Th1 Th2 Research Articles

In vitro cytokine responses of peripheral blood mononuclear cells from healthy dogs to distemper virus, Malassezia and Toxocara

Naïve CD4+ T cells may differentiate into a number of subsets including T helper 1 (Th1) Th2, Th3, Th17 and T regulatory (Treg) cells depending on the type of antigen they encounter. These CD4+ families have been defined based on the array of cytokines they produce and the effects they have on adaptive immune responses. CD4+ subsets are cross regulatory and at times cooperative. The study of these adaptive immune modulators has revealed the important role that cytokines play in mounting effective as well as detrimental immune responses to pathogens. Examining the cytokine responses of lymphocytes in culture can provide important understanding of how immune responses to pathogens are orchestrated. For this purpose the in vitro cytokine production of peripheral blood mononuclear cells (PBMC) from healthy dogs was examined in response to stimulation with antigens from a common canine virus (canine distemper virus, CDV), a commensal skin yeast of dogs ( Malassezia pachydermatis) and a common canine helminth ( Toxocara canis ( T. canis)). Cell culture supernatants were removed from antigen stimulated and unstimulated control PBMC after 4, 24, 48 and 72 h and the concentration of Th1 type cytokines (IL-2, IFN-γ, TNF-α) and Th2 type cytokines (IL-4, IL-5, IL-10) was determined using sandwich ELISA assays. CDV induced low levels of cytokine production initially with a predominance of IL-10 at 24 h and a balanced response at 48 h of incubation. Malassezia antigen stimulated an early type 2 cytokine response with dramatic production of IL-4 at 24 h of incubation compared to the other stimulants examined. By 48 h of incubation, however, the cytokine mix in response to Malassezia had also moved toward a Th1 type response. T. canis induced early production of Th2 type cytokines with IL-5 predominating; however, with longer incubation (48–72 h) there was a switch to a balanced Th1/Th2 response. In conclusion, the cytokines produced in vitro by canine PBMC in response to prototypical Th1 and Th2 type pathogens were not clearly polarized and shifted over time. While the in vitro study of PBMC cytokine responses cannot be directly extrapolated to in vivo responses to the same antigens, the results do highlight the dynamic and fluctuating nature of cytokine production.

P16-31. Skewed HIV-1-specific CD4+ Th2 helper cell contribution in progressive HIV-1 infection

Background CD4+ T helper cells play a crucial role in the orchestration of the immune system. While CD4+ Th1 responses provide important helper signals to the survival and maturation of CD8+ T cells, CD4+ Th2 responses are involved in B cell maturation and antibody class switching. However, very little is known about the role and contribution of HIV-1-specific CD4+ T helper responses to the control of viral replication. Here we show that the presence of Gagspecific CD4+ Th1 responses and Th2 responses against Env are associated with viral control, while changes in this pattern are associated with disease progression.

Immunological and cytotoxicological characterization of tuberculin purified protein derivative (PPD) conjugated to single-walled carbon nanotubes

Tuberculosis (TB) represents one of the leading killers among all infectious disease. Protection against TB depends on the activation of T-helper type I (Th1) immune response. Carbon nanotubes (CNTs) have attracted considerable attention because of their potential applications as new nanovehicle. In the current study, tuberculin purified protein derivative (PPD) was conjugated to carboxylated single-walled carbon nanotubes (SWCNTs). Cytotoxicity of the carboxylated SWCNT and SWCNT-PPD conjugate was analyzed with MTT assay and by reactive oxygen species (ROS) and nitric oxide (NO) generation. Male BALB/c mice were immunized with BCG, PPD, SWCNT-PPD conjugate and PPD in complete Freund's adjuvant (CFA). Induction of cellular immune response was analyzed by measuring the levels of Th1 cytokines (IFN-γ and IL-12) and Th2 cytokines (IL-10 and IL-5). Immunization with non-conjugated PPD or PPD in Freund's adjuvant induced a Th2 cytokine response while immunization with BCG resulted to a mixed Th1/Th2 cytokine response. In contrast, PPD in conjugation with SWCNT generated preferentially a Th1-type cytokine response in the absence of potential cytotoxic effects.

Influence of the route of administration on immunomodulatory properties of bovine β-lactoglobulin-producing Lactobacillus casei

Because of their intrinsic immunomodulatory properties, some lactic acid bacteria were reported to modulate allergic immune responses in mice and humans. We recently developed recombinant strains of Lactobacillus casei that produce β-lactoglobulin (BLG), a major cow's milk allergen. Here, we investigated immunomodulatory potency of intranasal and oral administrations of recombinant lactobacilli on a subsequent sensitization of mice to BLG. Intranasal administration of the BLG-producing Lb. casei stimulated serum BLG-specific IgG2a and IgG1 responses, and fecal IgA response as well, but did not inhibit BLG-specific IgE production. In contrast, oral administration led to a significant inhibition of BLG-specific IgE production while IgG1 and IgG2a responses were not stimulated. After both oral and intranasal administrations, production of IL-17 cytokine by BLG-reactivated splenocytes was similarly enhanced, thus confirming the adjuvant effect of the Lb. casei strain. However, a mixed Th1/Th2 cell response was evidenced in BLG-reactivated splenocytes from mice intranasally pretreated, with enhanced secretions of Th1 cytokines (IFN-γ and IL-12) and Th2 cytokines (IL-4 and IL-5) whereas only production of Th1 cytokines, but not Th2 cytokines, was enhanced in BLG-reactivated splenocytes from mice orally pretreated. Our results show that the mode of administration of live bacteria may be critical for their immunomodulatory effects.

Polysaccharide Nucleic Acid of Bacillus Calmette Guerin Modulates Th1/Th2 Cytokine Gene Expression in Lipopolysaccharide-Induced Mastitis in Rats

The aim of this study was to evaluate, in rats, the changes in the T helper type 1 (Th1)/Th2 radio in mammary glands after an intramammary infusion of lipopolysaccharide (LPS) and to characterize the moderating effects of the polysaccharide nucleic acid of Bacillus Calmette Guerin (BCG-PSN) on the mammary gland. In the control group, the levels of IL-2 and INF-γmRNA expression increased, whereas IL-4 mRNA expression decreased after LPS challenge. As a consequence, the INF-[.gamma]/IL-4 mRNA ratio was significantly higher at 3, 6, and 9 h post-infusion (PI) compared to the control value (0 h; P< 0.01). BCG-PSN increased mRNA expression of both INF-γ and IL-4 before infusion of LPS. LPS challenge significantly the reduced Th1/Th2 cytokine ratio due to Th1 cytokine IFN-γ suppression and Th2 cytokine IL-4 upregulation compared with the control group. A significant reduction of N-acetyl-[.beta]-D-glucosaminidase (NAGase) was observed at 24 h PI in the BCG-PSN treatment group compared to the control group ( P < 0.05). Thus, it was demonstrated that level of BCG-PSN might change the Th1/Th2 ratio mainly by enhancing the Th2 immune response. This is the first report of a Th1/Th2 change induced by coliform mastitis and characterization of the effect of BCG-PSN on mammary gland inflammation. This study makes a better understanding of the mechanisms of coliform mastitis and provides a putative novel strategy for the prevention and/or treatment of mastitis.

Inhibitory effects of enhanced expression of CD40L in ovarian cancer OVHM cells on the liver metastases in mice

To examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murine ovarian cancer OVHM cells (CD40L-OVHM) injected into the spleen on liver metastasis in mice. OVHM cells were inoculated into the spleen of 6 to 8 week-old female B6C3F1 (C57BL/6N x C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVHM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-gamma, TNF-alpha, IL-12, IL-4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded. The mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rate in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-gamma, TNF-alpha and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (p < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05). The data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Th1 cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.

Enhancement of immune response and protection in BALB/c mice immunized with liposomal recombinant major surface glycoprotein of Leishmania (rgp63): The role of bilayer composition

Development of new generation vaccines requires adjuvants to elicit the type and intensity of immune response needed for protection. Liposomes have been shown to be an effective adjuvant formulation. In this study, the role of liposome bilayer composition with different phase transition temperature ( T c) to induce a T helper 1 (Th1) type of immune response and protection against leishmaniasis in BALB/c mice was assessed. Liposome formulations with different bilayer compositions consisting of egg phosphatidylcholine (EPC, T c < 0 °C), dipalmitoylphosphatidylcholine (DPPC, T c 41 °C), or distearoylphosphatidylcholine (DSPC, T c 54 °C) were prepared. All liposomes were contained rgp63 as a recombinant antigen and used to immunize mice subcutaneously 3 times in 3-week intervals. Evaluation of lesion development and splenic parasite burden after challenge with L. major, evaluation of Th1 cytokine (IFN-γ) and Th2 cytokine (IL-4), and titration of IgG isotypes were carried out to assess the type of generated immune response and extent of protection. The results indicated the generated immune response in mice was influenced by the bilayer composition of liposomes, so that mice immunized with liposomes consisting of EPC induced a Th2 type of immune response while liposome consisting of DPPC or DSPC induced Th1 type of immune response. It seems that liposomes prepared with higher Tm phospholipids are suitable formulation to induce Th1 type of immune response and protection, and so might be used for further investigations to develop an effective vaccine against leishmaniasis.

Control of T helper cell differentiation through cytokine receptor inclusion in the immunological synapse

The antigen recognition interface formed by T helper precursors (Thps) and antigen-presenting cells (APCs), called the immunological synapse (IS), includes receptors and signaling molecules necessary for Thp activation and differentiation. We have recently shown that recruitment of the interferon-γ receptor (IFNGR) into the IS correlates with the capacity of Thps to differentiate into Th1 effector cells, an event regulated by signaling through the functionally opposing receptor to interleukin-4 (IL4R). Here, we show that, similar to IFN-γ ligation, TCR stimuli induce the translocation of signal transducer and activator of transcription 1 (STAT1) to IFNGR1-rich regions of the membrane. Unexpectedly, STAT1 is preferentially expressed, is constitutively serine (727) phosphorylated in Thp, and is recruited to the IS and the nucleus upon TCR signaling. IL4R engagement controls this process by interfering with both STAT1 recruitment and nuclear translocation. We also show that in cells with deficient Th1 or constitutive Th2 differentiation, the IL4R is recruited to the IS. This observation suggest that the IL4R is retained outside the IS, similar to the exclusion of IFNGR from the IS during IL4R signaling. This study provides new mechanistic cues for the regulation of lineage commitment by mutual immobilization of functionally antagonistic membrane receptors.

Genotype-Dependent Tumor Regression in Marek’s Disease Mediated at the Level of Tumor Immunity

Marek’s disease (MD) of chickens is a unique natural model of Hodgkin’s and Non Hodgkin’s lymphomas in which the neoplastically-transformed cells over-express CD30 (CD30hi) antigen. All chicken genotypes can be infected with MD virus and develop microscopic lymphomas. From 21 days post infection (dpi) microscopic lymphomas regress in resistant chickens but, in contrast, they progress to gross lymphomas in susceptible chickens. Here we test our hypothesis that in resistant chickens at 21 dpi the tissue microenvironment is pro T-helper (Th)-1 and compatible with cytotoxic T lymphocyte (CTL) immunity but in susceptible lines it is pro Th-2 or pro T-regulatory (T-reg) and antagonistic to CTL immunity. We used the B2, non-MHC-associated, MD resistance/susceptibility system (line [L]61/line [L]72) and quantified the levels of key mRNAs that can be used to define Th-1 (IL-2, IL-12, IL-18, IFNγ), Th-2 (IL-4, IL-10) and T-reg (TGFβ, GPR-83, CTLA-4, SMAD-7) lymphocyte phenotypes. We measured gene expression in both whole tissues (represents tissue microenvironment and tumor microenvironment) and in the lymphoma lesions (tumor microenvironment) themselves. Gene ontology-based modeling of our results shows that the dominant phenotype in whole tissue as well as in microscopic lymphoma lesions, is pro T-reg in both L61 and L72 but a minor pro Th-1 and anti Th-2 tissue microenvironment exists in L61 whereas there is an anti Th-1 and pro Th-2 tissue microenvironment in L72. The tumor microenvironment per se is pro T-reg, anti Th-1 and pro Th-2 in both L61 and L72. Together our data suggests that the neoplastic transformation is essentially the same in both L61 and L72 and that resistance/susceptibility is mediated at the level of tumor immunity in the tissues.

Th17: The ascent of a new effector T‐cell subset

Th17: The ascent of a new effector T‐cell subset

Involvement of TH1/TH2 Cytokines in the Pathogenesis of Autoimmune Skin Disease—Pemphigus Vulgaris

Pemphigus vulgaris (PV) is classical example of antigen-driven severe autoimmune bullous skin disorder. Auto reactive T cells are critical for the induction and regulation of antibody production. With regard to cytokine production profiles, it has been reported that qualitative as well as quantitative alterations in cytokine production can result in activation of inefficacious effector mechanisms and therefore, complex and severe impairment in immune functions. The purpose of this study was to observe the alterations in the levels of TH1 [Interleukin-2 (IL-2), Interferon-gamma (IFN-γ)] and TH2 (IL-4 and IL-10) cytokines in the sera from patients affected with PV and compared with Pemphigus foliaceus and healthy subjects. This work is aimed to comprehend the involvement of TH1 and TH2 cells as inflammatory infiltrate in the modulation of acantholysis and production of pemphigus lesions. Seventy PV, 13 PF and 50 healthy, age-matched individuals without any generalized skin diseases were included in this study. The diagnosis of PV and PF patients was confirmed by histopathology (hematoxylin and eosin) and / direct immunofluorescence. The levels of TH1 cytokines (IL-2 and IFN-γ) and TH2 cytokine markers (IL-4 and IL-10) were estimated by high sensitivity ELISA kits. All patients with PV and PF showed significantly (p < 0.000) elevated levels of TH2 cytokines (IL-10 and IL-4) as compared with healthy controls. However, the mean concentration of TH1 cytokines (IL-2 and IFN-γ) was significantly decreased in patients as compared to healthy individuals. Both TH1 and TH2 cytokines did not show any significant difference between PV and PF cases. Current concepts support the idea that PV, induced by autoantibodies against Dsg3, is the consequence of an imbalance between Dsg3-reactive TH2 and TH1 cells that may be critical for the maintenance of tolerance against Dsg3. Cytokine profile for confirmed PV cases showed direct evidences for involvement of T cell responses. Increase in IL-4 and IL-10 shows induction of TH2 cells in the pathogenesis of autoimmune disorders Pemphigus vulgaris. The decreased levels of IL-2 and IFN-γ might demonstrate the inhibitory effects by IL-4 and IL-10, which suppress the expansion of TH1 population.

Retinoic acid inhibits dendritic cell differentiation driven by interleukin-4

All-trans-retinoic acid (atRA) appears to affect Th1–Th2 differentiation and its effects on immune responses might also be mediated by dendritic cell (DC). Nonetheless, studies have been showing contradictory results since was observed either induction or inhibition of DC differentiation. Our aim was to investigate atRA action on human monocyte derived DC differentiation. For this purpose we tested pharmacological and physiological doses of atRA with or without cytokines. Cell phenotypes were analyzed by flow cytometry and function was investigated by phagocytosis and respiratory burst. DC, positive control group, was differentiated with GM-CSF and IL-4 and maturated with TNF-α. We demonstrated that atRA effects depend on the dose used as pharmacological doses inhibited expression of all phenotypic markers tested while a physiological dose caused cell differentiation. However, atRA combined or not with cytokines did not promote DC differentiation. In fact, atRA was detrimental on IL-4 property as a DC inductor.

The Influence of the Preoperative Immune Response on Blood Transfusion Requirements in Patients Undergoing Cardiac Surgery

The purpose of this study was to evaluate the influence of preoperative type I and II immune responses on blood transfusion requirements. A prospective and observational trial. A postcardiac surgery unit of a university hospital. Seventy-one consecutive patients undergoing elective cardiac surgery. Blood samples drawn for laboratory analysis and immunologic study. Patients were divided into 2 groups according to blood transfusion requirements: < or = 2 units (n = 35) and >2 units of red blood cells (n = 36). The preoperative immune response was assessed by flow cytometry, measuring the proportion of CD4+ T helper cells producing cytokines, including Th1 response (interferon-gamma and tumor necrosis factor-alpha [TNF-alpha]) and Th2 response (interleukin 4 and 10). Two logistic regression analyses (including and not including immunologic variables) were used to select and weight perioperative variables associated with an increased risk of transfusion. Three variables were found to be independent predictors of transfusion requirements when immunologic variables were not included: preoperative platelet count, preoperative hemoglobin, and hypertension. When all the variables were included, preoperative hemoglobin, cardiopulmonary bypass time, and the preoperative proportion of CD4+ T cells producing TNF-alpha were associated with an increased risk of transfusion (Hosmer-Lemeshow, 0.33; c-index, 0.93), but preoperative platelet count and hypertension were not. A low preoperative Th1 immune response, as assessed by the proportion of CD4+ T-helper-producing TNF-alpha, was associated with a higher blood transfusion rate.

Targeting allergen to FcγRI: a strategy to treat allergic disease?

Targeting allergens to surface receptors on antigen presenting cells may provide a therapeutic strategy for allergic disease. This article discusses the immunomodulatory capacity of a molecule (H22-Fel d 1), which targets the major cat allergen, Fel d 1, to the high affinity IgG receptor, Fc gammaRI, on human dendritic cells. The fusion protein, H22-Fel d 1, induced a semi-mature phenotype in dendritic cells characterized by production of inflammatory cytokines with no change in surface markers, suggesting tolerogenic capacity. At the T-cell level, H22-Fel d 1 stimulated increased proliferation coupled with amplification of T cells expressing IL-5 and IL-10. Further analysis revealed induction of diverse T cell subtypes characteristic of Th0, regulatory Th1 and regulatory Th2 cells. Notably, this effect was restricted to T cells isolated from cat-allergic patients. Despite the increase in IL-5-expressing T cells, responses induced by H22-Fel d 1 appeared to be regulated by IL-10. Comparison with nonreceptor-targeted allergens from cat and house dust mite confirmed that qualitative T-cell changes induced by H22-Fel d 1 were unique. H22-Fel d 1 induces a novel variation of the Th2 response, which incorporates elements of a protective T-cell response. Exploiting Fc gammaRI-mediated pathways for allergen delivery may offer a new approach for treatment.

Cytokine responses in a severe case of glandular fever treated successfully with foscarnet combined with prednisolone and intravenous immunoglobulin

Viral loads and cytokine responses Epstein-Barr virus (EBV) were measured in an 18-year-old boy with severe glandular fever complicated by a mild anaemia, severe thrombocytopaenia and neutropaenia. Hepatosplenomegaly was detected by abdominal ultrasound in the presence of significant hepatitis. Cytokine testing demonstrated elevated cell-mediated Th1 (IFN-gamma, IL-12, sTNFR1, CXCL10, CXCL9 and CCL3) and humoral Th2 (IL-4) immune responses. Serum antibodies to EBV virus capsid antigen (VCA) IgM and IgG antibodies were detected, together with a raised EBV DNA level (up to about 70,000 DNA copies/mL) in the acute phase of the illness. This EBV DNA load decreased rapidly in response to treatment with a combination of foscarnet, intravenous immunoglobulin and prednisolone, and the boy's symptoms settled eventually after approximately 50 days of illness, following this combined antiviral and immune-modulating therapy. Detailed immunological, virological, haematological and biochemical laboratory parameters are presented to document this patient's severe EBV disease and eventual recovery.

Profiles of Th1, Th2, Th17 and Treg Cells in Patients with Chronic Idiopathic Thrombocytopenic Purpura.

Objective: To explore the profiles of Th1,Th2, Th17 and Treg cells in patients with chronic idiopathic thrombocytopenic purpura.Methods: Samples of peripheral blood were collected from 30 chronic ITP patients ( 9 males and 21 females), aged 41, 21 being in active stage, and 9 in remission stage, and 9 healthy persons in control (3 males and 6 females), aged 36. Peripheral blood was cultured, and activated with PMA/ionomycin when Th1, Th2 and Th17 cells were detected. Flow cytometry was used to measure the intracellular cytokines interferon (IFN)-γ, interleukin (IL)-4 and interleukin (IL)-17 so as to identify the Th1 cells (CD3+ CD8− IFN-γ+ IL-4− cells), Th2 cells (CD3+ CD8− IFN-γ − IL-4+ cells) and IL-17 cells (CD3+ CD8− IL-17+ cells); Treg cells were identified to CD4+ CD25+ Foxp3+ cells and uncultured peripheral blood was used to measured the CD4+ CD25+ Foxp3+ cells by flow cytometry. The ratios of Th1/Th2 were calculated.Results: The Th1/Th2 ratio for patients in active stage was 15.04±9.67, significantly higher than those for patients in remission stage (7.17±5.38, P <0.05) and in control (8.47±3.78, P <0.05); the percentage of Treg cells of the patients in active stage was 0.89±0.58%, significantly decreased than those of patients in remission stage (6.41±1.86%, P <0.001) and in control (6.06±0.85%, P <0.001); the percentage of Th17 cells was 1.94±0.77% for patients in active stage, 2.16±0.52% for patients in remission stage and 1.82±0.58% for patients in control, respectively, and there was no statistic significance between them.Conclusion: Chronic ITP is a Th1 predominant disease; decreased number and function of Treg cells might be one of mechanisms that cause immune regulation dysfunction in chronic ITP; Th17 cells might not play a role in the development of chronic ITP.

CD11b− Donor Dendritic Cells Enhance Donor T-Cells' Th1 Polarization and Graft Versus Leukemia Activity in Allogeneic Hematopoietic Stem Cell Transplantation

Background: Based on a clinical association of donor plasmacytoid dendritic cell (DC) content with leukemia relapses after allogeneic BMT (Waller, Blood 2001), we have previously reported that CD11b− donor DC added to a graft containing FACS-purified hematopoietic stem cells (HSC) and T-cells enhanced interferon-γ (IFN-γ) production and GvL activity in MHC-mismatched allogeneic transplant mouse models (Li, Blood 2007).Objective: In this study, we studied the mechanisms whereby donor DC in the graft modulate donor T-cell activity and the graft-versus-leukemia (GvL) effect in MiHA (C3H.SW → C57BL/6J)- and MHC (C57BL/6J → B10.BR)- mismatched models of allogeneic hematopoietic stem cell transplantation (HSCT).Methods: Mice irradiated to 11 Gy received 5 × 104 log-phase viable MMB3.19 myeloid lymphoma cells via intraperitoneal injection or intravenous injection of 1 x 105 LBRM T-cell lymphoma cells one day before transplant. Allografts consisted of 5 × 104 FACS-purified donor BM CD11b− DC or CD11b+ DC plus 3 × 103 FACS-purified c-kit+ sca-1+ lineage− hematopoietic stem cells (HSC) in combination with either 3 × 105 T-cells, 3 × 105 CD8+ T-cells or no additional T-cells transplanted via tail vein. Graft-versus-host disease (GvHD) clinical scores (based on body weight loss, posture, skin, fur texture, activity) were recorded twice weekly in non-tumor bearing recipients. In vitro proliferation and cytotoxic activity of donor-derived T-cells against tumor targets was assessed by CFSE staining and a caspase flow cytometry assay (CyToxiLux PLUS) using donor T-cells harvested from recipients on day 34 and day 82 post transplant. Serum and intracellular Th1 cytokines (IFN-γ, IL-2, and TNF-α) and Th2 cytokines (IL-4, IL-5, and IL-10) from recipients' peripheral blood and spleens day 3 and day 10 post-transplant was measured by ELISA and flow cytometry. IFN-γ direct killing of leukemia cells was tested by in vitro IFN-γ exposure.Results: In non-tumor bearing mice, recipients of all combinations of donor DC subsets, with and without donor T-cells had equivalent survival (75% – 85%) at 3 months post transplant without significant clinical signs of GvHD. Transplantation of tumor cells to recipients of HSC alone, HSC plus donor T-cells, or HSC plus T-cells and CD11b+ DC in the MiHA- and the MHC-mismatched transplant models led to 0% or 5% 3 month survival, respectively. Strikingly, tumor-bearing mice transplanted with CD11b− DC had significantly enhanced 3 month survival (35% in the MiHA-mismatched model and 45% in the MHCmismatched model) without increased GvHD (p<0.001). There was no significant difference in survival between mice that received HSC plus CD11b− DC and a mixture of CD4+ and CD8+ donor T-cells versus mice that received HSC plus CD11b− DC and only CD8+ donor T-cells. Donor T-cells harvested from recipients of CD11b− DC 34 days after transplant in the MiHA-mismatched model as well as 82 days after transplant in the MHC-mismatched model displayed increased cell proliferation following co-culture with irradiated hosttype splenocytes as a source of alloantigen compared with donor T-cells harvested from recipients of CD11b+ DC or recipients of HSC plus T-cells without donor DC. Leukemia cell killing was greater following incubation of purified donor T-cells recovered from recipients of CD11b− DC with tumor targets compared to T-cells recovered from other treatment groups. Recipients of CD11b− DC had higher serum levels of Th1 cytokines IFN-γ and IL-2 and higher number of Th1 positive donor T-cells compared with recipients of other treatment groups. In contrast, recipients of CD11b+ DC had higher serum levels of Th2 cytokines IL-4, IL-5, and IL-10 and higher number of Th2 positive donor T-cells. IFN-γ added to in vitro cultures with MMB3.19, and LBRM, had no direct cell killing effect.Conclusion: CD11b− donor DC enhanced Th1 polarization of donor T-cells and GvL without increasing GvHD. Donor CD8+ T-cells mediated tumor killing effect. CD11b+ donor DC enhanced Th2 polarization of donor CD4+ T-cells and led to limited GvHD and GvL.

Antimicrobial and anti‐inflammatory activity of five Taxandria fragrans oils in vitro

The antimicrobial activity of five samples of Taxandria fragrans essential oil was evaluated against a range of Gram-positive (n= 26) and Gram-negative bacteria (n= 39) and yeasts (n= 10). The majority of organisms were inhibited and/or killed at concentrations ranging from 0.06-4.0% v/v. Geometric means of MIC were lowest for oil Z (0.77% v/v), followed by oils X (0.86%), C (1.12%), A (1.23%) and B (1.24%). Despite differences in susceptibility data between oils, oils A and X did not differ when tested at 2% v/v in a time kill assay against Staphylococcus aureus. Cytotoxicity assays using peripheral blood mononuclear cells demonstrated that T. fragrans oil was cytotoxic at 0.004% v/v but not at 0.002%. Exposure to one or more of the oils at concentrations of <or=0.002% v/v resulted in a dose responsive reduction in the production of proinflammatory cytokines IL-6 and TNF-alpha, regulatory cytokine IL-10, Th1 cytokine IFN-gamma and Th2 cytokines IL-5 and IL-13 by PHA stimulated mononuclear cells. Oil B inhibited the production of all cytokines except IL-10, oil X inhibited TNF-alpha, IL-6 and IL-10, oil A inhibited TNF-alpha and IL-6, oil C inhibited IL-5 and IL-6 and oil Z inhibited IL-13 only. IL-6 production was significantly inhibited by the most oils (A, B, C and X), followed by TNF-alpha (oils A, B and X). In conclusion, T. fragrans oil showed both antimicrobial and anti-inflammatory activity in vitro, however, the clinical relevance of this remains to be determined.

Th1–Th2 polarisation and autophagy in the control of intracellular mycobacteria by macrophages

Autophagy is a major intracellular pathway for the lysosomal degradation of long-lived cytoplasmic macromolecules and damaged or surplus organelles. More recently, autophagy has also been linked with innate and adaptive immune responses against intracellular pathogens, including Mycobacterium tuberculosis, which can survive within macrophages by blocking fusion of the phagosome with lysosomes. Induction of autophagy by the Th1 cytokine IFN-γ enables infected macrophages to overcome this phagosome maturation block and inhibit the intracellular survival of mycobacteria. Conversely, the Th2 cytokines IL-4 and IL-13 inhibit autophagy in murine and human macrophages. We discuss how differential modulation of autophagy by Th1 and Th2 cytokines may represent an important feature of the host response to mycobacteria.

Synthetic Glycolipid Ligands for Human iNKT Cells as Potential Therapeutic Agents for Immunotherapy

Invariant natural killer T (iNKT) cells are an attractive therapeutic target in autoimmune diseases, since they play a major role in immune regulation. iNKT cells recognize glycolipid antigens presented by CD1d molecules that resemble the non-polymorphic MHC class I protein. alpha-galactosylceramide (alpha-GalCer) isolated from marine sponge has long been used as a prototype iNKT cell ligand in the laboratory. As alpha-GalCer is the most efficacious ligand for iNKT cells, its potential to treat autoimmune disease has been evaluated in animal models. Previous studies showed that alpha-GalCer effectively suppressed disease in some autoimmunity models, but not in others. This inconsistency may be attributed to the ability of alpha-GalCer to induce the production of both proinflammatory Th1 and anti-inflammatory Th2 cytokines by iNKT cells. To overcome this issue, we and other groups have synthesized new, unnatural glycolipids by modifying the structure of alpha-GalCer. These efforts have led to an identification of glycolipid compounds that provoke the production of Th2 (but not Th1) cytokines by iNKT cells. Among these novel ligands, an alpha-GalCer analogue named OCH, which contains a truncated sphingosine chain, induces a Th2 biased response by murine iNKT cells. Here we describe that OCH also polarizes human iNKT cells towards Th2, which opens up a new avenue for the clinical application of glycolipid compounds in treating of autoimmune diseases such as multiple sclerosis. The pursuit of synthetic glycolipid antigens has the great potential to lead to a better understanding of the regulatory effects of human iNKT cells and development of a new therapeutic agent for autoimmune diseases.