Striking Activation of NALT and Nasal Passages Lymphocytes Induced by Intranasal Immunization with Cry1Ac protoxin

Cry1Ac protoxin from Bacillus thuringiensis is a potent mucosal immunogen and adjuvant. When delivered intranasally (i.n.) Cry1Ac elicits significant antibody response and is able to improve vaccination against Naegleria fowleri infection, but the functional effects occurring in nasal lymphocytes when this protein is administered alone have not been determined. Here, we investigated the effects of i.n. immunization with Cry1Ac on antibody production, lymphocyte activation and cytokine production in lymphocytes from nasal‐associated lymphoid tissue (NALT) and nasal passages (NP). Our results show that i.n. immunization with Cry1Ac induced significant specific IgA and IgG cell responses, especially in NP. Besides, it increased the proportion of lymphocytes expressing the activation markers CD25 and CD69 in both nasal tissues, but differently. CD25 was increased in B cells along with CD4 and CD8 T cells from NALT and NP, while CD69 was increased in B cells from both tissues but only in CD4 T cells from NP. Finally, we found that Cry1Ac augmented especially a Th2 profile of cytokines, as the proportion of T cells that spontaneously produced IL‐4, IL‐5 and IL‐10 was increased and this effect was higher in NP than in NALT. These data contribute to explain the potent immunogenicity of Cry1Ac via i.n. route.     

Intranasal bacteria induce Th1 but not Treg or Th2

Commensal microorganisms colonize the nasal mucosa without inducing inflammation. Pathogens perturbing the commensal flora often invade evading immune defenses. The different types of adaptive responses that drive the distinct behaviors of commensals and pathogens, allowing one to persist at mucosal surfaces and the other to survive within tissues, are not yet clear. In the present work we demonstrate that although crossing epithelial barriers, the commensal Lactobacillus murinus stimulates epitope-specific CD4⁺ T cells in nasal-associated lymphoid tissue (NALT) less efficiently than the pathogen Streptococcus pyogenes. In NALT antigen-presenting cells other than CCR6⁺ CD11c⁺ dendritic cells process and present the microbial antigens. Effector/memory CD4⁺ T cells generated after intranasal priming with L. murinus and S. pyogenes surprisingly express similar proinflammatory cytokines and are not CD25+/FoxP3+ T-regulatory cells when recirculating in the spleen. These findings suggest that when a commensal crosses the nasal epithelial barrier it induces a proinflammatory response similar to a pathogen but without causing disease.     

Interleukin 12 and innate molecules for enhanced mucosal immunity

Recent strategies for understanding the mechanisms underlying mucosal immune responses and subsequent development of mucosal vaccines for induction of targeted immunity now include cytokines and molecules of innate immunity. These studies have shown that cytokines influencing the development of T helper (Th) cells differentially affect the outcome of mucosal vs systemic immune responses to mucosal vaccines. Serum antigen-specific antibody (Ab) responses were enhanced when either IL-6 or IL-12 was mucosally administered with a protein antigen, while only IL-12 induced antigen-specific mucosal IgA Ab responses. Mucosal IL-6 and IL-12 also affected the type of Th cell responses induced by CD4⁺ T cells from mice that received IL-12 secreted larger amounts of IFN-γ and IL-6 when compared with mice nasally treated with IL-6. Discrepancies in the ability to enhance mucosal or systemic immune responses were also observed when human neutrophil peptide (HNP) defensins or lymphotactin were nasally coadministered with protein antigens. Only lymphotactin promoted mucosal secretory IgA (S-IgA) Ab responses while both lymphotactin and defensins enhanced systemic immunity to mucosally co-administered protein antigens. Mixed antigen-specific Th1-and Th2-type CD4⁺ T cell responses were induced in the systemic compartment by both lymphotactin and the mixture of HNP-1, HNP-2, and HNP-3 defensins. However, HNPs failed to significantly enhance cytokine secretion by mucosally derived, antigen-specific CD4⁺ T cells relative to those isolated from the systemic compartment. In summary, these studies clearly show that IL-12 and lymphotactin are able to trigger S-IgA Ab responses and provide new avenues for the design of safe and targeted mucosal vaccines.     

Phenotypic and functional differences between lymphocytes from NALT and nasal passages of mice

Nasal-associated lymphoid tissue (NALT) and nasal passages (NP) are considered as inductive and effector sites, respectively. The differences among lymphocyte populations of these nasal compartments have not been clearly established. The aim of this work was to contribute to the characterization of NALT and NP lymphocytes in mice. We isolated lymphocytes from both compartments, determined the frequencies of B220(+) cells as well as CD8(+), CD4(+) T cells; and analysed the expression of CD69 and CD25. Besides we analysed the proportion of T cells producing IL-2, IL-4, IL-5, IL-10, IFN-gamma and TNF-alpha. We found differences between NALT and NP. Two populations of B cells, B220+(hi) and B220+(low) were clearly distinguished only in NP, but not in NALT. Both (hi) and (low) B220(+) cells expressed CD19, but only a fraction of the B220+(low) population, expressed the plasma cell marker CD138(+). More B than T lymphocytes, as well as higher frequencies of CD4(+) than CD8(+) T cells were found in both compartments. A small fraction of NK cells (CD3(-)DX5(+)) along with a significant proportion of double negative CD4(-)CD8(-)CD3(+)DX5(-) T cells was detected in both nasal tissues. Furthermore, as expected for a mucosal effector site, NP contained major proportions of B220(+), T CD4(+) and T CD8(+) cells expressing CD25 and CD69 in comparison to NALT. Likewise, the proportion of T cells spontaneously producing IL-2, IFN-gamma, and IL-4, was higher in NP than in NALT. These data provide further evidence indicating that distinctive phenotypic and functional features exist in the lymphocyte populations residing at NALT and NP.     

Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus-infected macaques

Loss of CD4⁺ T cells in the gut is necessary but not sufficient to cause AIDS in animal models, raising the possibility that a differential loss of CD4⁺ T-cell subtypes may be important. We found that CD4⁺ T cells that produce interleukin (IL)-17, a recently identified lineage of effector CD4⁺ T-helper cells, are infected by SIV_mac251in vitro and in vivo, and are found at lower frequency at mucosal and systemic sites within a few weeks from infection. In highly viremic animals, Th1 cells predominates over Th17 T cells and the frequency of Th17 cells at mucosal sites is negatively correlated with plasma virus level. Because Th17 cells play a central role in innate and adaptive immune response to extracellular bacteria, our finding may explain the chronic enteropathy in human immunodeficiency virus (HIV) infection. Thus, therapeutic approaches that reconstitute an adequate balance between Th1 and Th17 may be beneficial in the treatment of HIV infection.     

T lymphocytes from the normal human peritoneum contain high frequencies of Th2-type CD8+ T cells

The majority of peritoneal T lymphocytes have been shown to be CD8⁺ and to co-express CDw60. Expression of CDw60 characterizes CD8 T cells capable of secreting interleukin (IL)-4 and supporting IgG production by B cells. We analyzed at the clonal level the functional cytokine profile of CD8⁺ T lymphocytes from the normal human peritoneum. While the majority of the clones produced interferon (IFN)-γ and exhibited high alloantigen-specific cytolytic activity, some clones secreted IL-4 and IL-5 but no detectable IFN-γ. These Th2-type CD8⁺ T cell clones provided substantial B cell help for IgG and IgA synthesis and exhibited reduced cytolytic activity. Our results suggest that distinct subsets of CD8⁺ T cell may occur in different immune compartments.     

Down-modulation of CD4+ T helper type 2 and type 0 cells by T helper type 1 cells via Fas/Fasligand interaction

Fas was recently demonstrated to be the major target molecule engaged by CD4⁺ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4⁺ or CD8⁺ CTL. A reciprocal relationship in Fas and Fas-ligand expression was observed in CD4⁺ T helper (Th)1- and Th2-type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas-ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas-ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4-mediated lysis. A functional consequence of the inverse Fas-Fas-ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4⁺ CTL and a CD8⁺ CTL clone in a Fas-dependent manner. These results suggest that cytolytic CD4⁺ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas-mediated lysis.     

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.     

Early IL-4 Production Driving Th2 Differentiation in a Humanin VivoAllergic Model Is Mast Cell Derived

IL-4 is central to the formation of IgE and the development of Th2 effector cells, both key features of an allergic response. We have examined IL-4 production early in the formation of an allergic response by using a previously established humanin vivomodel of allergic rhinitis where allergic subjects are challenged internasally with allergen and the particulate pollutant diesel exhaust particles (DEP). This model is characterized by enhanced IgE production and deviation to a Th2-type cytokine profile in nasal lavage fluid from these subjects. In this model, IL-4 protein and IL-4-positive cells could first be detected 4 h after challenge and maximal production was observed after 18 h. Two-color flow cytometric analysis for the detection of intracellular IL-4 and surface markers was performed on nasal cells recovered 4 h after challenge. At this time, CD117⁺(c-kit⁺) cells constituted between 65 and 100% of the IL-4⁺cells, while 0–12% of the IL-4⁺cells were CD3 positive. No IL-4⁺CD19/CD20⁺or IL-4⁺CD56⁺cells were detected at 4 h. As the allergic response progressed the primary source of IL-4 changed. At the peak of IL-4 production, 18 h after challenge, CD3⁺comprised the majority of cells staining for intracellular IL-4 (73 to 100%). Thus we show an initial role for cells of the mast cell/basophil lineage residing in the nasal mucosa in the initial production of IL-4, which frames the subsequent immune response by expanding the repertoire of TH2 cytokine-producing cells in the local microvicinity.     

The Th1/Th2 balance in autoimmunity

The study of autoimmune disease in the context of T-helper type 1 (Th1) and T-helper type 2 (Th2) CD4⁺ T-cell responses demonstrates that the relative contribution of either T-cell type to the development of a particular autoimmune response can influence whether or not this response leads to clinical disease. Moreover, this influence can be quite different depending on whether the particular disease process is cell mediated or antibody mediated. Recent studies have demonstrated that the development of Th1 and Th2 responses may be significantly influenced by the costimulatory molecules recognized by responding CD4 T cells, and by other undefined factors in the genetic background. It has also been demonstrated that autoreactive Th2 CD4⁺ cells can regulate the activity of disease-causing Th1 CD4⁺ T cells in vivo. Control of autoimmune disease may thus be achieved by procedures that regulate the relative contribution of Th1/Th2 CD4 T cells to an autoimmune response.     

Thermal-sensitive hydrogel as adjuvant-free vaccine delivery system for H5N1 intranasal immunization

For H5N1 influenza immunization, we developed a thermal-sensitive hydrogel as intranasal vaccine delivery system, which was formulated with N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) and α, β-glycerophosphate (α, β-GP). The flowing solution of HTCC/GP under room temperature could gelate rapidly at body temperature, which significantly prolonged the H5N1 split antigen residence time in nasal cavity. This system also enhanced the transepithelial transport via the paracellular routes due to the disorganization of ZO-1 protein in nasal epithelial tissue. In comparison to naked H5N1 split antigen and MF59 adjuvanted antigen, as designed hydrogel/H5N1 vaccine induced greater antigen-specific systemic immune responses and mucosal IgA immunity without adjuvants. Furthermore, a boosted cellular and humoral response was also obtained by examination of IFN-γ and IL-4 cytokines, respectively. In addition, hydrogel based formulation promoted the antigen-specific CD8⁺ T cell immune memory as determined by the proportion of central and effector memory CD8⁺ T cells in nasal associated lymphoid tissue (NALT). These results demonstrate that the HTCC hydrogel has potential as an adjuvant-free platform for H5N1 split antigen intranasal vaccination.     

The imbalance of Th17/Th1/Tregs in patients with type 2 diabetes: relationship with metabolic factors and complications

Immune disorders are linked to the development of type 2 diabetes (T2D) and its complications. The relationship of CD4⁺CD25^hi T regulatory cells (Treg) and pro-inflammatory Th17 and Th1 subsets in T2D patients with metabolic disorders and complications need to be determined. The ratios of CD4⁺CD25^hi Treg/Th17 cells and CD4⁺CD25^hi Treg/Th1 cells, but not Th17/Th1 cells, were significantly decreased in T2D patients. The thymic output CD4⁺Foxp3⁺Helios⁺ Tregs were normal but peripheral induced CD4⁺Foxp3⁺Helios⁻ Tregs were decreased in T2D patients. The Bcl-2/Bax ratio decreased in CD4⁺CD25^hi Tregs in T2D patients, supporting the increased sensitivity to cell death of these cells in T2D. CD4⁺CD25^hiCD127⁻ Tregs in T2D patients with microvascular complications were significantly less than T2D patients with macrovascular complications. Importantly, CD4⁺CD25^hiCD127⁻ Tregs were positively correlated with plasma IL-6, whereas IL-17⁺CD4⁺cells were negatively related to high-density lipoprotein (HDL). Our data offered evidence for the skewed balance of anti- and pro-inflammatory T cell subsets in T2D patients and identified that HDL closely modulate T cell polarization. These results opened an alternative explanation for the substantial activation of immune cells as well as the development of T2D and complications, which may have significant impacts on the prevention and treatment of T2D patients.     

Fetal BM‐derived mesenchymal stem cells promote the expansion of human Th17 cells,but inhibit the production of Th1 cells

Th type 17 (Th17) cells have been identified as a proinflammatory T‐cell subset. Here, we investigated the regulation of human Th17 cells by fetal BM‐derived mesenchymal stem cells (FBM‐MSC). We cocultured FBM‐MSC with human PBMC or CD4⁺ T cells from healthy donors. FBM‐MSC significantly suppressed the proliferation of CD4⁺ T cells stimulated by PHA and recombinant IL‐2. Significantly higher levels of IL‐17 were observed in FBM‐MSC cocultured with either PBMC or CD4⁺ T cells than that in PBMC cultured alone or CD4⁺ T cells cultured alone. Flow cytometry analysis showed that the percentage of Th17 cells in coculture of FBM‐MSC and CD4⁺ T cells was significantly higher than that in CD4⁺ T‐cell cultured alone. FBM‐MSC did not express IL‐17 protein. Consistent with the augmentation of Th17 cells, significantly higher levels of IL‐6 and IL‐1 were observed in coculture of FBM‐MSC and CD4⁺ T cells than that in CD4⁺ T‐cell culture, while the levels of IL‐23 were similar between FBM‐MSC + PBMC coculture and PBMC alone, or FBM‐MSC + CD4⁺ T‐cell and CD4⁺ T‐cell alone. The presence of FBM‐MSC decreased the percentage of Th1 cells, but minimally affected the expansion of CD4⁺CD25⁺ T cells. In conclusion, our data demonstrate for the first time that FBM‐MSC promote the expansion of Th17 cells and decrease IFN‐γ‐producing Th1 cells. These data suggest that IL‐6 and IL‐1, instead of IL‐23, may be partly involved in the expansion of Th17 cells.     

Distinctive features of classic and nonclassic (Th17 derived) human Th1 cells

T helper17 (Th17) lymphocytes represent a third arm of the CD4⁺ T‐cell effector responses, in addition to Th1 and Th2 cells. Th17 cells have been found to exhibit high plasticity because they rapidly shift into the Th1 phenotype in inflammatory sites. In humans, Th1 cells derived from Th17 cells express CD161, whereas classic Th1 cells do not; these Th17‐derived Th1 cells have been termed nonclassic Th1 cells. In this study, we examined similarities and differences between classic and nonclassic human Th1 cells by assessing a panel of T‐cell clones, as well as CD161⁺ or CD161^? CD4⁺ T cells derived ex vivo from the circulation of healthy subjects or the synovial fluid of patients with juvenile idiopathic arthritis. The results show that nonclassic Th1 cells can be identified based on CD161 expression, as well as the consistent expression of retinoic acid orphan receptor C, IL‐17 receptor E, CCR6, and IL‐4‐induced gene 1, which are all virtually absent in classic Th1 cells. The possibility to distinguish these two‐cell subsets by using such a panel of markers may allow the opportunity to better establish the respective pathogenic roles of classic and nonclassic (Th17 derived) Th1 cells in different chronic inflammatory disorders.     

T regulatory cells and Th1/Th2 cytokines in peripheral blood from tuberculosis patients

About 10% of people infected with Mycobacterium tuberculosis develop active tuberculosis (TB), and Th1 effector cells and Th1 cytokines play key roles in controlling M. tuberculosis infection. Here, we hypothesise that this susceptibility to M. tuberculosis infection is linked to increased T regulatory (Treg) cells and Th2 cytokines in TB patients. To test this, we recruited 101 participants (71 TB patients, 12 non-TB pulmonary diseases and 18 healthy subjects) and investigated Treg cells and Th1/Th2 cytokines in peripheral blood. CD4⁺CD25⁺ T cells and CD4⁺CD25⁺FoxP3⁺ T cells significantly increased and IL-5 dramatically decreased in TB patients relative to healthy subjects. CD8⁺CD28⁻ T cells, IFN-γ, TNF-α, IL-10 and IL-4 significantly increased in patients with culture and sputum smear-positive pulmonary TB (PTB(+)) compared with healthy subjects. CD4⁺CD25⁺FoxP3⁺ and CD8⁺CD28⁻ T cells significantly decreased in PTB(+) after one month of chemotherapy. CD4⁺, CD4⁺CD25⁺ and CD8⁺CD28⁺ T cells significantly increased in extra-pulmonary TB patients after one month of chemotherapy. These findings suggest that M. tuberculosis infection induces circulating CD4⁺CD25⁺FoxP3⁺ and CD8⁺CD28⁻ T cell expansion, which may be related to the progression of M. tuberculosis infection, and that the balance between effector immune responses and suppression immune responses is essential to control M. tuberculosis infection.     

Increased Circulating Th22 and Th17 Cells are Associated with Tumor Progression and Patient Survival in Human Gastric Cancer

Although Th22 and Th17 cells have been reported to play critical roles during autoimmunity and inflammation, information on their role in cancer-immunity is limited. In this study, we investigated clinical relevance of circulating Th22 and Th17 cells in patients with gastric cancer (GC). Using multi-color flow cytometry and PMA stimulation, we determined the levels of Th22, Th17 and Th1 cells in the peripheral blood of 32 GC patients and 19 healthy donors, and evaluated their correlations with tumor stage and overall survival. Compared with healthy donors, the frequencies of circulating CD4⁺IL-22⁺ T cells, CD4⁺IL-17⁺ T cells, Th22 (CD4⁺IL-22⁺IL-17^-INF-γ^?) cells, Th17 (CD4⁺IL-17⁺INF-γ^?) cells were increased in patients with GC, but there was no significant differences in the frequencies of CD4⁺IFN-γ⁺ T cells and Th1 (CD4⁺IL-17^?INF-γ⁺) cells. Th22 cells showed positive correlation with Th17 cells and CD4⁺IL-17⁺ T cells in patients with GC. Furthermore, the frequencies of Th22 and Th17 cells were significantly higher in stage III–IV GC patients versus stage I–II and correlated with patients’ overall survival. These data suggest that circulating Th22 cells as well as Th17 cells are increased in the peripheral blood of GC patients with tumor progression, and that these cells may be promising novel clinical markers for GC.