Sepsis leads to a reduced antigen-specific primary antibody response

Immunosuppression, impaired cytokine production and high susceptibility to secondary infections are characteristic for septic patients, and for mice after induction of polymicrobial septic peritonitis by sublethal cecal ligation and puncture (CLP). Here, we demonstrate that CLP markedly altered subsequent B‐cell responses. Total IgG and IgM levels, as well as the memory B‐cell response, were increased in septic mice, but antigen‐specific primary antibody production was strongly impaired. We found that two days after CLP, CD11b⁺ splenocytes were activated as demonstrated by the increased expression of activation markers, expression of arginase and production of NO by immature myeloid cells. The in vivo clearance of a bacterial infection was not impaired. DCs demonstrated reduced IL‐12 production and altered antigen presentation, resulting in decreased proliferation but enhanced IFN‐γ production by CD4⁺ cells. CD4⁺ T cells from mice immunized on day 2 after CLP showed reduced Th1 and Th2 cytokine production. In addition, there was an increase in Treg cells. Interestingly, levels of immature B cells decreased but levels of mature B cells increased two days after CLP. However, adoptive transfer of naïve CD4⁺ T cells, naïve B cells, or naïve DCs did not rescue the antigen‐specific antibody response.     

OX40 and IL‐7 play synergistic roles in the homeostatic proliferation of effector memory CD4+ T cells

T‐cell homeostasis preserves the numbers, the diversity and functional competence of different T‐cell subsets that are required for adaptive immunity. Naïve CD4⁺ T (T_N) cells are maintained in the periphery via the common γ‐chain family cytokine IL‐7 and weak antigenic signals. However, it is not clear how memory CD4⁺ T‐cell subsets are maintained in the periphery and which factors are responsible for the maintenance. To examine the homeostatic mechanisms, CFSE‐labeled CD4⁺CD44^highCD62L^low effector memory T (T_EM) cells were transferred into sublethally‐irradiated syngeneic C57BL/6 mice, and the systemic cell proliferative responses, which can be divided distinctively into fast and slow proliferations, were assessed by CFSE dye dilution. We found that the fast homeostatic proliferation of T_EM cells was strictly regulated by both antigen and OX40 costimulatory signals and that the slow proliferation was dependent on IL‐7. The simultaneous blockade of both OX40 and IL‐7 signaling completely inhibited the both fast and slow proliferation. The antigen‐ and OX40‐dependent fast proliferation preferentially expanded IL‐17‐producing helper T cells (Th17 cells). Thus, OX40 and IL‐7 play synergistic, but distinct roles in the homeostatic proliferation of CD4⁺ T_EM cells.     

Human and mouse perforin are processed in part through cleavage by the lysosomal cysteine proteinase cathepsin L

Summary Infection of mice with the gastrointestinal nematode Trichuris muris represents a valuable tool to investigate and dissect intestinal immune responses. Resistant mouse strains respond to T. muris infection by mounting a T helper type 2 response. Previous results have shown that CD4⁺ T cells play a critical role in protective immunity, and that CD4⁺ T cells localize to the infected large intestinal mucosa to confer protection. Further, transfer of CD4⁺ T cells from immune mice to immunodeficient SCID mice can prevent the development of a chronic infection. In the current study, we characterize the protective CD4⁺ T cells, describe their chemokine receptor expression and explore the functional significance of these receptors in recruitment to the large intestinal mucosa post‐T. muris infection. We show that the ability to mediate expulsion resides within a subpopulation of CD4⁺ T cells marked by down‐regulation of CD62L. These cells can be isolated from intestine‐draining mesenteric lymph nodes (MLN) from day 14 post‐infection, but are rare or absent in MLN before this and in spleen at all times post‐infection. Among CD4⁺ CD62L^low MLN cells, the two most abundantly expressed chemokine receptors were CCR6 and CXCR3. We demonstrate for the first time that CD4⁺ CD62L^low T‐cell migration to the large intestinal mucosa is dependent on the family of Gα_i‐coupled receptors, to which chemokine receptors belong. CCR6 and CXCR3 were however dispensable for this process because neutralization of CCR6 and CXCR3 did not prevent CD4⁺ CD62L^low cell migration to the large intestinal mucosa during T. muris infection.     

Antigen presentation by B cells guides programing of memory CD4+ T‐cell responses to a TLR4‐agonist containing vaccine in mice

The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here, we sought to determine the role of B cells in the induction of T‐helper 1 (T_H1) CD4⁺ T cells upon vaccination with a tuberculosis (TB) antigen combined with a TLR4 agonist. We used B‐cell deficient mice (μMT^?/?), tetramer‐positive CD4⁺ T cells, markers of memory “precursor” effector cells (MPECs), and T‐cell adoptive transfers and demonstrated that the early antigen‐specific cytokine‐producing T_H1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory T_H1 response in μMT^?/? mice. We further show that antigen‐presentation by B cells is necessary for their role in MPEC generation using B‐cell adoptive transfers from wt or MHC class II knock‐out mice into μMT^?/? mice. Our study challenges the view that B‐cell deficiency exclusively alters the T_H1 response at memory time‐points. Collectively, our results provide new insights on the multifaceted roles of B cells that will have a high impact on vaccine development against several pathogens including those requiring T_H1 cell‐mediated immunity.     

Marked anti-tumor effects of CD8+CD62L+ T cells from melanoma-bearing mice

CD8⁺CD62L⁺ T cells have been shown to play pivotal roles in anti-viral immunity, chronic myeloid leukemia and renal cell carcinoma. Recently, CD8⁺CD62L⁺ T cells from naïve mice (nCD8⁺CD62L⁺ T cells) have shown superior anti-tumor properties in melanoma-bearing mice. Considering that antigen-specific memory T cells have shown to possess more potent immunity than non-specific memory T cells, we hypothesized that CD8⁺CD62L⁺ T cells from tumor-bearing individuals (mCD8⁺CD62L⁺ T cells) might have superior anti-tumor effect than nCD8⁺CD62L⁺ T cells. Therefore, we investigated phenotypes, functions and the in vivo distribution of mCD8⁺CD62L⁺ T cells in tumor-bearing mice. We found that, while keeping the features of central memory T cells, the frequency of mCD8⁺CD62L⁺ T cell in the spleen of tumor-bearing mice was significantly higher than that the one of nCD8⁺CD62L⁺ T cell in naive mice. Moreover, we demonstrated that mCD8⁺CD62L⁺ T cells had higher proliferation rate and IFN-γ production than nCD8⁺CD62L⁺ T cells, in vitro. We performed adoptive transfer of mCD8⁺CD62L⁺ T cells into melanoma-bearing mice and tracked them in spleen, lymph nodes and in melanoma tissues. Our results show that mCD8⁺CD62L⁺ T cells had stronger in vivo anti-tumoral activity than nCD8⁺CD62L⁺ T cells. This study highlights the therapeutic potential of mCD8⁺CD62L⁺ T cells in the immunotherapy of melanoma and possibly other tumors.     

c‐Rel is crucial for the induction of Foxp3+ regulatory CD4+ T cells but not TH17 cells

The NF‐κB/Rel family member c‐Rel was described to be required for the development of T_H1 responses. However, the role of c‐Rel in the differentiation of T_H17 and regulatory CD4⁺Foxp3⁺ T cells (Treg) remains obscure. Here, we show that in the absence of c‐Rel, in vitro differentiation of pro‐inflammatory T_H17 cells is normal. In contrast, generation of inducible Treg (iTreg) within c‐Rel‐deficient CD4⁺ T cells was severely hampered and correlated to reduced numbers of Foxp3⁺ T cells in vivo. Mechanistically, in vitro conversion of naive CD4⁺ T cells into iTreg was crucially dependent on c‐Rel‐mediated synthesis of endogenous IL‐2. The addition of exogenous IL‐2 was sufficient to rescue the development of c‐Rel‐deficient iTreg. Thus, c‐Rel is essential for the development of Foxp3⁺ Treg but not for T_H17 cells via regulating the production of IL‐2.     

n‐Butyrate Anergized Effector CD4+ T Cells Independent of Regulatory T cell Generation or Activity

CD4⁺ T cell anergy reflects the inability of CD4⁺ T cells to respond functionally to antigenic stimulation through proliferation or IL‐2 secretion. Histone deacetylase (HDAC) inhibitors have been shown to induce anergy in antigen‐activated CD4⁺ T cells. However, questions remain if HDAC inhibitors mediate anergy through direct action upon activated CD4⁺ T cells or through the generation and/or enhancement of regulatory T (T_reg) cells. To assess if HDAC inhibitor n‐butyrate induces anergy independent of the generation or expansion of FoxP3⁺ T_reg cells in vitro, we examine n‐butyrate‐treated murine CD4⁺ T cells for anergy induction and FoxP3⁺ T_reg activity. Whereas n‐butyrate decreases CD4⁺ T cell proliferation and IL‐2 secretion, n‐butyrate did not augment FoxP3 protein production or confer a suppressive phenotype upon CD4⁺ T cells. Collectively, these data suggest that HDAC inhibitors can facilitate CD4⁺ T cell functional unresponsiveness directly and independently of T_reg cell involvement.     

Effects of simvastatin on the function of splenic CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells in sepsis mice

Simvastatin may be beneficial for treating sepsis due to its immune-regulating properties, although the mechanisms remain elusive. Herein, we hypothesized simvastatin may attenuate T cell dysfunction induced by sepsis. To test this hypothesis, we used a model based on cecal ligation and puncture (CLP) to induce sepsis in mice. Male C57BL/6 mice were pre-treated with simvastatin (0.2 μg/g of body weight) before CLP. The expression of B and T lymphocyte attenuator (BTLA) on splenic CD4⁺ T cells and T cell apoptosis, CD4⁺ and CD8⁺ T cells were quantified by flow cytometry. Immunohistochemical staining was performed to evaluate the loss of immune effector cells. Formation of TNF-α and interleukin 10 (IL-10) in the spleen and plasma levels of presepsin, IL-1β, and IL-6 were determined using enzyme-linked immunosorbent assay. Simvastatin markedly inhibited the reduction in cytokine secretion from lipopolysaccharide (LPS)-stimulated splenocytes. Simvastatin-treated mice had significantly decreased the percentages of negative costimulatory receptor BTLA on CD4 T cell expression. Simvastatin markedly reduced T cell apoptosis through downregulating the Fas/FasL expression and decrease the percentage of caspase-3 activity in spleen tissue. There was significantly less depletion of splenic CD4⁺ and CD8⁺ T cells in simvastatin-treated mice. Simvastatin reduced plasma levels of presepsin, IL-1β, and IL-6. Simvastatin can be a powerful regulator of immune function under sepsis conditions by improving T cell function in sepsis.     

Skin resident memory CD8+ T cells are phenotypically and functionally distinct from circulating populations and lack immediate cytotoxic function

The in‐depth understanding of skin resident memory CD8⁺ T lymphocytes (T_RM) may help to uncover strategies for their manipulation during disease. We investigated isolated T_RM from healthy human skin, which expressed the residence marker CD69, and compared them to circulating CD8⁺ T cell populations from the same donors. There were significantly increased proportions of CD8⁺CD45RA^–CD27^– T cells in the skin that expressed low levels of killer cell lectin‐like receptor G1 (KLRG1), CD57, perforin and granzyme B. The CD8⁺ T_RM in skin were therefore phenotypically distinct from circulating CD8⁺CD45RA^–CD27^– T cells that expressed high levels of all these molecules. Nevertheless, the activation of CD8⁺ T_RM with T cell receptor (TCR)/CD28 or interleukin (IL)‐2 or IL‐15 in vitro induced the expression of granzyme B. Blocking signalling through the inhibitory receptor programmed cell death 1 (PD)‐1 further boosted granzyme B expression. A unique feature of some CD8⁺ T_RM cells was their ability to secrete high levels of tumour necrosis factor (TNF)‐α and IL‐2, a cytokine combination that was not seen frequently in circulating CD8⁺ T cells. The cutaneous CD8⁺ T_RM are therefore diverse, and appear to be phenotypically and functionally distinct from circulating cells. Indeed, the surface receptors used to distinguish differentiation stages of blood T cells cannot be applied to T cells in the skin. Furthermore, the function of cutaneous T_RM appears to be stringently controlled by environmental signals in situ .     

The protein tyrosine phosphatase PTPN22 controls forkhead box protein 3 T regulatory cell induction but is dispensable for T helper type 1 cell polarization

Protein tyrosine phosphatases (PTPs) regulate T cell receptor (TCR) signalling and thus have a role in T cell differentiation. Here we tested whether the autoimmune predisposing gene PTPN22 encoding for a PTP that inhibits TCR signalling affects the generation of forkhead box protein 3 (FoxP3)⁺ T regulatory (T_reg) cells and T helper type 1 (Th1) cells. Murine CD4⁺ T cells isolated from Ptpn22 knock‐out (Ptpn22^KO) mice cultured in T_reg cell polarizing conditions showed increased sensitivity to TCR activation compared to wild‐type (WT) cells, and subsequently reduced FoxP3 expression at optimal‐to‐high levels of activation. However, at lower levels of TCR activation, Ptpn22^KO CD4⁺ T cells showed enhanced expression of FoxP3. Similar experiments in humans revealed that at optimal levels of TCR activation PTPN22 knock‐down by specific oligonucleotides compromises the differentiation of naive CD4⁺ T cells into T_reg cells. Notably, in vivo T_reg cell conversion experiments in mice showed delayed kinetic but overall increased frequency and number of T_reg cells in the absence of Ptpn22. In contrast, the in vitro and in vivo generation of Th1 cells was comparable between WT and Ptpn22^KO mice, thus suggesting PTPN22 as a FoxP3‐specific regulating factor. Together, these results propose PTPN22 as a key factor in setting the proper threshold for FoxP3⁺ T_reg cell differentiation.     

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögren's syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)‐10 producing T regulatory type 1 (Tr1) cells and CD4⁺CD25⁺ regulatory T cells (T_reg) cells were determined by flow cytometry and serum cytokine levels of IL‐4, IL‐6, IL‐10, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were evaluated by enzyme‐linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of T_reg cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4⁺CD25⁺ T_reg cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4⁺CD25⁺ T_reg cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4⁺CD25⁺ T_reg cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4⁺CD25⁺ T_reg cells in patients compared to controls. Serum IL‐6 and TNF‐α levels were elevated, while IL‐10 was decreased in patients compared to controls. Negative correlation was found between IL‐10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.     

The transduction pattern of IL‐12‐encoding lentiviral vectors shapes the immunological outcome

In situ modification of antigen‐presenting cells garnered interest in cancer immunotherapy. Therefore, we developed APC‐targeted lentiviral vectors (LVs). Unexpectedly, these LVs were inferior vaccines to broad tropism LVs. Since IL‐12 is a potent mediator of antitumor immunity, we evaluated whether this proinflammatory cytokine could enhance antitumor immunity of an APC‐targeted LV‐based vaccine. Therefore, we compared subcutaneous administration of broad tropism LVs (VSV‐G‐LV) with APC‐targeted LVs (DC2.1‐LV)‐encoding enhanced GFP and ovalbumin, or IL‐12 and ovalbumin in mice. We show that codelivery of IL‐12 by VSV‐G‐LVs or DC2.1‐LVs augments CD4⁺ or CD8⁺ T‐cell proliferation, respectively. Furthermore, we demonstrate that codelivery of IL‐12 enhances the CD4⁺ T_H1 profile irrespective of its delivery mode, while an increase in cytotoxic and therapeutic CD8⁺ T cells was only induced upon VSV‐G‐LV injection. While codelivery of IL‐12 by DC2.1‐LVs did not enhance CD8⁺ T‐cell performance, it increased expression of inhibitory checkpoint markers Lag3, Tim3, and PD‐1. Finally, the discrepancy between CD4⁺ T‐cell stimulation with and without functional CD8⁺ T‐cell stimulation by VSV‐G‐ and DC2.1‐LVs is partly explained by the observation that IL‐12 relieves CD8⁺ T cells from CD4⁺ T‐cell help, implying that a T_H1 profile is of minor importance for antitumor immunotherapy if IL‐12 is exogenously delivered.     

Sublingual tolerance induction with antigen conjugated to cholera toxin B subunit induces Foxp3+CD25+CD4+ regulatory T cells and suppresses delayed-type hypersensitivity reactions

Although sublingual (s.l.) immunotherapy with selected allergens is safe and often effective for treating patients with allergies, knowledge of the immunological mechanisms involved remains limited. Can s.l. administration of antigen (Ag) induce peripheral immunological tolerance and also suppress delayed‐type hypersensitivity (DTH) responses? To what extent can s.l.‐induced tolerance be explained by the generation of Foxp3⁺CD25⁺CD4⁺ regulatory T cells (T_reg)? This study addressed these questions in mice and compared the relative efficacy of administering ovalbumin (OVA) conjugated to cholera toxin B (CTB) subunit with administration of the same Ag alone. We found that s.l. administration of a single or even more efficiently three repeated 40‐μg doses of OVA/CTB conjugate suppressed T‐cell proliferative responses to OVA by cervical lymph node (CLN), mesenteric lymph node (MLN) and spleen cells and concurrently strongly increased the frequency of Ag‐specific T_reg in CLN, MLN and spleen and also transforming growth factor‐β (TGF‐β) levels in serum. The CLN and splenic cells from OVA/CTB‐treated BALB/c mice efficiently suppressed OVA‐specific T‐cell receptor (TCR) transgenic (DO11.10) CD25^?CD4⁺ effector T‐cell proliferation in vitro. Further, s.l. treatment with OVA/CTB completely suppressed OVA‐specific DTH responses in vivo and T‐cell proliferative responses in mice immunized subcutaneously with OVA in Freund's complete adjuvant. The intracellular expression of Foxp3 was strongly increased in OVA‐specific (KJ1‐26⁺) CD4⁺ T cells from OVA/CTB‐treated mice. Thus, s.l. administration of CTB‐conjugated Ag can efficiently induce peripheral T‐cell tolerance associated with strong increases in serum TGF‐β levels and in Ag‐specific Foxp3⁺CD25⁺CD4⁺ T_reg cells.     

Deficient CCR7 signaling promotes TH2 polarization and B-cell activation in vivo

The chemokine receptor CCR7 has a central role in regulating homing and positioning of T cells and DCs to lymph nodes (LNs) and participates in T‐cell development and activation. In this study, we addressed the role of CCR7 signaling in T_H2 polarization and B‐cell activation. We provide evidence that the lack of CCR7 drives the capacity of naïve CD4⁺ T cells to polarize toward T_H2 cells. This propensity contributes to a lymph node environment in CCR7‐deficent mice characterized by increased expression of IL‐4 and increased frequency of T_H2 cells. We show that elevated IL‐4 levels lead to B‐cell activation characterized by up‐regulated expression of MHC class II, CD23 and CD86. Activated B cells are in turn highly efficient in presenting antigen to CD4⁺ T cells and thus potentially contribute to the T_H2 microenvironment. Taken together, our results support the idea of a CCR7‐dependent patterning of T_H2 responses, with absent CCR7 signaling favoring T_H2 polarization, dislocation of T helper cells into the B‐cell follicles and, as a consequence, B‐cell activation.     

Differential effects of interleukin-12 on the development of naive mouse CD4+ T cells

The influence of interleukin (IL)-12 and IL-4 on the differentiation of naive CD4⁺ T cells was studied in an accessory cell-free in vitro system. Dense CD4⁺ T cells were purified from unimmunized mice and activated using immobilized anti-CD3 monoclonal antibodies (mAb) in the presence of IL-4, IL-12, or a combination of both cytokines, and restimulated after 6 days by re-exposure to anti-CD3-coated culture wells. T cells initially activated in the presence of IL-4 produced substantial amounts of IL-4 and trace amounts of interferon (IFN)-γ after restimulation at day 6 with plate-bound anti-CD3 mAb. By contrast, T cells primed in the presence of IL-12 produced high levels of IFN-γ and only minimal amounts of IL-4, thus indicating that IL-12 and IL-4 by acting directly on stimulated naive CD4⁺ T cells support the development of T_H1 and T_H2 cells, respectively. When naive CD4⁺ T cells were stimulated in the presence of IL-12 together with IL-4 in comparable concentrations, the effect of IL-12 on T_H1 differentiation was largely inhibited by IL-4. On the other hand, IL-12 exerted no inhibitory effect on IL-4-induced T_H2 differentiation but rather enhanced the production of IL-4 after restimulation of the respective T cells. Decreasing amounts of IL-4 in combination with a high level of IL-12 led to an increasing production of IFN-γ by the emerging T cells and, simultaneously, to a relatively high production of IL-4. These data were confirmed by time-course experiments which revealed that the delayed addition of IL-4 to IL-12-primed T cell cultures resulted in a gradual restoration of IFN-γ production whereas in parallel the secretion of IL-4 was not reduced over a wide period of delay (6–72 h). These results, therefore, demonstrate that (a) IL-4 dominates the effect of IL-12, (b) IL-12 promotes the development of T_H1 cells; however, in the presence of IL-12 and relatively high levels of IL-4 also the development of T_H2-like cells is slightly but significantly enhanced by IL-12, and (c) high amounts of IL-12 in combination with relatively low levels of IL-4 give rise to a T cell population that upon rechallenge exhibited a cytokine profile resembling that of T_H0 cells.     

Antigen-activated T cells induce IL-12p75 production from dendritic cells in an IFN-γ-independent manner

The addition of IL‐12p75 to naïve CD4⁺ T cells promotes their differentiation towards a T_H1‐type cytokine pattern. Dendritic cells stimulated by LPS generate IL‐12p75, but only if the environment also contains IFN‐γ. Thus, it appears that IFN‐γ is needed to start the response that will result in further production of IFN‐γ. We previously reported that paradoxically DCs produce IL‐12p75 only after engaging primed, but not naïve T cells. This study examines the mechanism by which primed T cells trigger IL‐12p75 secretion and asks whether this induction is also dependent on the presence of IFN‐γ. Here, we show that, in contrast to LPS, primed T cells induce IL‐12p75 in an IFN‐γ‐independent manner. Addition of rIFN‐γ to cocultures of naïve T cells with DCs did not induce IL‐12p75. Moreover, antigen‐activated CD4⁺ T cells from wild type or IFN‐γ‐deficient mice both initiated IL‐12p75 production from DCs. Surprisingly, we found that synergies between three T‐cell‐derived factors – CD40 Ligand, IL‐4 and GM‐CSF – were necessary and sufficient for IL‐12p75 production. These results suggest that there are at least two distinct pathways for IL‐12p75 production in vivo. Furthermore, the T‐cell‐dependent pathway of IL‐12p75 production employs molecules that are not classically associated with a T_H1‐type response.     

Transforming growth factor beta 1 plays an important role in inducing CD4+CD25+forhead box P3+ regulatory T cells by mast cells

The role of mast cells (MCs) in the generation of adaptive immune responses especially in the transplant immune responses is far from being resolved. It is reported that mast cells are essential intermediaries in regulatory T cell (T_reg) transplant tolerance, but the mechanism has not been clarified. To investigate whether bone marrow‐derived mast cells (BMMCs) can induce T_regs by expressing transforming growth factor beta 1 (TGF‐β1) in vitro, bone marrow cells obtained from C57BL/6 (H‐2^b) mice were cultured with interleukin (IL)‐3 (10 ng/ml) and stem cell factor (SCF) (10 ng/ml) for 4 weeks. The purity of BMMCs was measured by flow cytometry. The BMMCs were then co‐cultured with C57BL/6 T cells at ratios of 1:2, 1:1 and 2:1. Anti‐CD3, anti‐CD28 and IL‐2 were administered into the co‐culture system with (experiment groups) or without (control groups) TGF‐β1 neutralizing antibody. The percentages of CD4⁺CD25⁺forkhead box P3 (FoxP3)⁺ T_regs in the co‐cultured system were analysed by flow cytometry on day 5. The T_reg percentages were significantly higher in all the experiment groups compared to the control groups. These changes were deduced by applying TGF‐β1 neutralizing antibody into the co‐culture system. Our results indicated that the CD4⁺ T cells can be induced into CD4⁺CD25⁺FoxP3⁺ T cells by BMMCs via TGF‐β1.     

Increased susceptibility of Fas ligand‐deficient gld mice to Trypanosoma cruzi infection due to a Th2‐biased host immune response

Infection of BALB / c mice with Trypanosoma cruzi resulted in up‐regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4⁺ T cells, activation‐induced CD4⁺ T cell death (AICD), and in Fas : FasL‐mediated cytotoxicity. When CD4⁺ T cells from infected mice were co‐cultured with T. cruzi‐infected macrophages, onset of AICD exacerbated parasite replication. CD4⁺ T cells from T. cruzi‐infected FasL‐deficient BALB gld / gld mice had no detectable AICD in vitro and their activation with anti‐TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld / gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild‐type mice. Secretion of Th2 cytokines IL‐10 and IL‐4 by CD4⁺ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti‐IL‐4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild‐type mice. These results indicate that, besides controlling CD4⁺ T cell AICD and parasite replication in vitro, an intact Fas : FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2‐type immune response to the parasite.     

Plasmacytoid dendritic cells and type 1 interferon promote peripheral expansion of forkhead box protein 3+ regulatory T cells specific for the ubiquitous RNA‐binding nuclear antigen La/Sjögren's syndrome (SS)‐B

RNA‐binding nuclear antigens are a major class of self‐antigen to which immune tolerance is lost in rheumatic diseases. Serological tolerance to one such antigen, La/Sjögren's syndrome (SS)‐B (La), is controlled by CD4⁺ T cells. This study investigated peripheral tolerance to human La (hLa) by tracking the fate of hLa‐specific CD4⁺ T cells expressing the transgenic (Tg) 3B5.8 T cell receptor (TCR) after adoptive transfer into lymphocyte‐replete recipient mice expressing hLa as a neo‐self‐antigen. After initial antigen‐specific cell division, hLa‐specific donor CD4⁺ T cells expressed forkhead box protein 3 (FoxP3). Donor cells retrieved from hLa Tg recipients displayed impaired proliferation and secreted interleukin (IL)?10 in vitro in response to antigenic stimulation. Transfer of highly purified FoxP3‐negative donor cells demonstrated that accumulation of hLa‐specific regulatory T cells (T_reg) was due primarily to expansion of small numbers of donor T_reg. Depletion of recipient plasmacytoid dendritic cells (pDC), but not B cells, severely hampered the accumulation of FoxP3⁺ donor T_reg in hLa Tg recipients. Recipient pDC expressed tolerogenic markers and higher levels of co‐stimulatory and co‐inhibitory molecules than B cells. Adoptive transfer of hLa peptide‐loaded pDC into mice lacking expression of hLa recapitulated the accumulation of hLa‐specific T_reg. Blockade of the type 1 interferon (IFN) receptor in hLa Tg recipients of hLa‐specific T cells impaired FoxP3⁺ donor T cell accumulation. Therefore, peripheral expansion of T_reg specific for an RNA‐binding nuclear antigen is mediated by antigen‐presenting pDC in a type 1 IFN‐dependent manner. These results reveal a regulatory function of pDC in controlling autoreactivity to RNA‐binding nuclear antigens.     

Critical role of IL‐33 receptor ST2 in experimental cerebral malaria development

Cerebral malaria, a severe complication of Plasmodium falciparum infection, can be modeled in murine Plasmodium berghei ANKA (PbA) infection. PbA‐induced experimental cerebral malaria (ECM) is CD8⁺ T‐cell mediated, and influenced by T_H1/T_H2 balance. Here, we show that IL‐33 expression is increased in brain undergoing ECM and we address the role of the IL‐33/ST2 pathway in ECM development. ST2‐deficient mice were resistant to PbA‐induced neuropathology. They survived >20 days with no ECM neurological sign and a preserved cerebral microcirculation, while WT mice succumbed within 10 days with ECM, brain vascular leakage, distinct microvascular pathology obstruction, and hemorrhages. Parasitemia and brain parasite load were similar in ST2‐deficient and WT mice. Protection was accompanied by reduced brain sequestration of activated CD4⁺ T cells and perforin⁺ CD8⁺ T cells. While IFN‐γ and T‐cell‐attracting chemokines CXCL9 and CXCL10 were not affected in the absence of functional ST2 pathway, the local expression of ICAM‐1, CXCR3, and LT‐α, crucial for ECM development, was strongly reduced, and this may explain the diminished pathogenic T‐cell recruitment and resistance to ECM. Therefore, IL‐33 is induced in PbA sporozoite infection, and the pathogenic T‐cell responses with local microvascular pathology are dependent on IL‐33/ST2 signaling, identifying IL‐33 as a new actor in ECM development.