The Cell Wall and Membrane of Cryptococcus neoformans Possess a Mitogen for Human T Lymphocytes

The mechanism of human T-lymphocyte activation by the pathogenic yeast Cryptococcus neoformans has not been established. Previous investigations have suggested that C. neoformans contains a mitogen for T lymphocytes, while other investigators have attributed lymphocyte proliferation in vitro to a recall antigen. Because of the potential importance of the mechanism of T-cell activation for our understanding of the immune response to C. neoformans, the present studies were performed to determine whether C. neoformans contains a mitogen for T lymphocytes. C. neoformans stimulates fetal blood lymphocytes to proliferate and stimulates proliferation of CD45RA⁺ cells from adults, indicating that it stimulates naive T cells. The T-cell response to C. neoformans was dependent upon the presence of accessory cells. However, allogeneic cells were sufficient for accessory cell function, indicating that the response was not major histocompatibility complex restricted. The percentage of T cells in the cell cycle was higher than that with the recall antigen tetanus toxoid but lower than that with the mitogenic lectin phytohemagglutinin A or the superantigen Staphylococcus enterotoxin B. Precursor frequency analysis established that 1 in 7,750 ± 2,270 T cells proliferated in response to the cryptococcal cell wall and membrane. Compared to the case for most mitogens or superantigens, the proliferative response is late and the number of T cells that enter the cell cycle and the precursor frequency are low, indicating that the mitogenic effect is modest. However, the mitogenic effect of C. neoformans should be considered when interpreting the immune response to C. neoformans, since even weak mitogens can have profound effects on host defense.     

Impact of Surfactant Protein D,Interleukin-5, and Eosinophilia on Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen that initiates infection following inhalation. As a result, the pulmonary immune response provides a first line of defense against C. neoformans. Surfactant protein D (SP-D) is an important regulator of pulmonary immune responses and is typically host protective against bacterial and viral respiratory infections. However, SP-D is not protective against C. neoformans. This is evidenced by previous work from our laboratory demonstrating that SP-D-deficient mice infected with C. neoformans have a lower fungal burden and live longer than wild-type (WT) control animals. We hypothesized that SP-D alters susceptibility to C. neoformans by dysregulating the innate pulmonary immune response following infection. Thus, inflammatory cells and cytokines were compared in the bronchoalveolar lavage fluid from WT and SP-D^−/− mice after C. neoformans infection. Postinfection, mice lacking SP-D have reduced eosinophil infiltration and interleukin-5 (IL-5) in lung lavage fluid. To further explore the interplay of SP-D, eosinophils, and IL-5, mice expressing altered levels of eosinophils and/or IL-5 were infected with C. neoformans to assess the role of these innate immune mediators. IL-5-overexpressing mice have increased pulmonary eosinophilia and are more susceptible to C. neoformans infection than WT mice. Furthermore, susceptibility of SP-D^−/− mice to C. neoformans infection could be restored to the level of WT mice by increasing IL-5 and eosinophils by crossing the IL-5-overexpressing mice with SP-D^−/− mice. Together, these studies support the conclusion that SP-D increases susceptibility to C. neoformans infection by promoting C. neoformans-driven pulmonary IL-5 and eosinophil infiltration.     

Encapsulation of Cryptococcus neoformans with Glucuronoxylomannan Inhibits the Antigen-Presenting Capacity of Monocytes

This report examines the effect of the major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), on the antigen-presenting capability of human monocytes treated with acapsular cells of C. neoformans. We found that pretreatment of acapsular cryptococci with GXM downregulates, in a dose-dependent manner, the antigen-presenting capacity of monocytes, leading to reduced proliferative T-lymphocyte responses. Similar levels of suppression occurred when monocytes were exposed to encapsulated cryptococci or acapsular cryptococci that were pretreated with GXM. The magnitude of the T-cell response correlated with the ability of monocytes to ingest the yeast. Supernatant fluids from cocultures of monocytes and T cells cultured with encapsulated cryptococci contained higher levels of interleukin-10 (IL-10) than supernatant fluids of cells with acapsular cryptococci. Addition of anti-IL-10 monoclonal antibodies to the incubation medium of monocytes and T cells cultured with encapsulated cryptococci restored proliferative T-cell responses to levels observed during culture with acapsular cryptococci. Finally, treatment of monocytes with encapsulated cryptococci or GXM-treated acapsular cryptococci suppressed expression of class II major histocompatibility complex (MHC) molecules in a manner consistent with previous reports of IL-10-mediated suppression of class II MHC molecules and suppression of proliferative T-cell responses. These results suggest a link between GXM encapsulation, increased IL-10 synthesis by monocytes, decreased expression of class II MHC molecules on monocytes, and reduced proliferative T-cell responses.Cryptococcus neoformans is an opportunistic fungus that produces a life-threatening meningoencephalitis in AIDS patients (10, 11, 25, 38). This fungus is believed to enter the body via inhalation of airborne yeast cells or conidia from the environment. During initial exposure to the microorganism, the capsule may be absent or small, but it is rapidly synthesized in vivo. The capsule represents a major virulence factor endowed with antiphagocytic and toleragenic properties (16, 17, 23, 31). Glucuronoxylomannan (GXM) is the major component of the capsule of C. neoformans, and studies focusing on capsular material have identified many regulatory effects on phagocytic cells (16, 19, 21, 24, 28, 39, 45). The large amount of this inhibitory substance found in the body fluids of patients with cryptococcosis (8, 36) raises the possibility that GXM exerts regulatory effects during a cryptococcal infection. Several reports indicate that GXM or products of C. neoformans that are rich in GXM induce or regulate the cell-mediated response to C. neoformans. Murphy and Cox found that intravenous injection of sera with high GXM titers from C. neoformans-infected mice into recipient mice suppresses the ability of the recipient mice to produce a normal delayed hypersensitivity response to cryptococcal antigens (30). Several studies from the Murphy laboratory found that intravenous injection of cryptococcal polysaccharides induces a cascade of suppressor cells and soluble factors that downregulate the anticryptococcal delayed hypersensitivity response (13, 29, 32–34). More recently, Blackstock reported that GXM stimulates an antigen-presenting cell (APC) to induce secretion by T cells of a T-suppressor factor that is specific for GXM (1).Collins and Bancroft were the first to report that encapsulation of C. neoformans greatly reduces the ability of C. neoformans-treated monocytes/macrophages to induce a T lymphoproliferative response (5). This report was followed by one presenting similar observations by Mody and Syme (26), as well as ours (43). Recently we found that GXM is a potent downregulator of proinflammatory cytokine release by human monocytes (45). This inhibitory effect is due, in part, to an increased secretion of interleukin-10 (IL-10) that appears, in turn, to be responsible for reduced secretion of tumor necrosis factor alpha (TNF-α), and/or IL-1β (6, 22, 46). A role for GXM encapsulation in suppression of the T lymphoproliferative response is supported by our observation that opsonization of encapsulated cryptococci with anti-GXM monoclonal antibodies (MAbs) enhances the ability of monocytes to process C. neoformans yeast cells, leading to an enhanced T proliferative response (44).The objectives of our study were (i) to provide further evidence for the central role that encapsulation with GXM plays in reducing the ability of monocytes to process C. neoformans yeast cells, leading to a proliferative response by T lymphocytes, (ii) to determine the effect of GXM encapsulation on expression of major histocompatibility complex (MHC) class II molecules by monocytes, and (iii) to assess the importance of autologous IL-10 in altered expression of MHC class II molecules by monocytes and suppressed T lymphoproliferation. Our results confirmed the role of GXM in suppression of the T lymphoproliferative response and provide a link between the capability of GXM encapsulation to perturb processing by APC and suppression of the cell-mediated response to acapsular cryptococci.     

Surfactant protein D facilitates Cryptococcus neoformans infection

Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Furthermore, Cryptococcus neoformans has become a primary human pathogen, causing infection in seemingly healthy individuals. Although numerous studies have elucidated the virulence properties of C. neoformans, less is understood regarding lung host immune factors during early stages of fungal infection. Based on our previous studies documenting that pulmonary surfactant protein D (SP-D) protects C. neoformans cells against macrophage-mediated defense mechanisms in vitro (S. Geunes-Boyer et al., Infect. Immun. 77:2783–2794, 2009), we postulated that SP-D would facilitate fungal infection in vivo. To test this hypothesis, we examined the role of SP-D in response to C. neoformans using SP-D^−/− mice. Here, we demonstrate that mice lacking SP-D were partially protected during C. neoformans infection; they displayed a longer mean time to death and decreased fungal burden at several time points postinfection than wild-type mice. This effect was reversed by the administration of exogenous SP-D. Furthermore, we show that SP-D bound to the surface of the yeast cells and protected the pathogenic microbes against macrophage-mediated defense mechanisms and hydrogen peroxide (H₂O₂)-induced oxidative stress in vitro and in vivo. These findings indicate that C. neoformans is capable of coopting host SP-D to increase host susceptibility to the yeast. This study establishes a new paradigm for the role played by SP-D during host responses to C. neoformans and consequently imparts insight into potential future preventive and/or treatment strategies for cryptococcosis.     

Cryptococcus neoformans infection in mice previously infected with LP-BM5 MuLV, the agent of murine AIDS (MAIDS)

We studied susceptibility to experimental systemic cryptococcosis in mice previously infected with the retroviral complex LP-BM5 (responsible for murine AIDS). LP-BM5 was inoculated to C57B1/6 mice by intravenous (i.v.) injection 8 weeks before an i.v. challenge with 4×l0³ CFU of Cryptococcus neoformans. Uninfected and singly infected mice were used as controls. LP-BM5 infection did not result in a significant increase in fungal burdens in the lungs or brains of co-infected animals compared to mice infected with C. neoformans alone. However, mortality was enhanced in the co-infected animals. The kinetics of splenocyte subsets differed in co-infected mice and LP-BM5-infected mice; the increase in CD4⁺, CD8⁺ and Ly5⁺ cells was only moderate in the former. Cytokine production by concanavalin A (Con A)-stimulated splenocytes from co-infected mice showed a marked decrease in the Thl response (IFN-γ, IL-2) and an increase in the Th2 response (IL-4, IL-10). Furthermore, cryptococcosis altered the course of MAIDS, inhibiting splenomegaly. This effect was not related to a decrease in ecotropic virus titres in the spleen or to improved in vitro responsiveness of spleen cells to Con A. The marked decrease in IFN-γ production in co-infected animals could partly explain the inhibition of LP-BM5-induced splenomegaly. This model of murine retroviral infection does not seem to be suitable for studying cryptococcosis in immunosuppressed animals, but remains valuable for investigating in vivo interactions between two pathogens.     

Specific Antibody to Cryptococcus neoformans Alters Human Leukocyte Cytokine Synthesis and Promotes T-Cell Proliferation

Addition of a monoclonal antibody which binds the Cryptococcus neoformans capsule to suspensions of human monocytes, T lymphocytes, and cryptococcal cells (i) enhances interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-2 production; (ii) reduces IL-10 secretion; and (iii) promotes T-cell proliferation. The ability of specific antibody to influence cytokine production and lymphoproliferation suggests a mechanism by which humoral immunity can influence cell-mediated immunity.     

Eosinophils elicit proliferation of naive and fungal-specific cells in vivo so enhancing a T helper type 1 cytokine profile in favour of a protective immune response against Cryptococcus neoformans infection

Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, because as in healthy humans, rats can effectively contain cryptococcal infection. Moreover, it has been shown that eosinophils are components of the immune response to C. neoformans infections. In a previous in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C. neoformans, thereby triggering their activation, as indicated by the up‐regulation of MHC and co‐stimulatory molecules and the increase in interleukin‐12, tumour necrosis factor‐α and interferon‐γ production. Furthermore, this work demonstrated that C. neoformans‐specific CD4⁺ and CD8⁺ T lymphocytes cultured with these activated C. neoformans‐pulsed eosinophils proliferated, and produced important amounts of T helper type 1 (Th1) cytokines in the absence of Th2 cytokine synthesis. In the present in vivo study, we have shown that C. neoformans‐pulsed eosinophils are also able to migrate into lymphoid organs to present C. neoformans antigens, thereby priming naive and re‐stimulating infected rats to induce T‐cell and B‐cell responses against infection with the fungus. Furthermore, the antigen‐specific immune response induced by C. neoformans‐pulsed eosinophils, which is characterized by the development of a Th1 microenvironment with increased levels of NO synthesis and C. neoformans‐specific immunoglobulin production, was demonstrated to be able to protect rats against subsequent infection with fungus. In summary, the present work demonstrates that eosinophils act as antigen‐presenting cells for the fungal antigen, hence initiating and modulating a C. neoformans‐specific immune response. Finally, we suggest that C. neoformans‐loaded eosinophils might participate in the protective immune response against these fungi.     

Interleukin-2 Induced Mitogenesis of Human Peripheral Blood T-Lymphocytes: Role of Accessory Cells

We and others have shown that Interleukin-2 (IL-2) is mitogenic to a subset of unstimulated T lymphocytes in human peripheral blood (1–7). We extend our work here in showing that prolonged continuous exposure of human peripheral blood lymphocytes to exogenous IL-2 throughout the 7–8 day culture is not necessary since mitogenesis occurs reproducibly after short term (2–3 hr) pulse exposure. The mitogenic effect of pulse exposure to IL-2 is not significantly reduced by inclusion of anti-Tac monoclonal antibody in the pulsing medium. However, anti-Tac monoclonal antibody markedly inhibits the response if present continuously throughout the 7 day culture.

The mitogenic effect of IL-2 is dependent on the presence of accessory cells (monocytes) but the accessory cell requirement can be replaced by the phorbol ester TPA (10^?8 to 10^?11 M). Purified monocytes subjected to short term pulse exposure to IL-2 can cause proliferative response in unprimed autologous lymphocytes in cocultures. The mitogenic effect of IL-2 pulsed monocytes can not be suppressed by inclusion of anti-Tac antibody in the pulsing medium although the same concentration of the antibody suppresses the effect if present throughout culture. The response of lymphocytes to IL-2 pulsed monocytes is not inhabitable by the continuous presence of a monoclonal antibody to human HLA-DR antigens (OK-Ial) in culture.     

Double positive CD4+CD8+ T cells are part of the adaptive immune response against Candida albicans

Although multiple immune cells participate in the innate and adaptive immune response against Candida albicans, the elucidation of cellular and inflammation kinetics may be a promising strategy to decipher events propitious to infection eradication. We used an in vitro Candida-human leucocyte coculture approach to study the dynamics of rare CD4+CD8+ double positive T lymphocytes (DP T) produced in response to this fungus. Our results highlight the presence of two phenotypically distinct subsets of DP T cells: CD4hiCD8lo and CD4loCD8hi, and that the different ratio of these cells correlates with infection outcome. The ratio of CD4hiCD8lo over CD4loCD8hi by day 6 was significantly higher in controlled infections and decreased when infection persisted due to a significant increase in the proportion of CD4loCD8hi. When infection was controlled, CD4hiCD8lo T cells secreted IFNγ, TNFα, IL-4 and IL-10 cytokines two days after challenge. By day 2, under conditions of persistent infection, CD4hiCD8lo and CD4loCD8hi T cells secreted significant levels of IL-4 and IL-10, respectively, compared to uninfected cultures. Frequency kinetics and original cytokine profiles detailed in this work indicate that DP T cells could participate in the adaptive immune response to C. albicans.     

Antigen-Induced Protective and Nonprotective Cell-Mediated Immune Components against Cryptococcus neoformans

Mice immunized with two different cryptococcal antigen preparations, one a soluble culture filtrate antigen (CneF) in complete Freund’s adjuvant (CFA) and the other heat-killed Cryptococcus neoformans cells (HKC), develop two different profiles of activated T cells. CneF-CFA induces CD4⁺ T cells responsible for delayed-type hypersensitivity (DTH) reactivity and for amplification of the anticryptococcal DTH response, whereas HKC induce CD4⁺ and CD8⁺ T cells involved in anticryptococcal DTH reactivity and activated T cells which directly kill C. neoformans cells. The main purpose of this study was to assess the level of protection afforded by each of the two different T-cell profiles against challenge with viable C. neoformans cells, thereby identifying which activated T-cell profile provides better protection. CBA/J mice immunized with CneF-CFA had significantly better protective responses, based on better clearance of C. neoformans from tissues, on longer survival times, and on fewer and smaller lesions in the brain, than HKC-immunized mice or control mice similarly infected with C. neoformans. Both immunization protocols induced an anticryptococcal DTH response, but neither induced serum antibodies to glucuronoxylmannan, so the protection observed in the CneF-CFA immunized mice was due to the activated T-cell profile induced by that protocol. HKC-immunized mice, which displayed no greater protection than controls, did not have the amplifier cells. Based on our findings, we propose that the protective anticryptococcal T cells are the CD4⁺ T cells which have been shown to be responsible for DTH reactivity and/or the CD4⁺ T cells which amplify the DTH response and which have been previously shown to produce high levels of gamma interferon and interleukin 2. Our results imply that there are protective and nonprotective cell-mediated immune responses and highlight the complexity of the immune response to C. neoformans antigens.     

Enhanced innate immune responsiveness to pulmonary Cryptococcus neoformans infection is associated with resistance to progressive infection

Genetically regulated mechanisms of host defense against Cryptococcus neoformans infection are not well understood. In this study, pulmonary infection with the moderately virulent C. neoformans strain 24067 was used to compare the host resistance phenotype of C57BL/6J with that of inbred mouse strain SJL/J. At 7 days or later after infection, C57BL/6J mice exhibited a significantly greater fungal burden in the lungs than SJL/J mice. Characterization of the pulmonary innate immune response at 3 h after cryptococcal infection revealed that resistant SJL/J mice exhibited significantly higher neutrophilia, with elevated levels of inflammatory cytokine tumor necrosis factor alpha (TNF-α) and keratinocyte-derived chemokine (KC)/CXCL1 in the airways, as well as increased whole-lung mRNA expression of chemokines KC/CXCL1, MIP-1α/CCL3, MIP-1β/CCL4, MIP-2/CXCL2, and MCP-1/CCL2 and cytokines interleukin 1β (IL-1β) and IL-1Ra. At 7 and 14 days after infection, SJL/J mice maintained significantly higher levels of TNF-α and KC/CXCL1 in the airways and exhibited a Th1 response characterized by elevated levels of lung gamma interferon (IFN-γ) and IL-12/IL-23p40, while C57BL/6J mice exhibited Th2 immunity as defined by eosinophilia and IL-4 production. Alveolar and resident peritoneal macrophages from SJL/J mice also secreted significantly greater amounts of TNF-α and KC/CXCL1 following in vitro stimulation with C. neoformans. Intracellular signaling analysis demonstrated that TNF-α and KC/CXCL1 production was regulated by NF-κB and phosphatidylinositol 3 kinase in both strains; however, SJL/J macrophages exhibited heightened and prolonged activation in response to C. neoformans infection compared to that of C57BL/6J. Taken together, these data demonstrate that an enhanced innate immune response against pulmonary C. neoformans infection in SJL/J mice is associated with natural resistance to progressive infection.     

A non-protective T helper 1 response against the intra-macrophage protozoan Theileria annulata

Theileria annulata is a protozoan parasite which infects and transforms bovine macrophages. Infected macrophages possess augmented antigen presentation capabilities, as they are able to activate the majority of T cells from unexposed animals. In vivo, T cells in the draining lymph node (principal site of parasite development) are activated ‘non-specifically’ by the parasite. This event is followed by failure of the immune response to control the infection. Protective immune responses against intra-macrophage protozoa are usually mediated by T helper 1 (Th1) T cell responses. Here we examine the cytokine responses made by T. annulata-activated T cells. We show that the outcome of in vitro activation of T cells by parasitized macrophages is a skewing of their cytokine responses towards preferential expression of interferon-gamma (IFN-γ) mRNA. The in vitro response is mirrored during in vivo infection, as greatly elevated amounts of IFN-γ protein are found in lymph efferent from infected lymph nodes, while expression of IL-4 mRNA within the node stops. IFN-γ production does not correlate with protection against the parasite, as infected cells flourish during peak IFN-γ production, and only very small amounts of IFN-γ are produced during the effective immune response of an immunized animal. Overproduction of IFN-γ and loss of IL-4 expression are also likely to account for the failure of B cells to reach the light zone of germinal centres, a developmental step which is tightly regulated by cytokines.     

B lymphocytes regulate airway granulocytic inflammation and cytokine production in a murine model of fungal allergic asthma

Sensitization to fungi often leads to a severe form of asthma that is particularly difficult to manage clinically, resulting in increased morbidity and hospitalizations in these patients. Although B lymphocytes might exacerbate asthma symptoms through the production of IgE, these cells might also be important in the protective response against inhaled fungi. Through cytokine release and T-cell interactions, these lymphocytes might also influence the development and maintenance of airway wall fibrosis. J_H^−/− mice lack the JH gene for the heavy chain component of antibodies, which is critical for B-cell function and survival. These animals have facilitated the elucidation of the role of B lymphocytes in a number of immune responses; however, J_H^−/− mice have not been used to study fungal allergy. In this study, we examined the role of B lymphocytes using an Aspergillus fumigatus murine fungal aeroallergen model that mimics human airway disease that is triggered by environmental fungal exposure. We compared disease progression in sensitized wild-type BALB/c and J_H^−/− mice that were exposed to repeated fungal exposure and found no differences in airway hyperresponsiveness, overall pulmonary inflammation or collagen deposition around the large airways. However, the levels of the Th2-type cytokines IL-4 and IL-13 were significantly attenuated in the airways of J_H^−/− mice relative to the BALB/c controls. By contrast, levels of the inflammatory cytokines IL-17A and IL-6 were significantly elevated in the J_H^−/− animals, and there was significantly more robust airway eosinophilia and neutrophilia than in control animals. Taken together, these findings demonstrate that B lymphocytes help to regulate granulocytic responses to fungal exposure in the pulmonary compartment.     

Lung–infiltrating T helper 17 cells as the major source of interleukin-17A production during pulmonary <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> infection

Background

IL-17A has emerged as a key player in the pathologies of inflammation, autoimmune disease, and immunity to microbes since its discovery two decades ago. In this study, we aim to elucidate the activity of IL-17A in the protection against Cryptococcus neoformans, an opportunistic fungus that causes fatal meningoencephalitis among AIDS patients. For this purpose, we examined if C. neoformans infection triggers IL-17A secretion in vivo using wildtype C57BL/6 mice. In addition, an enhanced green fluorescence protein (EGFP) reporter and a knockout (KO) mouse models were used to track the source of IL-17A secretion and explore the protective function of IL-17A, respectively.

Results

Our findings showed that in vivo model of C. neoformans infection demonstrated induction of abundant IL-17A secretion. By examining the lung bronchoalveolar lavage fluid (BALF), mediastinal lymph node (mLN) and spleen of the IL-17A–EGFP reporter mice, we showed that intranasal inoculation with C. neoformans promoted leukocytes lung infiltration. A large proportion (~?50%) of the infiltrated CD4⁺ helper T cell population secreted EGFP, indicating vigorous T_H17 activity in the C. neoformans–infected lung. The infection study in IL-17A–KO mice, on the other hand, revealed that absence of IL-17A marginally boosted fungal burden in the lung and accelerated the mouse death.

Conclusion

Therefore, our data suggest that IL-17A is released predominantly from T_H17 cells in vivo, which plays a supporting role in the protective immunity against C. neoformans infection.

     

Interleukin-4 suppresses the cytotoxic potential of in vitro generated,adaptive regulatory CD4+ T cells by down-regulation of granzyme B

Regulatory CD4⁺ T cells (Tregs) control immune responses using secretion of anti-inflammatory cytokines and/or cytotoxic mechanisms and play a central role in the outcomes of several immune pathologies. Previous studies suggest an impaired function of Tregs in allergy, especially during allergen seasons, but the underlying mechanism is not known. Therefore, we analysed the impact of the T helper type 2 cytokine interleukin (IL)-4 on in vitro generated adaptive Tregs (aTregs), which have been reported to use the granzyme B (GrB)/perforin pathway to kill autologous immune cells. aTregs were generated by co-ligation of CD3 and CD46 on CD4⁺ T lymphocytes and granzyme expression was analysed using flow cytometry. To quantify GrB and perforin expression as well as IL-10 secretion in response to IL-4, specific enzyme-linked immunosorbent assays were performed in cell lysates and/or culture supernatants. Using a flow cytometry-based cytotoxicity assay the impact of IL-4 on the cytotoxic potential of aTregs was investigated. While IL-4 did not affect IL-10 secretion and perforin expression in aTregs, a significant suppression of GrB synthesis was detected in the presence of IL-4. In addition, IL-4-mediated suppression of GrB led to impaired cytotoxicity of aTregs against K562 target cells. In conclusion, our data suggest that IL-4 might play a role in impaired aTreg function in allergy.     

Study on the Effect of Polysaccharides from Solanum Nigrum Linne on Cellular Immune Function in Tumour-Bearing Mice

We investigated the anti-tumour effect of polysaccharides from Solanum nigrum Linne, and its relationship with the immune function of tumour-bearing organisms. MTT assay was used to observe the effect of different doses of polysaccharides from Solanum nigrum Linne on proliferation of lymphocytes in tumour-bearing mice. ELISA assay was also used to detect the levels of IL-2 in mice, and a laser scanning confocal microscope was used to detect the effect of polysaccharides from Solanum nigrum Linne on intralymphocytic free calcium ion concentration in tumour-bearing mice. Different doses of polysaccharides from Solanum nigrum Linne significantly inhibited the growth of mouse H22 solid tumours, improved the survival time of tumour-bearing mice, increased the proliferation of lymphocytes, elevated the levels of IL-2, and increased the concentration of calcium ions in the lymphocytes. Polysaccharides from Solanum nigrum Linne have certain anti-tumour effect, which is related with the cellular immune function that regulates the body.     

Different Trypanosoma cruzi strains promote neuromyopathic damage mediated by distinct T lymphocyte subsets

The proliferative response of CD4 and CD8 T lymphocytes obtained from C3H/HeN mice chronically infected with Trypanosoma cruzi strains that differ in virulence, tropism and immunogenicity, was assayed against skeletal muscle, sciatic nerve and spinal cord homogenates. Although both CD4 and CD8 T lymphocytes from mice infected with the RA strain strongly proliferated against the nervous system, no response against skeletal muscle antigens was detected. CD4 and CD8 T lymphocytes from mice infected with the K-98 clone (from CA-I strain) showed low proliferative response against all the antigens assayed. To determine whether the proliferation patterns showed correlation with T cell-mediated neuromuscular damage, passive cell transfer studies were performed. Fifteen days after transfer of CD4 T cells from RA-infected donors (CD4-RA), normal syngeneic recipients displayed exclusively nervous tissue damage, such as perineural, endoneural and/or meningeal inflammatory infiltrates, with predominance of CD4 T cells. Fifteen days after transfer of CD4 T lymphocytes from mice infected with K-98 (CD4-K98), recipients showed inflammatory infiltrates only in skeletal muscle, where CD4 T lymphocytes and macrophages were predominant cells. Recipients of CD8 T cells from RA-infected mice (CD8-RA) showed lesions in both spinal cord and sciatic nerves. Higher percentages of CD8 T cells were observed in comparison with the recipients of CD4-RA or CD4-K98. In contrast, CD8 T cells from K-98-infected donors (CD8-K98) did not induce tissue damage. These results provide evidence that mice infected with T. cruzi populations that differ in their biological characteristics show diverse immune mechanisms that may be involved in the pathogenesis of peripheral nervous system damage.     

Intestine-derived Clostridium leptum induces murine tolerogenic dendritic cells and regulatory T cells in vitro

Patients with autoimmune and allergic diseases frequently present with reduced numbers and functionally impaired regulatory T cells (Tregs) and/or tolerogenic dendritic cells (tDCs). tDC-mediated regulation of Treg proliferation (numbers) and activation is crucial to establishing and maintaining an appropriate level of immune tolerance. Colonic colonization of Clostridium spp. is associated with accumulation of Tregs, which inhibits development of inflammatory lesions. To investigate whether infection with the Clostridium leptum sp. can specifically induce Tregs and/or tDCs bone marrow-derived dendritic cells were cultured in the presence or absence of C. leptum then co-cultured with CD4⁺CD25⁻ T cells or not. Changes in tDC numbers, Treg numbers, percentages of T cell subsets, and expression of cytokines related to Tregs (IL-10 and transforming growth factor-beta (TGF-β1)), DCs (IL-12p40 and IL-6) and effector T cells (IFN-γ, IL-4, IL-5, IL-13, and IL-17A) were measured. In the co-culture system, C. leptum-stimulated tDCs were able to increase the percentage and total number of Tregs attenuate activation of T helper cells (Th1, Th2, and Th17), and decrease the amount of secreted IL-4, IL-5, IL-13, IFN-γ and IL-17A. Thus, C. leptum exposure can induce the tDC-mediated stimulation of Tregs while disrupting the immune inflammatory response mediated by Th1, Th2 and Th17 cells.