Ligation of TLR7 on CD19+CD1dhi B cells suppresses allergic lung inflammation via regulatory T cells

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B‐cell population is defined as IL‐10‐producing CD19⁺CD1d^hi cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19⁺CD1d^hi B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19⁺CD1d^hi B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL‐10 reporter, Il10^?/? and Tlr7^?/‐ mice, we formally demonstrate that TLR7 ligation of CD19⁺CD1d^hi B cells increases their capacity to produce IL‐10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7‐elicited CD19⁺CD1d^hi B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3⁺ T regulatory cells in allergen‐sensitized mice, we show that that TLR7‐elicited CD19⁺CD1d^hi B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL‐10‐producing CD19⁺CD1d^hi B cells, which can suppress allergic lung inflammation via T regulatory cells.     

Suppression of type 2 immunity and allergic airway inflammation by secreted products of the helminth Heligmosomoides polygyrus

Allergic asthma is less prevalent in countries with parasitic helminth infections, and mice infected with parasites such as Heligmosomoides polygyrus are protected from allergic airway inflammation. To establish whether suppression of allergy could be mediated by soluble products of this helminth, we tested H. polygyrus excretory‐secretory (HES) material for its ability to impair allergic inflammation. When HES was added to sensitising doses of ovalbumin, the subsequent allergic airway response was suppressed, with ablated cell infiltration, a lower ratio of effector (CD4⁺CD25⁺Foxp3^?) to regulatory (CD4⁺Foxp3⁺) T (Treg) cells, and reduced Th1, Th2 and Th17 cytokine production. HES exposure reduced IL‐5 responses and eosinophilia, abolished IgE production and inhibited the type 2 innate molecules arginase‐1 and RELM‐α (resistin‐like molecule‐α). Although HES contains a TGF‐β‐like activity, similar effects in modulating allergy were not observed when administering mammalian TGF‐β alone. HES also protected previously sensitised mice, suppressing recruitment of eosinophils to the airways when given at challenge, but no change in Th or Treg cell populations was apparent. Because heat‐treatment of HES did not impair suppression at sensitisation, but compromised its ability to suppress at challenge, we propose that HES contains distinct heat‐stable and heat‐labile immunomodulatory molecules, which modulate pro‐allergic adaptive and innate cell populations.     

ICOS controls Foxp3+ regulatory T‐cell expansion,maintenance and IL‐10 production during helminth infection

Foxp3⁺ regulatory T (Treg) cells are key immune regulators during helminth infections, and identifying the mechanisms governing their induction is of principal importance for the design of treatments for helminth infections, allergies and autoimmunity. Little is yet known regarding the co‐stimulatory environment that favours the development of Foxp3⁺ Treg‐cell responses during helminth infections. As recent evidence implicates the co‐stimulatory receptor ICOS in defining Foxp3⁺ Treg‐cell functions, we investigated the role of ICOS in helminth‐induced Foxp3⁺ Treg‐cell responses. Infection of ICOS^?/? mice with Heligmosomoides polygyrus or Schistosoma mansoni led to a reduced expansion and maintenance of Foxp3⁺ Treg cells. Moreover, during H. polygyrus infection, ICOS deficiency resulted in increased Foxp3⁺ Treg‐cell apoptosis, a Foxp3⁺ Treg‐cell specific impairment in IL‐10 production, and a failure to mount putatively adaptive Helios^?Foxp3⁺ Treg‐cell responses within the intestinal lamina propria. Impaired lamina propria Foxp3⁺ Treg‐cell responses were associated with increased production of IL‐4 and IL‐13 by CD4⁺ T cells, demonstrating that ICOS dominantly downregulates Type 2 responses at the infection site, sharply contrasting with its Type 2‐promoting effects within lymphoid tissue. Thus, ICOS regulates Type 2 immunity in a tissue‐specific manner, and plays a key role in driving Foxp3⁺ Treg‐cell expansion and function during helminth infections.     

Regulatory T cells in human geohelminth infection suppress immune responses to BCG and Plasmodium falciparum

Chronic helminth infections induce T‐cell hyporesponsiveness, which may affect immune responses to other pathogens or to vaccines. This study investigates the influence of Treg activity on proliferation and cytokine responses to BCG and Plasmodium falciparum‐parasitized RBC in Indonesian schoolchildren. Geohelminth‐infected children's in vitro T‐cell proliferation to either BCG or pRBC was reduced compared to that of uninfected children. Although the frequency of CD4⁺CD25^hiFOXP3⁺ T cells was similar regardless of infection status, the suppressive activity differed between geohelminth‐infected and geohelminth‐uninfected groups: Ag‐specific proliferative responses increased upon CD4⁺CD25^hi T‐cell depletion in geohelminth‐infected subjects only. In addition, IFN‐γ production in response to both BCG and parasitized RBC was increased after removal of CD4⁺CD25^hi T cells. These data demonstrate that geohelminth‐associated Treg influence immune responses to bystander Ag of mycobacteria and plasmodia. Geohelminth‐induced immune modulation may have important consequences for co‐endemic infections and vaccine trials.     

Early recruitment of natural CD4+Foxp3+ Treg cells by infective larvae determines the outcome of filarial infection

Human helminth infections are synonymous with impaired immune responsiveness indicating suppression of host immunity. Using a permissive murine model of filariasis, Litomosoides sigmodontis infection of inbred mice, we demonstrate rapid recruitment and increased in vivo proliferation of CD4⁺Foxp3⁺ Treg cells upon exposure to infective L3 larvae. Within 7 days post‐infection this resulted in an increased percentage of CD4⁺T cells at the infection site expressing Foxp3. Antibody‐mediated depletion of CD25⁺ cells prior to infection to remove pre‐existing ‘natural’ CD4⁺CD25⁺Foxp3⁺ Treg cells, while not affecting initial larval establishment, significantly reduced the number of adult parasites recovered 60 days post‐infection. Anti‐CD25 pre‐treatment also impaired the fecundity of the surviving female parasites, which had reduced numbers of healthy eggs and microfilaria within their uteri, translating to a reduced level of blood microfilaraemia. Enhanced parasite killing was associated with augmented in vitro production of antigen‐specific IL‐4, IL‐5, IL‐13 and IL‐10. Thus, upon infection filarial larvae rapidly provoke a CD4⁺Foxp3⁺ Treg‐cell response, biasing the initial CD4⁺ T‐cell response towards a regulatory phenotype. These CD4⁺Foxp3⁺ Treg cells are predominantly recruited from the ‘natural’ regulatory pool and act to inhibit protective immunity over the full course of infection.     

Gastrointestinal nematode infection interferes with experimental allergic airway inflammation but not atopic dermatitis

Background Some helminth infections are negatively associated with the prevalence of allergic disorders, arguing for a modulation of allergic reactions by the parasites, depending on the worm species, intensity and phase of infection and the type of disease. Objective The aim of this study was to analyse the influence of a chronic infection with the gastrointestinal nematode Heligmosomoides polygyrus, in a murine model of allergic airway disease and of atopic dermatitis (AD), respectively. Methods Mice were infected with H. polygyrus and systemically sensitized with the model allergen ovalbumin. Subsequently, the animals were challenged with the allergen either via the airways for induction of airway disease, or via skin patches for induction of dermatitis. Results Mice concomitantly infected with H. polygyrus showed diminished eosinophil and lymphocyte recruitment into the lungs and decreased allergen‐specific IgE levels when compared with sensitized and airway challenged controls. In addition, animals showed a trend towards reduced airway hyper‐reactivity. In contrast, no significant differences in the severity of eczematous skin lesions were observed between infected and control animals in the AD model. Although H. polygyrus infection reduced CD8⁺ and CD4⁺ T‐cell infiltration into the skin and production of allergen‐specific IgE, mast cell recruitment was significantly increased in worm‐infected mice in the dermatitis model. The worm infection was associated with significantly elevated numbers of Foxp3⁺ regulatory T cells (Treg) in peribronchial lymph nodes in H. polygyrus‐infected sensitized and airway challenged mice. In contrast, Treg cells were basically absent in eczematous skin and their number was not increased in skin‐draining lymph nodes of mice with experimental dermatitis. Conclusion Infection with the gastrointestinal nematode used in our study leads to significant inhibition of mucosa‐associated but not cutaneous allergic reactions, pointing to a site specificity of the immunomodulation exerted by helminths. This finding might be an important aspect for future considerations of helminths for treatment of allergic diseases.     

Control of Schistosoma mansoni egg‐induced inflammation by IL‐4‐responsive CD4+CD25−CD103+Foxp3− cells is IL‐10‐dependent

Host protection to helminth infection requires IL‐4 receptor α chain (IL‐4Rα) signalling and the establishment of finely regulated Th2 responses. In the current study, the role of IL‐4Rα‐responsive T cells in Schistosoma mansoni egg‐induced inflammation was investigated. Egg‐induced inflammation in IL‐4Rα‐responsive BALB/c mice was accompanied with Th2‐biased responses, whereas T‐cell‐specific IL‐4Rα‐deficient BALB/c mice (iLck^creIl4ra^?/lox) developed Th1‐biased responses with heightened inflammation. The proportion of Foxp3⁺ Treg in the draining LN of control mice did not correlate with the control of inflammation and was reduced in comparison to T‐cell‐specific IL‐4Rα‐deficient mice. This was due to IL‐4‐mediated inhibition of CD4⁺Foxp3⁺ Treg conversion, demonstrated in adoptively transferred Rag2^?/? mice. Interestingly, reduced footpad swelling in Il4ra^?/lox mice was associated with the induction of IL‐4 and IL‐10‐secreting CD4⁺CD25^?CD103⁺Foxp3^? cells, confirmed in S. mansoni infection studies. Transfer of IL‐4Rα‐responsive CD4⁺CD25^?CD103⁺ cells, but not CD4⁺CD25^high or CD4⁺CD25^?CD103^? cells, controlled inflammation in iLck^creIl4ra^?/lox mice. The control of inflammation depended on IL‐10, as transferred CD4⁺CD25^?CD103⁺ cells from IL‐10‐deficient mice were not able to effectively downregulate inflammation. Together, these results demonstrate that IL‐4 signalling in T cells inhibits Foxp3⁺ Treg in vivo and promotes CD4⁺CD25^?CD103⁺Foxp3^? cells that control S. mansoni egg‐induced inflammation via IL‐10.     

The regulation of regulation: interleukin‐10 increases CD4+ CD25+ regulatory T cells but impairs their immunosuppressive activity in murine models with schistosomiasis japonica or asthma

CD4⁺ CD25⁺ Foxp3⁺ regulatory T (Treg) cells play an important role in maintaining immune homeostasis. Interleukin‐10 (IL‐10), a cytokine with anti‐inflammatory capacities, also has a critical role in controlling immune responses. In addition, it is well known that production of IL‐10 is one of the suppression mechanisms of Treg cells. However, the action of IL‐10 on Treg cells themselves remains insufficiently understood. In this study, by using a Schistosoma japonicum‐infected murine model, we show that the elevated IL‐10 contributed to Treg cell induction but impaired their immunosuppressive function. Our investigations further suggest that this may relate to the up‐regulation of serum transforming growth factor (TGF‐β) level but the decrease in membrane‐bound TGF‐β of Treg cells by IL‐10 during S. japonicum infection. In addition, similar IL‐10‐mediated regulation on Treg cells was also confirmed in the murine model of asthma. In general, our findings identify a previously unrecognized opposing regulation of IL‐10 on Treg cells and provide a deep insight into the precise regulation in immune responses.     

Intestinal inflammation abrogates the tolerogenic properties of MLN CD103+ dendritic cells

Intestinal CD103⁺ DC promote the differentiation of Foxp3⁺ Treg from naïve CD4⁺ T cells through mechanisms involving TGF‐β and the dietary metabolite, retinoic acid (RA). In this study, we have analysed whether the specialised features of CD103⁺ DC are conserved in colitis. Our results show that inflammation dampens the tolerogenic properties of MLN CD103⁺ DC, which is associated with lower expression of tgfβ2 and aldh1a2. Accordingly, CD103⁺ DC taken from colitic mice are impaired in their ability to induce Foxp3⁺ Treg and instead favour the emergence of IFN‐γ‐producing CD4⁺ T cells compared with their steady‐state counterparts. BrdU‐labelling studies and analysis of ontogeny markers show that CD103⁺ DC from steady‐state and colitic settings retain similar subset composition and developmental pathways. These results indicate that MLN CD103⁺ DC are not hard‐wired to promote tolerance but can adapt to environmental conditions. The inflammatory properties of MLN CD103⁺ DC in colitic mice may reflect defective gut tolerogenic conditioning or altered migratory pathways and raise the possibility that migratory DC populations contribute to the pathogenesis of inflammatory bowel disease.     

CD4+FoxP3+ regulatory T cells suppress fatal T helper 2 cell immunity during pulmonary fungal infection

The opportunistic fungal pathogen Cryptococcus neoformans causes lung inflammation and fatal meningitis in immunocompromised patients. Regulatory T (Treg) cells play an important role in controlling immunity and homeostasis. However, their functional role during fungal infection is largely unknown. In this study, we investigated the role of Treg cells during experimental murine pulmonary C. neoformans infection. We show that the number of CD4⁺FoxP3⁺ Treg cells in the lung increases significantly within the first 4 weeks after intranasal infection of BALB/c wild‐type mice. To define the function of Treg cells we used DEREG mice allowing selective depletion of CD4⁺FoxP3⁺ Treg cells by application of diphtheria toxin. In Treg cell‐depleted mice, stronger pulmonary allergic inflammation with enhanced mucus production and pronounced eosinophilia, increased IgE production, and elevated fungal lung burden were found. This was accompanied by higher frequencies of GATA‐3⁺ T helper (Th) 2 cells with elevated capacity to produce interleukin (IL)‐4, IL‐5, and IL‐13. In contrast, only a mild increase in the Th1‐associated immune response unrelated to the fungal infection was observed. In conclusion, the data demonstrate that during fungal infection pulmonary Treg cells are induced and preferentially suppress Th2 cells thereby mediating enhanced fungal control.     

CD8+ Treg cells suppress CD8+ T cell‐responses by IL‐10‐dependent mechanism during H5N1 influenza virus infection

Although Treg‐cell‐mediated suppression during infection or autoimmunity has been described, functions of Treg cells during highly pathogenic avian influenza virus infection remain poorly characterized. Here we found that in Foxp3‐GFP transgenic mice, CD8⁺ Foxp3⁺ Treg cells, but not CD4⁺ Foxp3⁺ Treg cells, were remarkably induced during H5N1 infection. In addition to expressing CD25, the CD8⁺ Foxp3⁺ Treg cells showed a high level of GITR and produced IL‐10. In an adoptive transfer model, CD8⁺ Treg cells suppressed CD8⁺ T‐cell responses and promoted H5N1 virus infection, resulting in enhanced mortality and increased virus load in the lung. Furthermore, in vitro neutralization of IL‐10 and studies with IL‐10R‐deficient mice in vitro and in vivo demonstrated an important role for IL‐10 production in the capacity of CD8⁺ Treg cells to inhibit CD8⁺ T‐cell responses. Our findings identify a previously unrecognized role of CD8⁺ Treg cells in the negative regulation of CD8⁺ T‐cell responses and suggest that modulation of CD8⁺ Treg cells may be a therapeutic strategy to control H5N1 viral infection.     

Role of IL‐10‐producing regulatory B cells in control of cerebral malaria in Plasmodium berghei infected mice

Cerebral malaria (CM) is a neurological syndrome often occurring in severe malaria. Although CM is known as an immunopathology in brain tissue mediated by excessive proinflammatory cytokines, the immunoregulatory mechanism is poorly understood. Here, we investigated the role of IL‐10‐producing regulatory B (Breg) cells in modulating CM development in a murine model of Plasmodium berghei ANKA infection. We observed that blood‐stage P. berghei induced expansion of IL‐10‐producing Breg cells in C57BL/6 mice. Adoptive transfer of IL‐10⁺ Breg cells to P. berghei infected mice significantly reduced the accumulation of NK and CD8⁺ T cells and hemorrhage in brain tissue, and improved the survival of the mice compared with control groups, although parasitemia levels were not altered. Treatment of Breg‐cell recipient mice with anti‐IL‐10 receptor mAb blocked the protective effect of Breg cells. Adoptive transfer of CD4⁺CD25⁺ Treg cells failed to prevent CM in infected mice. Spleen cells from Breg‐cell recipient mice produced increased levels of IL‐10 in vitro. Cell co‐culture showed that purified IL‐10⁺ B cells, but not IL‐10^? B cells, promoted IL‐10 production by CD4⁺ T cells. These results demonstrate that IL‐10‐producing Breg cells may represent an important mechanism for controlling the immunopathology and prevention of CM associated with P. berghei infection.     

Influenza matrix 1‐specific human CD4+FOXP3+ and FOXP3− regulatory T cells can be detected long after viral clearance

Control and termination of infection with Influenza A virus is associated with increased IL‐10 production in mouse models. Notably, IL‐10 can be produced by Treg. Therefore, we investigated whether the population of IL‐10‐producing influenza‐specific CD4⁺ T cells comprised Treg as they are potent suppressors of the adaptive immune response. Influenza‐specific IL‐10‐producing T cells were detected in all human donors displaying influenza‐specific immunity. Isolation of Matrix 1 protein‐specific IL‐10‐producing T‐cell clones revealed that a substantial proportion of these T‐cell clones displayed the capacity to suppress effector cells, functionally identifying them as Treg. Both FOXP3⁺ and FOXP3^? CD4⁺ Treg were isolated and all were able to exert their suppressive capacity when stimulated with cognate antigen, including influenza virus‐infected cells. In vitro suppression was not mediated by IL‐10 but involved interference with the IL‐2 axis. The isolated Treg suppressed amongst others the IL‐2 production of influenza‐specific T‐helper cells as well as partially prevented the upregulation of the high‐affinity IL‐2 receptor on CD8 effector cells. So far the induction of virus‐specific Treg has only been studied in the context of chronic viral infections. This study demonstrates that virus‐specific Treg can also be induced by viruses that are rapidly cleared in humans.     

CD4+ Foxp3+ regulatory T‐cell number increases in the gastric tissue of C57BL/6 mice infected with Helicobacter pylori

Helicobacter pylori (H. pylori), one of the most common infections, is associated with various clinical outcomes. In addition to inducing inflammation, immunological clearance of the pathogen is often incomplete. Regulatory T cells (Treg cells) have been recently demonstrated to play an important role in H. pylori infection and the final clinical outcome. The aim of this study was to investigate the number and localization of CD4⁺Foxp3⁺ Treg cells in stomachs and spleens of H. pylori‐infected mice. The expression levels of Foxp3 as well as anti‐ and pro‐inflammatory cytokines before and after H. pylori triple eradication therapy were examined. We found that the percentages of CD4⁺Foxp3⁺ Treg cells out of the lamina propria lymphocytes (LPLs) and spleen lymphocytes in the infection group were higher than the PBS negative control group and the treatment group. H. pylori antigen stimulation was associated with an increased number of Treg cells in vitro. Furthermore, compared with the PBS and treatment groups, a higher mRNA expression level of Foxp3 in the gastric tissue was detected in the infection group. IL‐10 and TGF‐β1 contents were increased significantly in the culture supernatant of spleen lymphocyte stimulated with H. pylori antigen. A marked elevation in serum IFN‐γ level was observed in H. pylori‐infected mice. In addition, gastric tissues of the infection group contained more Foxp3⁺ cells. These results indicate that the percentage of CD4⁺Foxp3⁺ Treg cells are increased in H. pylori‐infected mice, suggesting a role of Treg cells in H. pylori‐induced pathologies, even at the early stages of chronic gastritis and gastric tumorigenesis.     

UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells

Background In human asthma, and experimental allergic airways disease in mice, antigen‐presenting cells and CD4⁺ effector cells at the airway mucosa orchestrate, and CD4⁺CD25⁺ regulatory T cells attenuate, allergen immunity. UV irradiation of skin before sensitization with ovalbumin (OVA) causes significantly reduced asthma‐like responses in respiratory tissues. Objective To determine whether UV‐induced changes in CD11c⁺ cells, CD4⁺CD25⁺ effector cells or CD4⁺CD25⁺ regulatory cells in the trachea and airway draining lymph nodes (ADLNs) were responsible for reduced allergic airways disease. Methods The phenotype and function of CD11c⁺ cells and CD4⁺CD25⁺ cells in the trachea and ADLNs of UV‐ and non‐irradiated, OVA‐sensitized mice was examined 24 h after a single exposure to aerosolized OVA. Results No changes in the function of CD11c⁺ cells from UV‐irradiated mice were observed. CD4⁺CD25⁺ cells from UV‐irradiated, OVA‐sensitized mice harvested 24 h after OVA aerosol proliferated less in response to OVA in vitro and were unable to suppress the proliferation of OVA‐sensitized responder cells. This result suggested reduced activation of effector T cells in the airway mucosa of UV‐irradiated, OVA‐sensitized mice. To exclude regulatory cells of any type, there was similar proliferation in vivo to aerosolized OVA by CFSE‐loaded, OVA‐TCR‐specific CD4⁺ cells adoptively transferred into UV‐ and non‐irradiated, OVA‐sensitized mice. In addition, there was no difference in the expression of regulatory T cell markers (Foxp3, IL‐10, TGF‐β mRNA). To examine effector T cells, ADLN cells from UV‐irradiated, OVA‐sensitized and ‐challenged mice were cultured with OVA. There was reduced expression of the early activation marker CD69 by CD4⁺CD25⁺ cells, and reduced proliferation in the absence of the regulatory cytokine, IL‐10. Conclusion Reduced allergic airways disease in UV‐irradiated mice is due to fewer effector CD4⁺CD25⁺ cells in the trachea and ADLNs, and not due to UV‐induced regulatory cells. Cite this as: J. P. McGlade, D. H. Strickland, M. J. M. Lambert, S. Gorman, J. A. Thomas, M. A. Judge, J. T. Burchell, G. R. Zosky and P. H. Hart, Clinical & Experimental Allergy, 2010 (40) 772–785.     

CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL‐17 in a STAT3‐dependent manner

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell‐based clinical therapy. However, human Treg cells are “plastic”, and are able to produce IL‐17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL‐17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL‐17 in vitro when activated in the presence of IL‐1β, but not IL‐6. “IL‐17 potential” is restricted to population III (CD4⁺CD25^hiCD127^loCD45RA^?) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL‐17 induction. Importantly, we find that IL‐17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL‐17. Finally, we show that CD161⁺ population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL‐17‐producing Treg‐cell population at these sites. As IL‐17 production from this Treg‐cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.     

Anti‐CD25 monoclonal antibody Fc variants differentially impact regulatory T cells and immune homeostasis

Interleukin‐2 (IL‐2) is a critical regulator of immune homeostasis through its non‐redundant role in regulatory T (Treg) cell biology. There is major interest in therapeutic modulation of the IL‐2 pathway to promote immune activation in the context of tumour immunotherapy or to enhance immune suppression in the context of transplantation, autoimmunity and inflammatory diseases. Antibody‐mediated targeting of the high‐affinity IL‐2 receptor α chain (IL‐2Rα or CD25) offers a direct mechanism to target IL‐2 biology and is being actively explored in the clinic. In mouse models, the rat anti‐mouse CD25 clone PC61 has been used extensively to investigate the biology of IL‐2 and Treg cells; however, there has been controversy and conflicting data on the exact in vivo mechanistic function of PC61. Engineering antibodies to alter Fc/Fc receptor interactions can significantly alter their in vivo function. In this study, we re‐engineered the heavy chain constant region of an anti‐CD25 monoclonal antibody to generate variants with highly divergent Fc effector function. Using these anti‐CD25 Fc variants in multiple mouse models, we investigated the in vivo impact of CD25 blockade versus depletion of CD25⁺ Treg cells on immune homeostasis. We report that immune homeostasis can be maintained during CD25 blockade but aberrant T‐cell activation prevails when CD25⁺ Treg cells are actively depleted. These results clarify the impact of PC61 on Treg cell biology and reveal an important distinction between CD25 blockade and depletion of CD25⁺ Treg cells. These findings should inform therapeutic manipulation of the IL‐2 pathway by targeting the high‐affinity IL‐2R.     

Foxp3+ regulatory T cells are activated in spite of B7‐CD28 and CD40‐CD40L blockade

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)‐cell activity. We previously reported that the blockade of the B7‐CD28 and CD40L‐CD40 interaction efficiently suppresses allogeneic T‐cell activation in vivo. This was characterized by an initial rise in Foxp3⁺ cells, followed by depletion of host‐reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4⁺ cell activation. When CTLA‐4Ig and anti‐CD40L mAb (MR1) were added to the cultures, T‐cell proliferation and IL‐2 production were strongly reduced. However, Foxp3⁺ cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4⁺ cells much more efficiently than did freshly isolated Treg cells. CD4⁺ cells activated by allogeneic cells in the presence of MR1 and CTLA‐4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4⁺ cells when Foxp3⁺ Treg cells were removed. We conclude that natural Treg cells are less dependent on B7‐CD28 or CD40‐CD40L costimulation compared with Foxp3^? T cells. Reduced costimulation therefore alters the balance between Teff and Treg‐cell activation in favor of Treg‐cell activity.