon-producing killer dendritic cells (IKDCs) protect against EAE

Natural killer (NK) cells and dendritic cells (DCs) have been shown to link the innate and adaptive immune systems. Likewise, a new innate cell subset, interferon-producing killer DCs (IKDCs), shares phenotypic and functional characteristics with both DCs and NK cells. Here, we show IKDCs play an essential role in the resolution of experimental autoimmune encephalomyelitis (EAE) upon treatment with the tolerizing agent, myelin oligodendrocyte glycoprotein (MOG), genetically fused to reovirus protein σ1 (termed MOG-pσ1). Activated IKDCs were recruited subsequent MOG-pσ1 treatment of EAE, and disease resolution was abated upon NK1.1 cell depletion. These IKDCs were able to kill activated CD4⁺ T cells and mature dendritic DCs, thus, contributing to EAE remission. In addition, IKDCs were responsible for MOG-pσ1-mediated MOG-specific regulatory T cell recruitment to the CNS. The IKDCs induced by MOG-pσ1 expressed elevated levels of HVEM for interactions with cognate ligand-positive cells: LIGHT⁺ NK and T_eff cells and BTLA⁺ B cells. Further characterization revealed these activated IKDCs being MHC class II^high, and upon their adoptive transfer (CD11c⁺NK1.1⁺MHC class II^high), IKDCs, but not CD11c⁺NK1.1⁺MHC class II^{intermediate/low} (unactivated) cells, conferred protection against EAE. These activated IKDCs showed enhanced CD107a, PD-L1, and granzyme B expression and could present OVA, unlike unactivated IKDCs. Thus, these results demonstrate the interventional potency induced HVEM⁺ IKDCs to resolve autoimmune disease.     

Divergent effects of calcineurin Aβ on regulatory and conventional T-cell homeostasis

Calcineurin (CN) is a phosphatase that activates nuclear factor of activated T cells (NFAT). While the CN inhibitors cyclosporine A (CsA) and tacrolimus (FK506) can prevent graft rejection, they also cause inflammatory diseases. We investigated the role of calcineurin using mice deficient in the CN catalytic subunit Aβ (CNAβ). Cnab(-/-) mice exhibit defective thymocyte maturation, splenomegaly and hepatomegaly. Further, as Cnab(-/-) mice age, they exhibit spontaneous T-cell activation and enhanced production of proinflammatory cytokines (IL-4, IL-6, and IFNγ). FOXP3(+) T(reg) cells were significantly decreased in Cnab(-/-) mice likely contributing to increased T-cell activation. Interestingly, we found that CNAβ is critical for promotion of BCL-2 expression in FOXP3(+) T(reg) and for permitting TGFβ signaling, as TGFβ induces FOXP3 in control but not in Cnab(-/-) T-cells. Together, these data suggest that CNAβ is important for the production and maintenance of T(reg) cells and to ensure mature T-cell quiescence.     

Dynamic changes in CD4+ CD25+(high) T cell apoptosis after the diagnosis of type 1 diabetes

Because type 1 diabetes (T1D) is a chronic, autoimmune, T cell-mediated disease, interventions affecting T cells are expected to modulate the immune cascade and lead to disease remission. We propose that increased CD4(+) CD25(+high) T cell apoptosis, a trait we discovered in recent-onset T1D subjects, reflects T1D partial remission within the first 6 months after diagnosis. Apoptosis of forkhead box P3 (FoxP3)(+) CD4(+) CD25(+high) T cells, in addition to total daily doses of insulin (TDD), blood glucose, HbA1c and age, were measured in 45 subjects with T1D at various times after diagnosis. Sixteen healthy control subjects were also recruited to the study. Higher CD4(+) CD25(+high) T cell apoptosis levels were detected within the first 6 months of diagnosis (odds ratio = 1.39, P = 0.009), after adjustment for age, TDD and HbA1c. A proportional hazards model confirmed that the decline of apoptosis after diagnosis of T1D was related significantly to survival time (hazards ratio = 1.08, P = 0.014), with TDD and age also contributing to survival. During this time there was an inverse relationship between CD4(+) CD25(+high) T cell apoptosis with TDD (r = -0.39, P = 0.008). The CD4(+) CD25(+high) T cell apoptosis levels decline significantly after the first 6 months from diagnosis of T1D and may help in the close monitoring of autoimmunity. In parallel, there is an increase in TDD during this time. We also propose that CD4(+) CD25(+high) T cell apoptosis assay can be used to gauge the efficacy of the several immune tolerance induction protocols, now under way.     

FOXP3 expression and frequency of regulatory T cells in healed individuals from Leishmania major infection and the asymptomatic cases

Two groups of residents in an endemic area of Leishmania major infection in Iran with positive leishmanin skin tests who were either asymptomatic or had healed cutaneous leishmaniasis lesions were compared with respect to their T helper responses. The percentages of regulatory T cells (T_reg; CD4⁺CD25^high FoxP3⁺) from the peripheral blood and CD4⁺ T cells producing intracellular cytokines (IL-4, IL-10, IL-17 and IFN-γ) from the stimulated PBMCs were evaluated by flow cytometry and the expressions of RORC and FOXP3 genes were quantified by real-time RT-PCR. T responder (CD4⁺CD25⁻) and T_reg-enriched (CD4⁺CD25⁺) cells were isolated magnetically and the suppressive capacity of the latter and the cytokines (IFN-γ, TGF-β and IL-10) secreted from them were evaluated by in vitro assays. The results showed that the frequency of T_reg in the studied groups were similar and T_reg from both groups exhibited high yet similar suppressive capacities while significantly higher levels of FOXP3 expression was observed in the asymptomatic group. Taken together, similar frequency and suppressiveness of T_reg combined with high ratios of IFN-γ/IL-10 producing CD4⁺ T cells were common in both groups; however the members of the asymptomatic group appeared to require higher expression of FOXP3 to maintain their immunity to re-infection.     

Thyrotropic hormone (TSH) regulation of triiodothyronine (T3) concentration in immune cells

Objective:  Cells of the immune system (peritoneal lymphocytes, monocytes, granulocytes and mast cells as well as thymocytes) contain triiodothyronine (T₃). The aim of the present experiments was to study whether thyrotropic hormone (TSH) regulates or not the T₃ concentration of these cells. Methods:  Peritoneal fluid and thymus cells of adult rats were studied by immunocytochemistry, combined with flow cytometry for triiodothyronine content with or without in-vitro TSH treatment. In addition, adult female CD1 mice were treated in vivo with 10 or 40 mU TSH and after 1 hour peritoneal immune cells were studied using the above mentioned method. Results:  Both in vitro (in rat) and in vivo (in mice) TSH treatments significantly elevated the T₃ content in each cell type. In vitro TSH 0.1 mU/ml cell suspension was enough to provoke about 50 % increase in T₃ production. Conclusion:  T₃ concentration in immune cells seems to be regulated by TSH, similarly to the T₃ in the thyroid. Considering the large number of immune cells in an organism, TSH regulation of their T₃ content could have an important physiological and pathological role, both in and beyond the immune system. Received 15 April 2008; returned for revision 19 May 2008; received from final revision 20 May 2008; accepted by I. Ahnfeld-R?nne 23 June 2008     

TRH-refraktäre TSH-Werte,Trijodthyroninspiegel und T3/T4-Quotienten bei mit T3/T4-Kombinationspräparaten behandelten hypothyreoten Kindern

Zusammenfassung Bei 23 Kindern mit primärer Hypothyreose, die mit Thyroxin (T₄) und Trijodthyronin (T₃) enthaltenden Kombinationspräparaten behandelt worden waren, wurden TRH-Teste, TBI, T₃- und T₄-Bestimmungen durchgeführt. Die T₃/T₄-Quotienten wurden berechnet. 7 von 10 Patienten mit TRH-refraktären TSH-Werten und normalen T₄-Spiegeln wiesen überhöhte T₃-Werte auf. Bei 6 von diesen Patienten konnten pathologisch überhöhte T₃/T₄-Quotienten beobachtet werden. Eine TRH-refraktäre TSH-Suppression wurde nur bei hohen T₄-Werten gefunden. Nach einer Reduktion der Substitutionsdosis normalisierten sich die überhöhten T₃-Spiegel. Die Befunde zeigen, daß nicht nur die T₄-Werte sondern auch die TSH- und T₃-Spiegeln unter der Behandlung mit Schilddrüsenhormonen kontrolliert werden sollten. Die Durchführung der Substitutionstherapie der Hypothyreose mit T₃/T₄-Kombinationspräparaten im Verhältnis 1:4 sollte überdacht werden.     

KLRG1 expression identifies short-lived Foxp3+ Treg effector cells with functional plasticity in islets of NOD mice

A progressive waning in Foxp3⁺ regulatory T (T_reg) cell function provokes autoimmunity in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D), a cellular defect rescued by prophylactic IL-2 therapy. We showed that most islet-infiltrating T_reg cells express inducible T-cell co-stimulator (ICOS) in pre-diabetic NOD mice, and that ICOS⁺ T_reg cells display enhanced fitness and suppressive function in situ. Moreover, T1D progression is associated with decreased expansion and suppressive activity of ICOS⁺Foxp3⁺ T_reg cells, in islets, an observation consistent with the exacerbated T1D seen in NOD.BDC2.5 mice in which the ICOS pathway is abrogated. Here, we show that a large proportion of islet-resident T_reg cells express the KLRG1 marker of terminally differentiation, in contrast to islet-infiltrating ICOS^? T_reg or T_eff cells. We hypothesized that KLRG1 expression designates a subpopulation of ICOS⁺ T_reg cells in islets that progressively loses function, and contributes to the immune dysregulation observed at T1D onset. Indeed, KLRG1-expressing ICOS⁺ T_reg cells are prone to apoptosis, and have an impaired proliferative capacity and suppressive function in vitro and in vivo. T1D protective low-dose IL-2 treatment in vivo could not rescue the loss of KLRG1-expressing T_reg cells in situ. While the global pool of Foxp3⁺ T_reg cells displays some degree of functional plasticity in vivo, the KLRG1⁺ ICOS⁺ T_reg cell subset is particularly susceptible to lose Foxp3 expression and reprogram into Th1- or Th17-like effector T (T_eff) cells in the pancreas microenvironment. Overall, KLRG1 expression delineates a subpopulation of dysfunctional T_reg cells during T1D progression in autoantigen-specific TCR transgenic NOD mice.     

Early dynamics of T helper cell cytokines and T regulatory cells in response to treatment of active Mycobacterium tuberculosis infection

Biomarkers that can identify tuberculosis (TB) disease and serve as markers for efficient therapy are requested. We have studied T cell cytokine production [interferon (IFN)-γ, interleukin (IL)-2, tumour necrosis factor (TNF)-α] and degranulation (CD107a) as well as subsets of CD4⁺ T regulatory cells (T_regs) after in-vitro Mycobacterium tuberculosis (Mtb) antigen stimulation [early secretory antigenic target (ESAT)-6, culture filtrate protein (CFP)-10, antigen 85 (Ag85)] in 32 patients with active tuberculosis (TB) disease throughout 24 weeks of effective TB treatment. A significant decline in the fraction of Mtb-specific total IFN-γ and single IFN-γ-producing T cells was already observed after 2 weeks of treatment, whereas the pool of single IL-2⁺ cells increased over time for both CD4⁺ and CD8⁺ T cells. The T_reg subsets CD25^highCD127^low, CD25^highCD147⁺⁺ and CD25^highCD127^lowCD161⁺ expanded significantly after Mtb antigen stimulation in vitro at all time-points, whereas the CD25^highCD127^lowCD39⁺ T_regs remained unchanged. The fraction of CD25^highCD127^low T_regs increased after 8 weeks of treatment. Thus, we revealed an opposing shift of T_regs and intracellular cytokine production during treatment. This may indicate that functional signatures of the CD4⁺ and CD8⁺ T cells can serve as immunological correlates of early curative host responses. Whether such signatures can be used as biomarkers in monitoring and follow-up of TB treatment needs to be explored further.     

Involvement of T helper type 17 and regulatory T cell activity in Citrobacter rodentium invasion and inflammatory damage

Citrobacter rodentium is a murine pathogen that transiently colonizes the lumen of the large intestine. C. rodentium induces colitis, but the relative importance and temporal induction of the T helper type 17 (Th17) and regulatory T cell (T_reg) pathways in protection from the infection and inflammation have not been assessed. Our aim was to investigate the key immunological signalling events associated with successful clearance of C. rodentium. Mice were challenged with luminescent-tagged C. rodentium and killed at days 3 (early infection), 10 (peak infection) and 21 (late infection) post-infection. Bioluminescent imaging and bacterial culture determined levels of C. rodentium. Distal colon mRNA expression of interleukin (IL)-17, IL-6, IL-1β, tumour necrosis factor (TNF)-α, forkhead box P3 (FoxP3) and ghrelin were assessed using real-time polymerase chain reaction. Results were compared with age-matched non-infected mice. Low levels of C. rodentium were found at day 3, high levels at day 10, with clearance from the majority of the mice by day 21. In the distal colon, there was up-regulation of TNF-α and FoxP3 throughout the study and increases in IL-6 and IL-17 during the peak and late stages of infection. Ghrelin expression was increased at the peak and late stages of infection. This study has characterized changes to the T helper cell pathways, following the course of C. rodentium infection in mice. There were significant immunological changes, with up-regulation of the Th17 and T_reg pathways in the distal colon and an increase in ghrelin expression compared with non-infected control mice. These changes may play a role in the pathology and clearance of C. rodentium.     

Chemokine responses distinguish chemical-induced allergic from irritant skin inflammation: memory T cells make the difference

BACKGROUND: As clinical and histological features of allergic and irritant contact dermatitis share common characteristics, the differentiation between them in the preclinical and clinical evaluations of chemicals remains difficult. OBJECTIVE: To identify the differences in the underlying immunological mechanisms of chemical-induced allergic or irritant skin responses. METHODS: We systematically studied the involvement of chemokines in both diseases by quantitative real-time polymerase chain reaction in mice and humans. The cellular origin of relevant chemokines and receptors was determined using immunohistochemistry; functional relevance was demonstrated in vitro by transwell chemotaxis and in vivo by adoptive transfer experiments using a model of hapten-induced murine contact hypersensitivity. RESULTS: Independent of overall skin inflammation, chemical-induced allergic and irritant skin responses showed distinct molecular expression profiles. In particular, chemokine genes predominantly regulated by T-cell effector cytokines demonstrated differential upregulation in hapten-specific skin inflammation. Notably, the expression of CXCR3 ligands, such as CXCL9 (Mig) and CXCL10 (IP-10), was upregulated in chemical-induced allergic skin responses when compared with irritant skin responses. Furthermore, we showed that inflammatory chemokines such as CXCL10 prime leukocytes to respond to CXCL12 (SDF-1), increasing their recruitment both in vitro and in vivo. CONCLUSION: We provide important insights into the molecular basis of chemical-induced allergic and irritant contact dermatitis, identify novel markers suitable for their differentiation, and demonstrate the cooperation of inflammatory and homeostatic chemokines in the recruitment of pathogenic leukocyte subsets. CLINICAL IMPLICATIONS: Molecular differences between both diseases represent the basis for new approaches to diagnostics and therapy.