Indirubin ameliorates imiquimod-induced psoriasis-like skin lesions in mice by inhibiting inflammatory responses mediated by IL-17A-producing γδ T cells.

OBJECTIVES: Indirubin (IR) is a bisindole compound extracted from the leaves of Chinese herb Indigo Naturalis. Indigo Naturalis has been widely used in traditional Chinese medicine to treat inflammatory and autoimmune diseases. Psoriasis is a chronic immune-mediated inflammatory skin disease in which γδ T cells play an important role. This study aims to determine the immunoregulatory effects and the underlying mechanisms of Indirubin in psoriasis-related inflammatory responses. METHODS: BALB/c mice with imiquimod (IMQ)-induced psoriasis-like dermatitis were treated with saline (Model), 1 mg/kg methotrexate (MTX) that serves as a positive control, or 12.5, 25 and 50 mg/kg Indirubin(IR) intragastrically. Keratinocytes proliferation, inflammatory cells infiltration, the expression of inflammatory cytokines and Jak/Stat pathway-related proteins in the skin lesion were examined. The abundance of γδ T cells in lymph nodes and spleen was determined by flow cytometry. The IL-17 expression and secretion, and the activation of Jak3/Stat3 pathways in in vitro cultured γδ T cell were tested. RESULTS: Indirubin ameliorated keratinocyte proliferation, reduced the infiltration of CD3+ T cells, IL-17 A-producing γδ T cells, and CD11b+ neutrophils, inhibited the mRNA expression of Il1, Il6, Il23, Il17a and Il22, and the protein expression of Jak/Stat pathway-related molecules in the skin lesion. Indirubin also reduced the abundance of γδ T cell and CCR6+ γδ T cells (the major IL-17 A producer) in spleen and lymph nodes. In cultured γδ T cells, Indirubin inhibited the mRNA expression of Il17a and Ifng, and the secretion of IL-17 A, while suppressed the activation of Jak3/Stat3 pathways. CONCLUSION: Indirubin alleviates IMQ-induced psoriasis-like dermatitis mainly through reducing the inflammatory responses mediated by IL-17 A-producing γδ T cells involving Jak3/Stat3 activation. Our results highlighted the novel mechanisms by which Indirubin ameliorates psoriasis-related inflammatory responses, supporting its therapeutic potential.

Three-Dimensional Ameliorated Biologics Elicit Thymic Renewal in Tumor-Bearing Hosts.

Cancer-initiating/sustaining stem cell subsets (CSCs) have the potential to regenerate cancer cell populations and are resistant to routine therapeutic strategies, thus attracting much attention in anticancer research. In this study, an innovative framework of endogenous microenvironment-renewal for addressing such a dilemma has been just developed. CSCs in three-dimensional multipotent spheroid-engineered biologics were prepared with 150 Gy radiation and inoculated into 15-mo-old BALB/c and C57BL/6 mice bearing diverse advanced tumors covering Mammary 4T1, liver Hepa, lung LL/2, and colon C26 tumors and distant metastases. Subsequently, the systematic microenvironment of tumor-bearing hosts was rapidly remodeled to resettle thymic cortex and medulla rudiment as an endogenous foxn1-thymosin reprogramming TCR-repertoire for resetting MHC-unrestricted multifunction renewal. Postrenewal Vγ4γδT-subsets would bind and lead migrating CSCs into apoptosis. Moreover, TCR repertoire multifunction renewal could reverse tumor metastases from tumoricidal resistance into eventual regression as a blockade of cancer-sustaining Bmi-1/Nanog-Oct4-Sox2 renewal loop with sequent multivalent depletion of both migrating/in situ CSCs and non-stem terminal cancer cell subsets. This study represents a promising start to set up a generalizable strategy of three-dimensional biologics evoking an endogenous integral microenvironment into pluripotent renewal versus advanced cancer.

Highly specific and potently activating Vγ9Vδ2-T cell specific nanobodies for diagnostic and therapeutic applications.

Vγ9Vδ2-T cells constitute the predominant subset of γδ-T cells in human peripheral blood and have been shown to play an important role in antimicrobial and antitumor immune responses. Several efforts have been initiated to exploit these cells for cancer immunotherapy, e.g. by using phosphoantigens, adoptive cell transfer, and by a bispecific monoclonal antibody based approach. Here, we report the generation of a novel set of Vγ9Vδ2-T cell specific VHH (or nanobody). VHH have several advantages compared to conventional antibodies related to their small size, stability, ease of generating multispecific molecules and low immunogenicity. With high specificity and affinity, the anti-Vγ9Vδ2-T cell receptor VHHs are shown to be useful for FACS, MACS and immunocytochemistry. In addition, some VHH were found to specifically activate Vγ9Vδ2-T cells. Besides being of possible immunotherapeutic value, these single domain antibodies will be of great value in the further study of this important immune effector cell subset.

Oral administration of acarbose ameliorates imiquimod-induced psoriasis-like dermatitis in a mouse model.

Psoriasis is a chronic autoimmune disease of undefined etiology that involves dysregulated interplay between immune cells and keratinocytes. Acarbose was found to decrease inflammatory parameters in diabetic patients in addition to its anti-diabetic effects. Here, we report that imiquimod (IMQ)-induced epidermal hyperplasia and psoriasis like-inflammation were significantly inhibited by acarbose treatment. Real-time PCR showed that mRNA levels of the cytokines TNF-α, IL-6, IL-1ß IL-17A, and IL-22 in skin were also decreased significantly by acarbose. In addition, we found that acarbose reduced infiltration of CD3(+) T cells and GR-1(+) neutrophils in lesional skin and also reduced the percentage of IL-17-producing CD4(+) T cells (Th17) and IL-17- and IL-22-producing γδ T cells in the spleen. In contrast, acarbose increased the frequency of IL-10-producing CD4(+) regulator Tr1 T cells in the spleen and small intestine. These results indicate that oral administration of acarbose can attenuate the severity of imiquimod-induced psoriasis with local and systemic anti-inflammatory and immune modulation effects, thus suggesting that acarbose is an effective therapeutic strategy for psoriasis regulation.

IFN-λ resolves inflammation via suppression of neutrophil infiltration and IL-1ß production.

The most studied biological role of type III interferons (IFNs) has so far been their antiviral activity, but their role in autoimmune and inflammatory diseases remains largely unexplored. Here, we show that treatment with IFN-λ2/IL-28A completely halts and reverses the development of collagen-induced arthritis (CIA) and discover cellular and molecular mechanisms of IL-28A antiinflammatory function. We demonstrate that treatment with IL-28A dramatically reduces numbers of proinflammatory IL-17-producing Th17 and γδ T cells in the joints and inguinal lymph nodes, without affecting T cell proliferative responses or levels of anticollagen antibodies. IL-28A exerts its antiinflammatory effect by restricting recruitment of IL-1b-expressing neutrophils, which are important for amplification of inflammation. We identify neutrophils as cells expressing high levels of IFN-λ receptor 1 (IFNLR1)-IL-28 receptor α (IL28RA) and targeted by IL-28A. Our data highlight neutrophils as contributors to the pathogenesis of autoimmune arthritis and present IFN-λs or agonists of IFNLR1-IL28RA as putative new therapeutics for neutrophil-driven inflammation.

[Amplification efficency and optimization of culture conditions of γδ T cells in peripheral blood by different phosphate compounds].

OBJECTIVE: To compare the efficiency of pamidronate (PAM) and isopentenyl pyrophosphate (IPP) to stimulate γδ T cell expansion from human peripheral blood and explore the optimized expansion conditions. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque gradient centrifugation, and then cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, IPP (1.0, 5.0, 10.0, 15.0 µg/mL) or PAM (2.0, 5.0, 8.0, 12.0 µg/mL), and IL-2 (100.0, 200.0, 500.0 IU/mL). The cells were observed and collected. The number and proportion of CD3⁺TCRδ2⁺ γδ T cells stimulated by PAM or IPP in total lymphocytes were evaluated by flow cytometry and the expansion efficiency was calculated. RESULTS: After 14 days, the ratios of γδ T cells in total lymphocytes in IPP group and PAM group increased to 81.3% and 78.5%, respectively. This indicated that both IPP and PAM could effectively stimulate γδ T cell expansion and there was no significant difference in the efficiency of expansion between the two groups (P>0.05). CONCLUSION: PAM has the similar ability with IPP to stimulate γδ T cell expansion in vitro. PAM could become more economical and practical choice for stimulating γδ T cell expansion.

Antioxidant and anti-inflammatory activities of extracts from Cassia alata, Eleusine indica, Eremomastax speciosa, Carica papaya and Polyscias fulva medicinal plants collected in Cameroon.

BACKGROUND: The vast majority of the population around the world has always used medicinal plants as first source of health care to fight infectious and non infectious diseases. Most of these medicinal plants may have scientific evidence to be considered in general practice. OBJECTIVE: The aim of this work was to investigate the antioxidant capacities and anti-inflammatory activities of ethanol extracts of leaves of Cassia alata, Eleusine indica, Carica papaya, Eremomastax speciosa and the stem bark of Polyscias fulva, collected in Cameroon. METHODS: Chemiluminescence was used to analyze the antioxidant activities of plant extracts against hydrogen peroxide or superoxide anion. Comet assays were used to analyze the protection against antioxidant-induced DNA damage induced in white blood cells after treating with hydrogen peroxide. Flow cytometry was used to measure γδ T cells proliferation and anti-inflammatory activity of γδ T cells and of immature dendritic cells (imDC) in the presence of different concentrations of plant extracts. RESULTS: Ethanol extracts showed strong antioxidant properties against both hydrogen peroxide and superoxide anion. Cassia alata showed the highest antioxidant activity. The effect of plant extracts on γδ T cells and imDC was evidenced by the dose dependent reduction in TNF-α production in the presence of Cassia alata, Carica papaya, Eremomastax speciosa Eleusine indica, and Polyscias fulva. γδ T cells proliferation was affected to the greatest extent by Polyscias fulva. CONCLUSION: These results clearly show the antioxidant capacity and anti-inflammatory activities of plant extracts collected in Cameroon. These properties of leaves and stem bark extracts may contribute to the value for these plants in traditional medicine and in general medical practice.

Metabolic engineering of Salmonella vaccine bacteria to boost human Vγ2Vδ2 T cell immunity.

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a metabolite in the 2-C-methyl-D-erythritol-4-phosphate pathway used by most eubacteria and apicomplexan parasites, and self isopentenyl pyrophosphate, a metabolite in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with prenyl pyrophosphates or aminobisphosphonates elicit short-term Vγ2Vδ2 expansion with rapid anergy and deletion upon subsequent immunizations. We hypothesized that a live, attenuated bacterial vaccine that overproduces HMBPP would elicit long-lasting Vγ2Vδ2 T cell immunity by mimicking a natural infection. Therefore, we metabolically engineered the avirulent aroA(-) Salmonella enterica serovar Typhimurium SL7207 strain by deleting the gene for LytB (the downstream enzyme from HMBPP) and functionally complementing for this loss with genes encoding mevalonate pathway enzymes. LytB(-) Salmonella SL7207 had high HMBPP levels, infected human cells as efficiently as did the wild-type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells from human donors. Importantly, vaccination of a rhesus monkey with live lytB(-) Salmonella SL7207 stimulated a prolonged expansion of Vγ2Vδ2 T cells without significant side effects or anergy induction. These studies provide proof-of-principle that metabolic engineering can be used to derive live bacterial vaccines that boost Vγ2Vδ2 T cell immunity. Similar engineering of metabolic pathways to produce lipid Ags or B vitamin metabolite Ags could be used to derive live bacterial vaccine for other unconventional T cells that recognize nonpeptide Ags.

Colostral antibody-mediated and cell-mediated immunity contributes to innate and antigen-specific immunity in piglets.

Immunoglobulins and immune cells are critical components of colostral immunity; however, their transfer to and function in the neonate, especially maternal lymphocytes, is unclear. Cell-mediated and antibody-mediated immunity in sow blood and colostrum and piglet blood before (PS) and after (AS) suckling were assessed to investigate transfer and function of maternal immunity in the piglet. CD4, CD8, and γδ lymphocytes were found in sow blood and colostrum and piglet blood PS and AS; each had a unique T lymphocyte profile. Immunoglobulins were detected in sow blood, colostrum, and in piglet blood AS; the immunoglobulin profile of piglet serum AS mimicked that of sow serum. These results suggest selectivity in lymphocyte concentration into colostrum and subsequent lymphocyte transfer into the neonate, but that immunoglobulin transfer is unimpeded. Assessment of colostral natural killer activity and antigen-specific proliferation revealed that colostral cells are capable of influencing the innate and specific immune response of neonatal pigs.

Neutrophil uptake of nitrogen-bisphosphonates leads to the suppression of human peripheral blood γδ T cells.

Nitrogen-bisphosphonates (n-BP), such as zoledronate, are the main class of drugs used for the prevention of osteoporotic fractures and the management of cancer-associated bone disease. However, long-term or high-dose use has been associated with certain adverse drug effects, such as osteonecrosis of the jaw and the loss of peripheral of blood Vγ9Vδ2 T cells, which appear to be linked to drug-induced immune dysfunction. In this report we show that neutrophils present in human peripheral blood readily take up zoledronate, and this phenomenon is associated with the potent immune suppression of human peripheral blood Vγ9Vδ2 T cells. Furthermore, we found this zoledronate-mediated inhibition by neutrophils could be overcome to fully reconstitute Vγ9Vδ2 T cell proliferation by concomitantly targeting neutrophil-derived hydrogen peroxide, serine proteases, and arginase I activity. These findings will enable the development of targeted strategies to mitigate some of the adverse effects of n-BP treatment on immune homeostasis and to improve the success of immunotherapy trials based on harnessing the anticancer potential of peripheral blood γδ T cells in the context of n-BP treatment.

Retinoic acid inhibits CD25+ dendritic cell expansion and γδ T-cell activation in experimental autoimmune uveitis.

PURPOSE: We determined the mechanism by which all-trans retinoic acid (ATRA) inhibits experimental autoimmune uveitis (EAU) and determined the role of γδ T cells in this autoimmune disease. METHODS: C57BL/6 (B6) mice were immunized with the uveitogenic, interphotoreceptor retinoid-binding protein1-20 peptide (IRBP1-20) in complete Freund's adjuvant (CFA), with or without a preceding ATRA treatment. Responses and pathogenic activity of Th1- and Th17-autoreactive T cells were compared, and the effects of ATRA on γδ T cells and CD25(+) dendritic cell (DC) subset were determined. Interactions among uveitogenic T cells, DC subsets, and γδ T cells were investigated. RESULTS: Administration of ATRA to B6 mice in which EAU was induced suppressed the response of Th17 autoreactive T cells, which was associated with decreased generation of the CD25(+) DC subset and suppressed activation of γδ T cells. Adoptively transferred γδ T cells isolated from ATRA-treated mice showed a diminished ability to promote the activation of Th17 autoreactive T cells in vitro and in vivo compared to γδ T cells from untreated donors. CONCLUSIONS: ATRA inhibits the expansion of CD25(+) DCs and γδ T-cell activation, thereby restraining the Th17 autoreactive T-cell response.

Mechanisms of the antitumor activity of human Vγ9Vδ2 T cells in combination with zoledronic acid in a preclinical model of neuroblastoma.

Low expression of surface major histocompatibility complex (MHC) class I molecules and defects in antigen processing machinery make human neuroblastoma (NB) cells appropriate targets for MHC unrestricted immunotherapeutic approaches. Human T-cell receptor (TCR) Vγ9Vδ2 lymphocytes exert MHC-unrestricted antitumor activity and are activated by phosphoantigens, whose expression in cancer cells is increased by aminobisphosphonates. With this background, we have investigated the in vivo anti-NB activity of human Vγ9Vδ2 lymphocytes and zoledronic acid (ZOL). SH-SY-5Y human NB cells were injected in the adrenal gland of immunodeficient mice. After 3 days, mice received ZOL or human Vγ9Vδ2 T cells or both agents by intravenous administration once a week for 4 weeks. A significantly improved overall survival was observed in mice receiving Vγ9Vδ2 T cells in combination with ZOL. Inhibition of tumor cell proliferation, angiogenesis and lymphangiogenesis, and increased tumor cell apoptosis were detected. Vγ9Vδ2 T lymphocytes were attracted to NB-tumor masses of mice receiving ZOL where they actively modified tumor microenvironment by producing interferon-γ (IFN-γ), that in turn induced CXCL10 expression in NB cells. This study shows that human Vγ9Vδ2 T cells and ZOL in combination inhibit NB growth in vivo and may provide the rationale for a phase I clinical trial in patients with high-risk NB.

Supplementation with aged garlic extract improves both NK and γδ-T cell function and reduces the severity of cold and flu symptoms: a randomized, double-blind, placebo-controlled nutrition intervention.

BACKGROUND & AIMS: Earlier studies show that dietary bioactive compounds can modify proliferation of γδ-T cells. Garlic contains numerous compounds that have this potential and, in addition, has been shown to influence NK cell function. Our primary aim was to demonstrate that aged garlic extract could modify these immune cells. METHODS: A randomized, double-blind, placebo-controlled parallel intervention study recruited 120 healthy subjects (60 per group) to determine the effect of aged garlic extract supplementation (2.56 g/d) on immune cell proliferation and cold and flu symptoms. RESULTS: After 45 d of consuming an encapsulated aged garlic extract, γδ-T cells (p = 0.039, n = 56) and NK cells (p = 0.043, n = 56) were shown to proliferate better compared to placebo. After 90 d of supplementation, illness diary entries showed that the incidence of colds and flu, a secondary outcome, were not statistically different; however, the group consuming the aged garlic extract appeared to have reduced severity as noted by a reduction in the number of symptoms reported (21% fewer, p < 0.001, z-test of proportions), a reduction in the number of days (61% fewer, p < 0.001, z-test) and incidences (58% fewer p < 0.001, z-test) where the subjects functioned sub-optimally and the number of work/school days missed due to illness (58% fewer, p = 0.035, z-test). CONCLUSIONS: These results suggest that supplementation of the diet with aged garlic extract may enhance immune cell function and that this may be responsible, in part, for reduced severity of colds and flu.

Prophylactic application of bovine colostrum ameliorates murine colitis via induction of immunoregulatory cells.

Epidemiologic studies suggest a relationship between early weaning and the incidence of inflammatory bowel disease. Herein, we addressed the question whether bovine colostrum, the first milk produced by mammals, is able to prevent dextran sulfate sodium (DSS)-induced colitis in mice. Prior to induction of colitis with 5% DSS, Naval Medical Research Institute mice were fed bovine colostrum [BV-20 or 200 mg/kg body weight (BW)], BSA (20 mg/kg BW), or water (100 µL) daily by oral gavage for 2 wk. The clinical severity of colitis was determined by scoring changes in BW and colon length reduction. Following 2 wk of observation, the colons were removed for histologic and immunohistochemical evaluation of inflammation. Flow cytometric phenotyping of leukocyte subsets was performed from peripheral blood, mesenteric lymph nodes, and spleens. Administration of bovine colostrum improved the clinical and histologic severity of colorectal inflammation. Compared with BSA-fed and water-fed controls, BV-20 pretreated mice had significantly less severe weight loss and decreased colon shortening. Beneficial effects were accompanied by redistribution of immunoregulatory, peripheral and splenic γδ TCR(+) cells, and CD11b(+)Gr1(+) cells. Higher colostrum doses did not affect disease activity. In summary, prophylactic administration of colostrum improved clinical symptoms of colorectal inflammation in a well-established mouse model of DSS-induced colitis. Further investigations will target the underlying immunomodulatory mechanisms to our approach.

In vivo manipulation of Vgamma9Vdelta2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients.

The potent anti-tumour activities of gammadelta T cells have prompted the development of protocols in which gammadelta-agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vgamma9Vdelta2 T cell agonist, plus low-dose interleukin (IL)-2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vgamma9Vdelta2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vgamma9Vdelta2 T cell numbers emerged, as seven patients who failed to sustain Vgamma9Vdelta2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vgamma9Vdelta2 cell populations showed declining CA15-3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vgamma9Vdelta2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low-dose IL-2 offers a novel, safe and feasible approach to enhance this in a subset of treatment-refractory patients with advanced breast cancer.

Interleukin-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates interleukin-17A and RORgammat in gammadelta T cells.

INTRODUCTION: Interleukin (IL)-23 is essential for the development of various experimental autoimmune models. However, the role of IL-23 in non-autoimmune experimental arthritis remains unclear. Here, we examined the role of IL-23 in the non-autoimmune antigen-induced arthritis (AIA) model. In addition, the regulatory potential of IL-23 in IL-17A and retinoic acid-related orphan receptor gamma t (RORgammat) expression in CD4+ and TCRgammadelta+ T cells was evaluated systemically as well as at the site of inflammation. METHODS: Antigen-induced arthritis was induced in wild-type, IL-23p19-deficient and IL-17 Receptor A - knockout mice. At different time points, synovial cytokine and chemokine expression was measured. At days 1 and 7 of AIA, splenocytes and joint-infiltrating cells were isolated and analyzed for intracellular IL-17A and interferon (IFN)-gamma ex-vivo by flow cytometry. In splenic CD4+ and TCRgammadelta+ T cells gene expression was quantified by flow cytometry and quantitative PCR. RESULTS: IL-23 was critical for full-blown AIA. Lack of IL-23 did not prevent the onset of joint inflammation but stopped the progression to a destructive synovitis. IL-23 regulated IL-17A expression in CD4+ T cells in the spleen. Of note, IL-17A and IFN-gamma expression was reduced in CD4+ T cells in the inflamed joints of IL-23p19-deficient mice. Interestingly, IL-23 was also critical for the induction of IL-17A and RORgammat but not IFN-gamma in TCRgammadelta+ T cells in the inflamed joints. The importance of the IL-23/IL-17 axis was further confirmed using IL-17 Receptor A knockout mice showing significantly milder AIA compared to control mice, with a disease course comparable to that of IL-23p19-deficient mice. CONCLUSIONS: These data show that IL-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates the proportion of IL-17A and IFN-gamma-positive CD4+ T cells at the site of inflammation. Furthermore, IL-23 regulates IL-17A and RORgammat expression in TCRgammadelta T cells in arthritis. These findings indicate that regulating the IL-23 pathway may have therapeutic potential in non-autoimmune arthritis.

Select plant tannins induce IL-2Ralpha up-regulation and augment cell division in gammadelta T cells.

Gammadelta T cells are innate immune cells that participate in host responses against many pathogens and cancers. Recently, phosphoantigen-based drugs, capable of expanding gammadelta T cells in vivo, entered clinical trials with the goal of enhancing innate immune system functions. Potential shortcomings of these drugs include the induction of nonresponsiveness upon repeated use and the expansion of only the Vdelta2 subset of human gammadelta T cells. Vdelta1 T cells, the major tissue subset, are unaffected by phosphoantigen agonists. Using FACS-based assays, we screened primary bovine cells for novel gammadelta T cell agonists with activities not encompassed by the current treatments in an effort to realize the full therapeutic potential of gammadelta T cells. We identified gammadelta T cell agonists derived from the condensed tannin fractions of Uncaria tomentosa (Cat's Claw) and Malus domestica (apple). Based on superior potency, the apple extract was selected for detailed analyses on human cells. The apple extract was a potent agonist for both human Vdelta1 and Vdelta2 T cells and NK cells. Additionally, the extract greatly enhanced phosphoantigen-induced gammadelta T cell expansion. Our analyses suggest that a tannin-based drug may complement the phosphoantigen-based drugs, thereby enhancing the therapeutic potential of gammadelta T cells.

Bacillus Calmette-Guérin-pulsed dendritic cells stimulate natural killer T cells and gammadeltaT cells.

BACKGROUND: Immunotherapy with bacillus Calmette-Guérin (BCG) for bladder cancer is successful, although the precise mechanism is unclear. Natural killer (NK) cells are a candidate for BCG-activated killer cells, but the roles of other T lymphocytes, such as NKT cells and gammadeltaT cells, are not fully understood. Mycobacterium tuberculosis is a potent activator of both NKT cells and gammadeltaT cells. However, it is known that the patient's prognosis is good if there are increased numbers of dendritic cells (DCs) in the urine after BCG therapy. Therefore, we investigated whether DCs are matured by BCG and whether BCG-pulsed DCs stimulate NKT cells and gammadeltaT cells. METHODS: Naïve Pan T cells were isolated form peripheral blood mononuclear cells (PBMCs) and DCs were obtained by culturing CD14(+) monocytes with granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs were pulsed with BCG and their maturation was measured by fluorescence-activated cell sorter analysis using the CD86 antibody. Naïve T lymphocytes were stimulated by coculture with BCG-pulsed DCs in vitro, followed by FACS analysis to estimate the ratio and activation of NKT cells and the ratio of gammadeltaT cells. The (51)Cr (chromium) release assay was used to estimate the cytotoxic activity of the stimulated T cells. Cytolytic proteins in the patient's PBMCs were measured during BCG therapy using semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: The DCs were matured by BCG stimulation and the number of NKT cells and gammadeltaT cells increased after culturing with BCG-pulsed DCs. The BCG-pulsed DCs also activated the NKT cells and gammadeltaT cells. Also, the lymphocytes that were cocultured with the BCG-pulsed DCs showed unspecific cytotoxic activity against a bladder cancer cell line. CONCLUSION: Sensitization of NKT cells and gammadeltaT cells by BCG-pulsed DCs might be one of the mechanisms of BCG immunotherapy.

Recognition of pollen-derived phosphatidyl-ethanolamine by human CD1d-restricted gamma delta T cells.

BACKGROUND: Evidences from mice and human beings indicate that gammadelta T cells could be relevant in recognition of stress-induced self and/or yet unidentified inhaled foreign antigens. Their specificity differs from classic MHC-restricted alphabeta T cells and involves the immunoglobulin-like structure of the gammadelta T-cell receptor with the recognition of small organic molecules, alkylamines, and self lipid compounds presented by CD1+ dendritic cells. OBJECTIVE: Because CD1 receptors are mainly devoted to lipid antigen presentation, we sought to determine whether exogenous pollen membrane lipids may act as allergens for CD1-restricted gammadelta T cells. METHODS: Peripheral blood and nasal mucosa-associated gammadelta T cells were cloned from normal controls and cypress-sensitive subjects and tested for their antigen specificity and CD1-restriction with phospholipids extracted from tree pollen grains, as well with other natural or synthetic compounds. Phospholipid reactivity of cloned gammadelta T cells was measured by mean of proliferative response and cytokine release as well as by testing their helper activity on IgE production in vitro and in vivo. RESULTS: Cloned gammadelta T lymphocytes from subjects with allergy, but not normal controls, were found to recognize pollen-derived phosphatidyl-ethanolamine (PE) in a CD1d-restricted fashion. Only 16:0/18:2 and 18:2/18:2 PE were stimulatory, whereas no response was recorded for disaturated PE, phosphatidylcholine, neutral lipids, or protein extract. Proliferating clones secreted both T(H)1-type and T(H)2-type cytokines and drove IgE production in vitro and in vivo. CONCLUSION: CD1d-restricted gammadelta T cells specific for phospholipids can represent a key mucosal regulatory subset for the control of early host reactivity against tree pollens. CLINICAL IMPLICATIONS: By knowing how lipid allergen constituents interact with mucosal immune system, we can expand our possibilities in diagnostic and therapeutic interventions.

Potentiation of antigen-stimulated V gamma 9V delta 2 T cell cytokine production by immature dendritic cells (DC) and reciprocal effect on DC maturation.

Vgamma9Vdelta2 T cells, a major gammadelta PBL subset in human adults, have been previously implicated in dendritic cell (DC) licensing, owing to their high frequency in peripheral tissues and their ability to produce inflammatory cytokines upon recognition of a broad array of conserved Ags. Although these observations implied efficient recognition of Ag-expressing immature DC (iDC) by Vgamma9Vdelta2 T cells, the role played by DC subsets in activation of these lymphocytes has not been carefully studied so far. We show that iDC, and to a lesser extent mature DC, potentiated Th1 and Th2 cytokine, but not cytolytic or proliferative responses, of established Vgamma9Vdelta2 T cell clones and ex vivo memory Vgamma9Vdelta2 PBL stimulated by synthetic agonists. The ability of iDC to potentiate Vgamma9Vdelta2 production of inflammatory cytokines required for their own maturation suggested that Vgamma9Vdelta2 T cells, despite their strong lytic activity, could promote efficient iDC licensing without killing at suboptimal Ag doses. Accordingly Vgamma9Vdelta2 cells induced accelerated maturation of Ag-expressing iDC but not "bystander" DC, even within mixed cell populations comprising both Ag-expressing and nonexpressing iDC. Furthermore Vgamma9Vdelta2 cells induced full differentiation into IL-12-producing cells of iDC infected by Vgamma9Vdelta2-stimulating mycobacteria that were otherwise unable to induce complete DC maturation. In conclusion the ability of iDC to selectively potentiate cytokine response of memory Vgamma9Vdelta2 T cells could underlie the adjuvant effect of these lymphocytes, and possibly other natural memory T cells, on conventional T cell responses.