Th2-driven Immune Response Research Articles

Determinants of activity at human Toll-like receptors 7 and 8: quantitative structure-activity relationship (QSAR) of diverse heterocyclic scaffolds.

Toll-like receptor (TLR) 7 and 8 agonists are potential vaccine adjuvants, since they directly activate APCs and enhance Th1-driven immune responses. Previous SAR investigations in several scaffolds of small molecule TLR7/8 activators pointed to the strict dependence of the selectivity for TLR7 vis-à-vis TLR8 on the electronic configurations of the heterocyclic systems, which we sought to examine quantitatively with the goal of developing “heuristics” to define structural requisites governing activity at TLR7 and/or TLR8. We undertook a scaffold-hopping approach, entailing the syntheses and biological evaluations of 13 different chemotypes. Crystal structures of TLR8 in complex with the two most active compounds confirmed important binding interactions playing a key role in ligand occupancy and biological activity. Density functional theory based quantum chemical calculations on these compounds followed by linear discriminant analyses permitted the classification of inactive, TLR8-active, and TLR7/8 dual-active compounds, confirming the critical role of partial charges in determining biological activity.

Does therapeutic intervention in atopic dermatitis normalize epidermal Notch deficiency?

This viewpoint presents a unifying concept for the treatment of atopic dermatitis (AD) that is based on the improvement of deficient Notch signalling, which appears to represent the fundamental epithelial defect of AD resulting in epidermal and immunological barrier dysfunction. One study of AD patients demonstrated a marked epidermal deficiency of Notch receptors and several mouse models with genetically suppressed Notch signalling exhibit dry skin, signs of scratching, skin barrier abnormalities, increased transepidermal water loss and Th2 cell-mediated immunological changes closely resembling human AD. Notch signalling is critically involved in the differentiation of regulatory T cells, in the feedback inhibition of activated innate immunity, in the repression of activating protein-1 (AP-1), the regulation of late epidermal differentiation associated with filaggrin- and stratum corneum barrier lipid processing, in aquaporin 3- and claudin-1 expression and in keratinocyte-mediated release of thymic stromal lymphopoietin (TSLP), which promotes Th2-driven immune responses with TSLP- and IL-31-mediated stimulation of cutaneous sensory neurons involved in the induction of itch. Translational evidence will be provided that all major therapeutic regimens employed for the treatment of AD such as glucocorticoids, calcineurin inhibitors and UV radiation may converge in the upregulation of impaired Notch signalling, the proposed pathogenic defect of AD.

Fasciola hepatica Tegumental Coat Impairs Mast Cells’ Ability To Drive Th1 Immune Responses

The parasitic worm Fasciola hepatica induces strong Th2 and T-regulatory immune responses while simultaneously suppressing Th1-driven immune responses to bystander microbial infections. It also prevents the initiation of Th1-mediated autoimmune disorders in mice through the suppression of Th17 and Th1 immune responses, and this can be mimicked by parasite-derived molecules. We have isolated F. hepatica tegumental coat Ag (FhTeg) and demonstrated its suppressive effect in vivo by directly targeting dendritic cells, impairing their ability to drive Th1 responses. Mast cells are critical in promoting Th1 protective immunity during bacterial infection and in driving Th1-mediated pathological conditions in autoimmune diseases. In this article, we show that FhTeg inhibits the ability of mast cells to drive the Th1 immune response by suppressing cytokine secretion (TNF-α, IL-6, IFN-γ, and IL-10) and ICAM1 expression in mast cells stimulated with LPS or heat-inactivated Bordetella pertussis Ag. These heat-inactivated B. pertussis Ag/LPS-stimulated mast cells fail to promote Th1 immune responses in CD4(+) T cells when pretreated with FhTeg, and a role for ICAM1 in this process was demonstrated. FhTeg suppresses the activation of transcription factors in the TLR signaling pathway, which explains the decrease in cytokine production and cell surface marker expression. We demonstrated that FhTeg suppresses MAPK and NF-κB activation and enhances SOCS3 expression, which could explain its negative effect on the TLR pathways. We conclude that FhTeg targets innate immune cells, inhibiting their ability to drive Th1 immune responses.

AMP Affects Intracellular Ca2+ Signaling, Migration, Cytokine Secretion and T Cell Priming Capacity of Dendritic Cells

The nucleotide adenosine-5′-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A1 and A2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4+CD45RA+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5′-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.

Tregs

Transfer of human regulatory T cells (Tregs) has become an attractive therapeutic alternative to improve the long-term outcome in transplantation and thus reduce the side-effects of conventional immunosuppressive drugs. Here, we summarize the recent findings on human Treg subsets, their phenotype and in-vivo function. In the last 2 years, it has become apparent that several Treg subsets exist that specifically regulate Th1-driven, Th2-driven, or Th17-driven immune responses; these subsets are very unstable and rapidly change their phenotype, for example, there is loss of Foxp3 expression upon extensive ex-vivo expansion and only the administration of rapamycin has been shown to be able to interfere reproducibly. New humanized mouse models incorporating human solid-organ grafts have been developed, which have been used to test the human Treg in-vivo function, and the first human Treg-cell products have been tested for safety and efficacy in stem cell transplantation. With the recent findings, we have gained a better understanding of Treg heterogeneity, plasticity and function. Using the outcomes of clinical trials in stem cell transplantation, we have learned that adoptive therapy of Tregs is well tolerated and we are now awaiting the first result in solid-organ transplantation from the 'ONE Study'.

Allergic diseases, gene–environment interactions

Allergic asthma develops in part from dysregulation of the innate and adaptive immune functions, particularly an imbalance in the Th2-driven adaptive immune response. This dysregulation is the result of complex interactions between genes and environment. These interactions occur both pre- and postnatally, providing opportunities for early interventions in immunological programming.

Age-related cytokine profile in uncomplicated Plasmodium malaria infection

Objective: Malaria infection is severe in children who are believed to be more at risk because of their relative poor immunity against the disease. Some cytokine levels (IFN-g, IL-2, IL-4, and IL-10) of children, adolescents, and adults were assessed in this study. Methods: Cytokine levels were assayed by using Enzyme Linked Immunosorbent Assay (ELISA). Malaria diagnosis and blood parameters were carried out by using standard parasitological and haematological techniques. Results: The mean cytokine levels were significantly elevated in children, adolescent, and adult subjects when compared to their respective healthy controls (p<0.05). Also, mean IFN-g and IL-2 levels were significantly higher in children than in adults (IFN-g: 57.31±77.79 pg/ml vs. 20.37± 2.95 pg/ml, and IL-2: 108.75±63.53 pg/ml vs. 66.09±45.34 pg/ml) (p<0.05) and adolescents (IFN-g: 20.37± 2.95 pg/ml and IL-2: 66.09±45.34 pg/ml) respectively. Furthermore, mean IL-10 level was significantly lower in children (7.39±15.08 pg/ml) than mean level in adults (22.73±13.89 pg/ml). The mean haematological parameters revealed significant increase in total white blood cell, CD4, and CD8 count and significant decrease in the hematocrit of children in relation to adolescent and adult subjects (p<0.05). However, mean monocyte count was significantly higher in subjects than in their respective healthy controls (p<0.05). Conclusion: Findings in this study revealed better Th1 driven immune response in children than in adolescents and adults.

Immunomodulatory Effects of Adipose-Derived Stem Cells in Airway Allergic Diseases

Allergic rhinitis and asthma are inflammatory airway allergic diseases caused by Th2-driven immune response. Several studies have shown that multipotent adipose-derived stem cells (ASCs) can exert profound immunosuppressive effects via modulation of both cellular and innate immune pathway, especially immunosuppressive effect on T cell activities. ASCs' ability to be readily isolated from a number of adipose tissues and expanded ex vivo makes them attractive candidate for use in clinical therapy in the context of allogeneic transplantation, in particular to modulate graft-versus-host disease and graft rejection. The authors have investigated whether ASCs can inhibit Th2-dependent airway allergic disease in the mouse model. In this article we review recent experimental data and discuss about the mechanisms by which ASCs inhibit allergic airway inflammation via immunomodulation from a Th2 to a Th1-biased response in the mouse model.

Experimental tuberculosis: Designing a better model to test vaccines against tuberculosis

Experimental models of infection are good tools for establishing immunological parameters that have an effect on the host-pathogen relationship and also for designing new vaccines and immune therapies. In this work, we evaluated the evolution of experimental tuberculosis in mice infected with increasing bacterial doses or via distinct routes. We showed that mice infected with low bacterial doses by the intratracheal route were able to develop a progressive infection that was proportional to the inoculum size. In the initial phase of disease, mice developed a specific Th1-driven immune response independent of inoculum concentration. However, in the late phase, mice infected with higher concentrations exhibited a mixed Th1/Th2 response, while mice infected with lower concentrations sustained the Th1 pattern. Significant IL-10 concentrations and a more preeminent T regulatory cell recruitment were also detected at 70 days post-infection with high bacterial doses. These results suggest that mice infected with higher concentrations of bacilli developed an immune response similar to the pattern described for human tuberculosis wherein patients with progressive tuberculosis exhibit a down modulation of IFN-gamma production accompanied by increased levels of IL-4. Thus, these data indicate that the experimental model is important in evaluating the protective efficacy of new vaccines and therapies against tuberculosis.

T-helper 2 cells are essential for modulation of vascular repair by allogeneic endothelial cells

Endothelial cells (ECs) embedded within 3-dimensional matrices (MEEC) control lumenal inflammation and intimal hyperplasia when placed in the vascular adventitia. Matrix embedding alters endothelial immunogenicity in vitro. T-helper (Th) cell-driven host immunity is an impediment of allogeneic grafts. We aimed to identify if modulation of Th balance would affect immune compatibility and endothelial regulation of vascular repair in vivo. Pigs (n = 4/group) underwent carotid artery balloon injury and were left untreated (Group 1) or received perivascular porcine MEEC implants (Group 2), 12 days of cyclosporine A (CsA; Group 3), or MEEC and CsA (Group 4). Host immune reactivity was analyzed after 28 and 90 days. MEEC treatment induced formation of EC-specific immunoglobulin (Ig) G(1) antibodies (41 +/- 6 mean fluorescence intensity [MFI]) and differentiation of host splenocytes into Th2, but not Th1, cytokine-producing cells (interleukin [IL]-4, 242 +/- 102; IL-10, 273 +/- 114 number of spots). Concomitant CsA therapy reduced IgG(1) antibody frequency (25 +/- 2 MFI; p < 0.02) and Th2-cytokine producing splenocytes upon MEEC treatment (IL-4, 157 +/- 19; IL-10, 124 +/- 26 number of spots; p < 0.05). MEECs inhibited luminal occlusion 28 and 90 days after balloon injury (12 +/- 7%) vs untreated controls (68 +/- 14%; p < 0.001) but to a lesser extent with concomitant CsA treatment (34 +/- 13%; p < 0.02 vs Group 2). MEECs do not induce a significant Th1-driven immune response but do enhance differentiation of splenocytes into cells producing Th2 cytokine. Reduction in this Th2 response reduces the vasoregulatory effects of allogeneic ECs after injury.

A Functional Polymorphism inIL-18Is Associated with Severity of Bronchial Asthma

IL-18 is a unique cytokine that enhances innate immunity and both Th1- and Th2-driven immune responses. Recent murine and human genetic studies have shown its role in the pathogenesis of asthma. We conducted an association study in a Japanese population to discover variants of IL-18 that might have an effect on asthma susceptibility and/or progression and conducted functional analyses of the related variants. The IL-18 gene locus was resequenced in 48 human chromosomes. Asthma severity was determined according to the 2002 Global Initiative for Asthma Guidelines. Association and haplotype analyses were performed using 1,172 subjects. Although no polymorphisms differed significantly in frequency between the control and adult asthma groups, rs5744247 C>G was significantly associated with the severity of adult asthma (steps 1, 2 vs. steps 3, 4; P = 0.0034). We also found a positive association with a haplotype (P = 0.0026). By in vitro functional analyses, the rs5744247 variant was found to increase enhancer-reporter activity of the IL-18 gene in bronchial epithelial cells. Expression levels of IL-18 in response to LPS stimulation in monocytes were significantly greater in subjects homozygous for the susceptibility G allele at rs5744247 C>G. Furthermore, we found a significant correlation between the serum IL-18 level and the genotype of rs5744247 (P = 0.031). Although the association results need to be replicated by other studies, IL-18 variants are significantly associated with asthma severity, and the rs5744247 variant reflects higher transcriptional activity and higher expression of IL-18 in LPS-stimulated monocytes and a higher serum IL-18 level.

Cyclosporine A modifies immune-mediating and therapeutic efficacy of allogeneic perivascular porcine endothelial cell implants (141.51)

Abstract Non-syngeneic endothelial cell embedded within 3D-matrices (MEEC) when placed in the vascular adventitia control vascular repair processes. Host immunity directed against engrafted allogeneic tissue is a predominant Th1-driven immune response and a major impediment for graft survival. We aimed to identify if cyclosporine A (CsA) would enhance immune compatibility and therapeutic efficacy of allogeneic MEEC. Pigs (n=4/group) underwent balloon injury of both carotid arteries and received a 12 days course of CsA (group 1), perivascular implants of porcine MEEC (group 2), combination of CsA and MEEC (group 3) or were left untreated (group 4). Host immune reactivity was analyzed after 28 and 90 days in 2 pigs/group respectively. No immune reactivity against MEEC was observed in treatment groups 1 and 4. MEEC treatment alone (group 2) induced formation of IgG1 antibodies specific for the allogeneic EC and differentiation of host splenocytes into Th2 but not Th1 cytokine-producing cells. CsA-therapy reduced the frequency of IgG1 antibodies and Th2-cytokine producing splenocytes upon MEEC treatment (group 3). Compared with groups 1 and 4 treatment with MEEC (group 2) significantly inhibited luminal occlusion 28 and 90 days after balloon injury. CsA-treatment reduced the ability of MEEC to inhibit luminal occlusion. MEEC are powerful regulators of vascular injury. MEEC failed to induce a significant Th1-driven allo-immune response but induced differentiation of splenocytes into Th2-cytokine producing cells. This induction and their therapeutic efficacy were alleviated by a 12 day course of CsA. Adventitial MEEC implants -even of allogeneic sources and without immunosuppressive treatment- may be useful in decreasing luminal narrowing in a clinical setting.

Crohn’s disease: The hot hypothesis

The peculiar geographic distribution of inflammatory bowel disease is a puzzle for researchers. A low vitamin D status has now been linked to several Th1-mediated autoimmune diseases, including multiple sclerosis, type 1 diabetes and rheumatoid arthritis, with the strongest evidence for the vitamin's protective role in multiple sclerosis. Sunlight and vitamin D may be potent immunomodulatory agents by down-regulating Th1-driven immune responses and inducing the synthesis of antimicrobial peptides considered as natural antibiotics of the immune system. Similarly to multiple sclerosis, we propose in CD the so-called north-south gradient may be partly explained by variations in the degree of sun exposure, with vitamin D being a "seasonal stimulus". These observations may yield a better understanding of the pathophysiology of Crohn's disease and pave the way for developing new therapeutic approaches for an incurable disease. Whether a low vitamin D status is associated with an increased risk of Crohn's disease in the general population and whether vitamin D and heliotherapy may be effective in treating Crohn's disease will require additional investigations.

Humoral Immune Responses against the Immature Laminin Receptor Show Prognostic Significance in Patients with Multiple Myeloma.

Multiple myeloma (MM) is characterized by a highly variable clinical course. The role of an autologous tumor-specific immune control contributing to the variable length of survival in MM is poorly understood. In the current study, we investigated whether humoral immunity specific for the tumor-associated oncofetal antigen/immature laminin receptor (OFA/iLR) has a prognostic value in MM. In patients with monoclonal gammopathy of unknown significance (MGUS) we found significant levels of anti-OFA/iLR antibodies in 21 of 30 sera (70%) and in 7 of 27 (26%) patients with asymptomatic MM (AMM). In contrast, in sera from patients with progressive MM (n=67) and in normal healthy donors (HD, n=60) no humoral reactivity against OFA/iLR was present. To look on long-term persistence of humoral anti-OFA/iLR immune responses, significant and stable titers of anti-OFA/iLR-specific IgG antibodies in follow-up samples of MGUS patients were detected. IgG subclass analyses showed a predominant IgG1 and IgG3 response suggestive for a Th1-driven immune response in the vast majority of patients with MGUS and to smaller extent in patients with AMM. OFA/iLR-antibodies were capable of recognizing and selectively killing OFA/iLR-expressing myeloma cells in complement-mediated and antibody-dependent cellular cytotoxicity (ADCC) assays. To determine whether the presence of anti-OFA/iLR antibodies influenced progression-free survival (PFS), patients were analyzed according to the presence/absence of a humoral immune response against OFA/iLR protein. In a univariate analysis, the group of patients with reactive sera (n=28) showed a significantly lower probability of disease progression compared to patients with non-reactive sera (n=29). In the patient group with significant anti-OFA/iLR titers only 2 of 28 patients developed disease progression, but 18 of 29 patients with OFA/iLR non-reactive sera (p=0.001). Our data suggest that spontaneous tumor-specific humoral immune responses against OFA/iLR exist in a significant proportion of MM patients and that superior PFS in those patients could reflect autologous immune control.

Interferon Gamma: Friend or Foe?

Interferon gamma (IFN-gamma) can elicit an inflammatory TH1-driven immune response but has also been found to be necessary for long-term allograft survival induced by costimulation blockade. Recently, we have found that regulatory T cells rapidly and transiently produce IFN-gamma creating a microenvironment that can influence the function of antigen presenting cells (APCs), T-cell proliferation and activation as well as T-cell effector mechanisms, thereby controlling immune responses locally. Moreover, addition of IFN-gamma to cocultures of T cells and APCs can drive the generation of T cells with regulatory activity. Thus, the influence of IFN-gamma on the immune response to a transplant is likely to be context dependent.

Effect of Both Ultraviolet B Irradiation and Histamine Receptor Function on Allergic Responses to an Inhaled Antigen

Exposure of skin to UVB radiation (290-320 nm) modulates the immune system, with most studies showing a suppression of Th1-driven immune responses. This study investigated the effects of UVB on Th2-associated immune responses using a murine model of allergic respiratory inflammation. C57BL/6, histamine receptor-1 knockout (H1RKO), and histamine receptor-2 knockout (H2RKO) mice were exposed to a single 4 kJ/m(2) dose of UVB (twice a minimal edemal dose) on shaved dorsal skin 3 days before intranasal sensitization with papain, a cysteine protease homologue of the dust mite allergen Der p 1. H1RKO mice demonstrated enhanced papain-specific inflammatory responses in the lung-draining lymph nodes (LDLNs), whereas the responses of H2RKO mice closely mimicked those of C57BL/6 mice. UVB irradiation 3 days before sensitization reduced in vitro papain-specific proliferation of LDLN cells of C57BL/6 and H1RKO mice but not H2RKO mice 24 h after challenge. The regulatory effect of UVB was transferred by adoptive transfer of unfractionated LDLN cells from UVB-irradiated, papain-sensitized C57BL/6 and H1RKO donor mice in naive recipients of the corresponding strain that were subsequently sensitized and challenged with papain. Additionally, UVB exposure suppressed papain-induced IL-5 and IL-10 production in vitro by LDLN cells from H1RKO mice but not from C57BL/6 mice or H2RKO mice. The results of this study demonstrate systemic immunomodulation of responses to intranasally delivered Ag by UVB irradiation and implicate a role for the H2 receptor in UVB-induced suppression of Ag-specific responses in the draining lymph nodes.

Effect of thymoquinone on cyclooxygenase expression and prostaglandin production in a mouse model of allergic airway inflammation

Prostaglandins (PGs) are potent proinflammatory mediators generated through arachidonic acid metabolism by cyclooxygenase-1 and -2 (COX-1 and COX-2) in response to different stimuli and play an important role in modulating the inflammatory responses in a number of conditions, including allergic airway inflammation. Thymoquinone (TQ) is the main active constituent of the volatile oil extract of Nigella sativa seeds and has been reported to have anti-inflammatory properties. We examined the effect of TQ on the in vivo production of PGs and lung inflammation in a mouse model of allergic airway inflammation. Mice sensitized and challenged through the airways with ovalbumin (OVA) exhibited a significant increase in PGD2 and PGE2 production in the airways. The inflammatory response was characterized by an increase in the inflammatory cell numbers and Th2 cytokine levels in the bronchoalveolar lavage (BAL) fluid, lung airway eosinophilia and goblet cell hyperplasia, as well as the induction of COX-2 protein expression in the lung. Intraperitoneal injection of TQ for 5 days before the first OVA challenge attenuated airway inflammation as demonstrated by the significant decrease in Th2 cytokines, lung eosinophilia, and goblet cell hyperplasia. This attenuation of airway inflammation was concomitant to the inhibition of COX-2 protein expression and PGD2 production. However, TQ had a slight inhibitory effect on COX-1 expression and PGE2 production. These findings suggest that TQ has an anti-inflammatory effect during the allergic response in the lung through the inhibition of PGD2 synthesis and Th2-driven immune response.

The role of the T cell in autoimmune inflammation

T cells, in particular CD4+ T cells, have been implicated in mediating many aspects of autoimmune inflammation. However, current evidence suggests that the role played by CD4+ T cells in the development of rheumatoid inflammation exceeds that of activated proinflammatory T-helper (Th)1 effector cells that drive the chronic autoimmune response. Subsets of CD4+ T cells with regulatory capacity, such as CD25+ regulatory T (Treg) cells and Th2 cells, have been identified, and recent observations suggest that in rheumatoid arthritis the function of these regulatory T cells is severely impaired. Thus, in rheumatoid arthritis, defective regulatory mechanisms might allow the breakdown of peripheral tolerance, after which the detrimental Th1-driven immune response evolves and proceeds to chronic inflammation. Here, we review the functional abnormalities and the contribution of different T cell subsets to rheumatoid inflammation.

Viewpoint 3

Viewpoint 3

Depletion of the CD1d-Restricted NKT Cells Suppresses In Vitro Alloreactivity: A Possible Means To Prevent aGVHD.

NKT cells constitute a small (<0.1% of blood and marrow T cells) but potent subset of regulatory T cells. Upon engagement of their unique TCR by the glycolipid presenting, MHC-like, non-polymorphic molecule CD1d, they are activated and secrete copious amounts of TH1 and TH2 cytokines. Activated NKT have a pivotal role in modulating all aspects of the innate and adaptive immune responses mainly through their interaction with antigen presenting cells (APC). Mice deficient in NKT or CD1d have diminished TH1 responses against a variety of pathogens and tumours. Conversely, administration of the model glycolipid α-galactosylceramide (αGC) to wild type mice considerably enhances TH1 immune responses in an NKT- and CD1d-dependent manner. In this work we sought to study the in situ role of NKT in alloreactivity. For this purpose, allogeneic mixed lymphocyte reactions (MLR) were performed using 3H incorporation assays. Purified, negatively selected CD3+ cells and irradiated allogeneic peripheral blood mononuclear cells from normal individuals were used as responders and stimulators respectively. After rigorous NKT cell (as identified by staining with the anti-TCRVα24 and -Vβ11 mAbs specific to the NKT TCR α and β chains) depletion by flow sorting we compared MLR reactivity in the presence and absence of NKT. In a series of MLR that included a panel of 4 different responders and 3–5 stimulators, depletion of NKT profoundly suppressed the proliferation by 68.5%±16.9 compared to baseline (i.e., NKT-replete MLR). This suppressive effect was mirrored by a reduction (45.3%±8) of IFNγ secretion in the supernatants of the NKT-depleted MLR compared to baseline. IL-4, IL-10 and TGFβ were not detected in either NKT replete or NKT depleted MLR. When freshly flow-sorted NKT cells were placed against the allogeneic stimulators they did not proliferate indicating that the decrease in the proliferation after NKT cell depletion is due to a decrease in the proliferation of the alloreactive T cells. Taken together, these findings indicate that NKT cells positively regulate the alloresponse, a TH1-driven immune response. Consistent with this, in MLR performed in the presence of αGC (100ng/ml), proliferation was significantly enhanced as compared to baseline MLR (i.e., performed in the presence of the αGC diluent). In a panel of 5 responders against a panel of 3–5 stimulators treatment of the MLR with αGC resulted in a 43.2%±15.2 increase in proliferation. In accordance with the proliferation data, IGNγ production was significantly increased (mean of 53%), whereas IL-4 was undetectable under both conditions. Furthermore, the enhancing effect of αGC was NKT-dependent, as in NKT-depleted MLR proliferation was equally suppressed in the presence of αGC and its diluent (82.6±6.8 vs 82±8.5 respectively, n=3).In summary, we have demonstrated that NKT cells exert an enhancing effect on the alloreactive response and NKT cell depletion effectively suppresses in vitro alloreactivity. These findings set the scene for exploring on one hand the potential for reducing the risk of severe aGVHD by using NKT depletion in allogeneic haemopoietic stem cell transplantation and on the other hand for exploring the adoptive transfer of purified NKT cells to improve immune reconstitution post transplant. In either case, the rapid, accurate identification and physical isolation of the NKT cells is possible either with the use of mAbs highly specific for the TCR of the NKT cells or with the use of the CD1d/α GC tetramer.