Anergy in vivo: down-regulation of antigen-specific CD4+ Th1 but not Th2 cytokine responses

Efficient immunologic tolerance, defined as antlgen-speclflcunresponslveness, can be peripherally induced by the l.v. Injectionof syngenelc splenocytes coupled with antigen using ethylenecarbodilmlde (ECDI). We have previously reported that unresponslvenessinduced via l.v. Injection of syngenelc splenocytes coupledwith intact, UV-lnactlvated Theiler's murine encephalomyelitisvirus (TMEV-SP) resulted in ‘split tolerance’. Bothvtrus-speclflc delayed-type hypersensltlvlty and lgG2a levelswere inhibited, whereas lgG1 levels were increased when comparedwith sham tolerized controls. In the present report we demonstratethat tolerance induced by l.v. Injection of TMEV-coupled splenocytesresulted in antigen-specific inhibition of T cell proliferation,as well as IL-2 and IFN- production in response to both wholeTMEV and the immunodomlnant viral epitope. Additionally, toleranceinduction resulted in abrogation of T_h1 -derived [IL-2, IFN-and LT/tumor necrosis factor-ß (TNF-ß)]cytokine mRNA expression in response to In vitro stimulationwith UV-inactlvated TMEV as determined by reverse transcrlptasepolymerase chain reaction. In contrast, expression of T_h2-derived(IL-4, IL-6 and IL-10) cytokine mRNA was not affected in tolerizedmice. Tolerance functioned directly at the level of CD4⁺ T_h1cells at both the induction and effector limbs as depletionof CD8⁺ T cells both prior to in vivo tolerizatlon or in vitroculture had no effect on inhibition of T_h1-specific responses.The mechanism of In vivo tolerance induction appeared to beanergy of CD4⁺ T_h1 cells since IL-2, IFN- and LT/TNF-ßmRNA expression as well as virus-specific prollferatlve responsescould be restored by addition of rlL-2 to In vitro culturesof tolerant, CD4⁺ T_h1 populations. These results suggest thatin vivo ‘split tolerance’ Induced by l.v. Injectionof ECDI-flxed, antigen-coupled splenocytes involves anergy ofTMEV-speclflc, CD4⁺ T_h1 lymphocytes and concomitant primingof T_h2 cells. The induction of antlgen-speclflc, in vivo anergyhas important implications in the design of therapeutic strategiesfor immunopathologic diseases mediated by T_h1 lymphocytes, especiallyT cell-mediated autoimmune disorders.     

IL-23-dependent IL-17 drives Th1-cell responses following Mycobacterium bovis BCG vaccination

The generation of effective type 1 T helper (Th1)-cell responses is required for immunity against intracellular bacteria. However, some intracellular bacteria require interleukin (IL)-17 to drive Th1-cell immunity and subsequent protective host immunity. Here, in a model of Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccination in mice, we demonstrate that the dependence on IL-17 to drive Th1-cell responses is a host mechanism to overcome bacteria-induced IL-10 inhibitory effects. We show that BCG-induced prostaglandin-E2 (PGE2) promotes the production of IL-10 which limits Th1-cell responses, while simultaneously inducing IL-23 and Th17-cell differentiation. The ability of IL-17 to downregulate IL-10 and induce IL-12 production allows the generation of subsequent Th1-cell responses. Accordingly, BCG-induced Th17-cell responses precede the generation of Th1-cell responses in vivo, whereas the absence of the IL-23 pathway decreases BCG vaccine-induced Th17 and Th1-cell immunity and subsequent vaccine-induced protection upon M. tuberculosis challenge. Importantly, in the absence of IL-10, BCG-induced Th1-cell responses occur in an IL-17-independent manner. These novel data demonstrate a role for the IL-23/IL-17 pathway in driving Th1-cell responses, specifically to overcome IL-10-mediated inhibition and, furthermore, show that in the absence of IL-10, the generation of BCG-induced Th1-cell immunity is IL-17 independent.     

Differential regulation of Th1 responses and CD154 expression in human CD4+ T cells by IFN-alpha

Like interleukin (IL)-12, interferon (IFN)-alpha has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-alpha on CD154 expression in human CD4+ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4+ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-alpha and IL-12 in the complete absence of accessory cells. IFN-alpha suppressed CD154 protein and mRNA expression in CD4+ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-alpha by itself did not enhance IFN-gamma production or mRNA expression in CD4+ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-alpha enhanced IL-12Rbeta2 mRNA expression in anti-CD3-stimulated CD4+ T cells. Neither IFN-alpha nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4+ T cells. These results indicate that IFN-alpha by itself enhances CD154 expression in CD4+ T cells independently of the induction of IFN-gamma mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-alpha might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4+ T cells might be regulated through different mechanisms.     

Induction of Th1 and Th2 CD4+ T cell responses by oral or parenteral immunization with ISCOMS

We examined the ability of oral or parenteral immunization with immune stimulating complexes containing ovalbumin (ISCOMS-OVA) to prime T cell proliferative and cytokine responses. A single subcutaneous immunization with ISCOMS-OVA primed potent antigen-specific proliferative responses in the draining popliteal lymph node, which were entirely dependent on the presence of CD4⁺ T cells. CD8⁺ T cells did not proliferate in vitro even in the presence of the appropriate peptide epitope and exogenous interleukin (IL)-2. Primed popliteal lymph node cells produced IL-2, IL-5 and interferon (IFN)-γ, but not IL-4 when restimulated with OVA in vitro. Serum antigen-specific IgG1 and IgG2a antibody responses were also primed by subcutaneous immunization with ISCOMS-OVA, confirming the stimulation of both Th1 and Th2 cells in vivo. Spleen cells from subcutaneously primed mice produced a similar pattern of cytokines, indicating that disseminated priming had occurred. Oral immunization with ISCOMS-OVA also primed local antigen-specific proliferative responses in the mesenteric lymph node and primed an identical pattern of systemic cytokine responses in the spleen. The ability of ISCOMS to prime both Th1 and Th2 CD4⁺ T cell responses may be central to their potent adjuvant activities and confirm the potential of ISCOMS as future oral vaccine vectors.     

DNA vaccination elicits protective immune responses against pandemic and classic swine influenza viruses in pigs

Swine influenza is a highly contagious viral infection in pigs that significantly impacts the pork industry due to weight loss and secondary infections. There is also the potential of a significant threat to public health, as was seen in 2009 when the pandemic H1N1 influenza virus strain emerged from reassortment events among avian, swine, and human influenza viruses within pigs. As classic and pandemic H1N1 strains now circulate in swine, an effective vaccine may be the best strategy to protect the pork industry and public health. Current inactivated-virus vaccines available for swine influenza protect only against viral strains closely related to the vaccine strain, and egg-based production of these vaccines is insufficient to respond to large outbreaks. DNA vaccines are a promising alternative since they can potentially induce broad-based protection with more efficient production methods. In this study we evaluated the potentials of monovalent and trivalent DNA vaccine constructs to (i) elicit both humoral and gamma interferon (IFN-γ) responses and (ii) protect pigs against viral shedding and lung disease after challenge with pandemic H1N1 or classic swine H1N1 influenza virus. We also compared the efficiency of a needle-free vaccine delivery method to that of a conventional needle/syringe injection. We report that DNA vaccination elicits robust serum antibody and cellular responses after three immunizations and confers significant protection against influenza virus challenge. Needle-free delivery elicited improved antibody responses with the same efficiency as conventional injection and should be considered for development as a practical alternative for vaccine administration.     

Altered Th1/Th2 commitment in human CD4+ T cells with ageing

The human immune system undergoes continuous remodelling with the advancement of age. Since age-associated functional alterations in the immune system could be caused by a possible change in helper T cell regulation in elderly subjects, we comparatively studied the function of CD4+ T cells in peripheral blood obtained from both young and old healthy volunteers. Upon cell activation by phorbol myristate acetate and ionomycin, the proportion of CD4+ T cells containing interferon-gamma (IFN-gamma) was found to be greater in the old subjects. Utilizing a co-culture system, which activated CD4+ T cells via the TCR/CD3 complex and CD28, we found that CD4+ T cells from the old subjects secreted more IFN-gamma and IL-2, but less IL-4, than those from the young subjects. Upon cell activation by co-culture, CD4+ T cells from the old subjects expressed more CD26, CD40L, and LFA-1, but less CD30, than those from the young. These results together suggest that the microenvironment in which CD4+ T cells develop in older people may cause production of more cells committed to Th1 than that in younger subjects.     

Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases

CD4⁺ T cells play a key role in regulating immune system function. When these regulatory processes go awry, organ-specific autoimmune diseases may develop. Here, Roland Liblau, Steven Singer and Hugh McDevitt explore the thesis that a particular subset of CD4⁺ T cells, namely T helper 1 (Th1) cells, contributes to the pathogenesis of organ-specifc autoimmune diseases, while another subset, Th2 cells, prevents them.     

Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4+ and CD8+ T cells

The pathogenesis of pulmonary sarcoidosis has been related to an increased production of Th1-like cytokines. However, cytokine expression in sarcoidosis has not been systematically studied at a single-cell level. We therefore investigated the expression of IL-2, IL-4, IL-13, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) intracellularly in bronchoalveolar lavage (BAL) and peripheral blood CD3+ T lymphocytes from patients with pulmonary sarcoidosis (radiologic stage II-III, n = 8) and normal controls (n = 9) by flow cytometry. In contrast to IL-4 and IL-13, the percentage of T lymphocytes expressing intracellular IL-2 (49.3 +/- 21.3% versus 14.5 +/- 15.6%), IFN-gamma (75.5 +/- 14.9% versus 32.6 +/- 18.7%) and TNF-alpha (68.3 +/- 18.7% versus 36.8 +/- 20.8%) was significantly higher in patients with sarcoidosis than in normal controls (each P < 0.005). In contrast to BAL lymphocytes, expression of these cytokines in peripheral blood lymphocytes did not differ between patients with sarcoidosis and normal controls. Close correlations were observed between the percentages of BAL lymphocytes expressing intracellular IL-2, IFN-gamma and TNF-alpha, but not for IL-4 or IL-13. Analysis of the expression of these cytokines in T lymphocyte subsets revealed IL-2, IFN-gamma, and TNF-alpha in CD4+ as well as CD8+ T lymphocytes, suggesting a contribution of TC1 cells to the production of proinflammatory cytokines in sarcoidosis. We conclude that a Th1-like cytokine pattern can be observed in CD4+ as well as in CD8+ BAL T lymphocytes in patients with pulmonary sarcoidosis.     

T helper 1 (Th1)/Th2 cytokine expression shift of peripheral blood CD4+ and CD8+ T cells in patients at the post-acute phase of stroke

Local humoral and cellular immune responses modulate the inflammatory processes involved in the development of atherosclerotic lesions, as well as in the evolution of brain infarcts in stroke patients. The role of systemic adaptive immunity on the progression of such disease manifestations is less clear. In the current study, we evaluated the percentages of T helper 1 (Th1) [interleukin (IL)-2, interferon (IFN)-gamma] and Th2 (IL-4, IL-10) cytokine-producing peripheral blood CD4+ and CD8+ T cells in 23 patients with a history of ischaemic stroke (IS) at the chronic stable phase of the disease (median post-stroke time 34.5 months). Seven stroke-free individuals matched for age and vascular risk factors (matched controls, MC) were collected for comparison. To measure cytokine values at baseline and after stimulation, we used a flow cytometry method of intracellular cytokine staining. Intrinsic Th1 and Th2 cytokine production in unstimulated T cells was negligible in all study participants. Following mitogenic stimulation with phorbol 12-myristate13-acetate/ionomycin, both the IS and the MC groups exhibited a similarly strong Th1 response; IL-2 production predominated in the CD4+ T cells and IFN-gamma in the CD8+ T cells. However, when measuring the Th2 cytokine-production capacity post-stimulation, a significant increase in the percentage of IL-4-producing T cells was observed in the IS groups, compared with the MC group, resulting in a significantly lower ratio of IFN-gamma-/IL-4-producing T cells. No such Th2 enhancement could be confirmed for the case of IL-10. We propose that in IS patients there is a systemic shift of the immune system towards Th2 responses at the late post-acute phase of stroke.     

Human 4-1BB regulates CD28 co-stimulation to promote Th1 cell responses

Our present study provides evidence that the 4-1BB signal is critical to CD28 co-stimulation in maintaining T cell activation when CD28 has been down-regulated because of repeated stimulation. The 4-1BB signal synergized with CD28 co-stimulation by lowering the threshold of anti-CD28 required to sustain proliferation and IL-2 production. The 4-1BB signal also modulated CD28-mediated cytokine profiles by markedly enhancing Th1 but suppressing Th2-type cytokine production. The 4-1BB signal generated Th1-type cells, as identified by intracellular IFN-γ production. IFN-γ induction was detected preferentially in 4-1BB-expressing cells, but not in those expressing CD30. 4-1BB and CD30 were induced in both CD4⁺ and CD8⁺ cells, but the location of the two molecules was mutually exclusive in each T cell subset. Our study suggests that the 4-1BB signal regulates CD28 co-stimulation in the targeted subset cells to favor Th1 development and maintain long-term cell growth.     

支气管哮喘患者外周血Th17、CD4~+CD25~+Treg细胞表达特征

目的:探讨外周血Th17和CD4+CD25+调节性T细胞(Treg)在支气管哮喘患者中的表达特征。方法:41例慢性持续期哮喘患者,分为间歇-轻度组(n=23)和中重度组(n=18),行肺功能检查和哮喘控制问卷(ACQ)调查,20例正常人作为对照。通过流式细胞术检测外周血Th17和CD4+CD25+Treg细胞的比例。ELISA检测血浆以及植物血凝素刺激24小时后外周血单个核细胞(PBMC)上清液中的IL-17、IL-10、TGF-β水平。结果:中重度哮喘组外周血Th17细胞比例及血浆IL-17水平高于间歇-轻度哮喘和正常人组,而外周血CD4+CD25+Foxp3+Treg细胞比例及血浆IL-10、TGF-β水平则降低。中重度哮喘组PBMC上清液中IL-17水平增高。哮喘患者FEV1(%预计值)与Th17细胞及血浆IL-17表达成负相关,与CD4+CD25+Treg表达成正相关。ACQ平均得分与Th17细胞和血浆IL-17表达成正相关,与外周血CD4+CD25+Treg表达成负相关。结论:中重度哮喘中外周血Th17细胞应答增强,而CD4+CD25+Treg细胞缺乏,哮喘的严重程度及症状控制与外周血Th17/Treg免疫应答失衡密切相关。     

LIGHT is dispensable for CD4+ and CD8+ T cell and antibody responses to influenza A virus in mice

The tumor necrosis factor family ligands, LIGHT (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes), 4-1BBL and CD70, are found in the same gene cluster on mouse chromosome 17. Although the roles of 4-1BB-4-1BBL and CD27-CD70 interactions in anti-viral T cell responses have been well established, the role of LIGHT in T cell activation/expansion in vivo is less clear. Under conditions that were previously employed to demonstrate a role for 4-1BBL in CD8+ T cell memory, wild-type and LIGHT-/- mice were infected with influenza A virus and primary and memory/recall responses were measured at various time points thereafter. Neither primary expansion nor memory/recall CD8+ T cell responses were affected by the absence of LIGHT, as measured up to 2 months post-infection. CD4+ T cell responses were also unaffected by LIGHT deficiency. Furthermore, we found that LIGHT played no role in the induction of influenza-specific IgG1 and IgG2a serum antibodies. Taken together, these data suggest that LIGHT is dispensable for the acquired immune response to influenza virus in mice with no effect on the induction, maintenance or reactivation of CD8+ T cell memory.     

CD4+ T-cell immunity after pandemic influenza vaccination cross-reacts with seasonal antigens and functionally differs from active influenza infection

Antigen-specific antibodies are well characterized after vaccination with pandemic H1N1 or seasonal influenza vaccines. However, knowledge on cellular immunity toward pandemic H1N1 after vaccination and infection and cross-reactivities toward seasonal antigens is limited. Nineteen individuals were vaccinated with the pandemic H1N1 vaccine. Among those, ten had been prevaccinated against seasonal influenza. CD4(+) T cells specific for pandemic H1N1 and for seasonal vaccine, and antibodies were monitored using flow cytometry and ELISA/neutralization assays, respectively. In addition, seven patients with acute pandemic influenza infection were analyzed. Pandemic H1N1 vaccination induced a strong 4.63-fold (IQR 4.16) increase in antigen-specific CD4(+) T cells that was more pronounced in individuals not prevaccinated with seasonal influenza (p = 0.01). T-cell levels toward seasonal vaccine concomitantly rose by 2.71-fold (IQR 2.26). Likewise, prevaccination with seasonal influenza induced a less pronounced increase in specific antibodies. Influenza-specific T cells in vaccinees had a Th1 phenotype mainly coexpressing IFN-γ and IL-2, whereas patients with active pandemic influenza showed a shift toward cells predominantly expressing IFN-γ. In conclusion, T cells toward seasonal influenza antigens cross-react with pandemic H1N1 antigens and affect induction of specific T cells after pandemic influenza vaccination. In addition, the cytokine patterns of specific T cells during acute H1N1 infection and after vaccination differ, and the predominantly dual-positive cytokine profile of vaccine-induced T cells suggests sufficient functionality to confer successful virus control.     

桥本氏甲状腺炎患者Th17与调节性T细胞免疫失衡的研究

目的 检测桥本氏甲状腺炎(HT)患者甲状腺组织及外周静脉血中Th17细胞和调节性T 细胞(Treg)细胞相关细胞因子的表达水平,分析Th17/Treg免疫平衡在HT发病机制中的作用.方法 采用逆转录-聚合酶链反应(RT-PCR)法,检测10例HT甲功正常组、10例HT甲低组和10例正常甲状腺组织(对照组)中IL-17A...     

CD4+ CD45RA+ and CD4+ CD45RO+ T cells differ in their TCR-associated signaling responses.

Peripheral CD4+ T cells can be divided into two different functional populations based on the expression of distinct isoforms of the surface molecule CD45. We have investigated the differences in the proximal signaling induced by anti-CD3 monoclonal antibody in purified populations of "naive" CD45RA+ and "memory" CD45RO+ human CD4+ T cells. Expression of cell surface CD3, CD4 and CD28 was comparable between RA+ and RO+ cells. However, TCR-directed stimulation in the form of anti-CD3 produced markedly different patterns of intracellular signaling. Greater inositol triphosphate generation occurred in naive cells and the rise in intracellular free calcium was also substantially greater in naive than in memory cells. Cells with the naive phenotype were considerably more active in TCR-dependent tyrosine phosphorylation, both at an overall level and specifically in terms of TCR-zeta and ZAP-70 phosphorylation. Despite these differences in phosphorylation, the amounts of TCR-zeta, ZAP-70 and Ick were equivalent between the two subsets. These findings suggest that the TCR-dependent signaling is differentially regulated in naive and memory CD4+ T cells. This may be due to differences in the way that the two isoforms of the CD45 phosphatase regulate the activity of proximal kinases in the TCR signaling pathway, and could be an important means by which the unique functions of differentiated T cell populations are maintained.     

Differential regulation of IL-13 and IL-4 production by human CD8+ and CD4+ Th0, Th1 and Th2 T cell clones and EBV-transformed B cells

In the present study, the requirements and characteristics forthe production of IL-13 by human T cells, T cell clones andB cells were determined and compared with those of IL-4. IL-13was produced by human CD4⁺ and CD8⁺ T lymphocyte subsets isolatedfrom peripheral blood mononuclear cells and by CD4⁺ and CD8⁺T cell clones. CD4⁺ T cell clones belonging to T_h0, T_h1-likeand T_h2-like subsets produced IL-13 following antigen-specificor polyclonal activation. In addition, EBV-transformed B celllines expressed IL-13 mRNA and produced small amounts of IL-13protein. Expression of IL-13 mRNA and production of IL-13 proteinby peripheral blood T cells and T cell clones was induced rapidlyand was relatively long lasting, whereas IL-4 production bythese cells was transient In addition, IL-13 mRNA expressionwas induced by modes of activation that failed to induce IL-4mRNA expression. IL-13 shares many biological activities withIL-4 which Is compatible with the notion that the IL-13 andIL-4 receptors share a common component required for signaltransduction. However, IL-13 lacks the T cell-activating propertiesof IL-4. Here we have shown that this is related to the factthat T cells fall to bind radiolabeled IL-13 and do not expressthe IL-13-speclflc receptor component Taken together, theseresults indicate that the differences In expression and biologicalactivities of IL-4 and IL-13 on T cells may have consequencesfor the relative roles of these cytokines In the immune response.     

Interleukin-11 modulates Th1/Th2 cytokine production from activated CD4+ T cells.

Recombinant human interleukin-11 (rHuIL-11) is a pleiotropic cytokine with effects on multiple cell types. rHuIL-11 reduces activated macrophage activity and downregulates production of proinflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). In vitro and in vivo, rHuIL-11 inhibits production of key immunostimulatory cytokines, including IL-12 and interferon-gamma (IFN-gamma). rHuIL-11 has recently demonstrated immunomodulatory activity to downregulate IFN-gamma production, increase IL-4 production, and reduce inflammatory tissue injury in a human psoriasis clinical trial. The cellular mechanisms of these effects are not fully elucidated. We demonstrate here that expression of gp130 and IL-11 receptor (IL-11R) alpha mRNA, components of the IL-11R complex, are detected in human and murine CD4(+) and CD8(+) lymphocytes, suggesting that rHuIL-11 can directly interact with T cells. In a cell culture model of murine T cell differentiation, rHuIL-11 acts to inhibit IL-2 production as well as IL-12-induced IFN-gamma production and enhances IL-4 and IL-10 production. rHuIL-11 had no effect on T cell proliferation. The ability of rHuIL-11 to modulate cytokine production from activated CD4(+) T cells provides a mechanism through which rHuIL-11 may ameliorate such inflammatory diseases as psoriasis.     

Staphylococcus aureus specific lung resident memory CD4+ Th1 cells attenuate the severity of influenza virus induced secondary bacterial pneumonia

Staphylococcus aureus is a major cause of severe pulmonary infections. The evolution of multi-drug resistant strains limits antibiotic treatment options. To date, all candidate vaccines tested have failed, highlighting the need for an increased understanding of the immunological requirements for effective S. aureus immunity. Using an S. aureus strain engineered to express a trackable CD4⁺ T cell epitope and a murine model of S. aureus pneumonia, we show strategies that lodge Th1 polarised bacterium specific CD4⁺ tissue resident memory T cells (Trm) in the lung can significantly attenuate the severity of S. aureus pneumonia. This contrasts natural infection of mice that fails to lodge CD4⁺ Trm cells along the respiratory tract or provide protection against re-infection, despite initially generating Th17 bacterium specific CD4⁺ T cell responses. Interestingly, lack of CD4⁺ Trm formation after natural infection in mice appears to be reflected in humans, where the frequency of S. aureus reactive CD4⁺ Trm cells in lung tissue is also low. Our findings reveal the protective capacity of S. aureus specific respiratory tract CD4⁺ Th1 polarised Trm cells and highlight the potential for targeting these cells in vaccines that aim to prevent the development of S. aureus pneumonia.