Essential role for both CD80 and CD86 costimulation, but not CD40 interactions, in allergen-induced Th2 cytokine production from asthmatic bronchial tissue: role for alphabeta, but not gammadelta, T cells

CD80 and CD86 interact with CD28 and deliver costimulatory signals required for T cell activation. We demonstrate that ex vivo allergen stimulation of bronchial biopsy tissue from mild atopic asthmatic, but not atopic nonasthmatic, subjects induced production of IL-5, IL-4, and IL-13. Explants from both study groups did not produce IFN-gamma, but secreted the chemokine RANTES without any overt stimulation. In addition to allergen, stimulation of asthmatic explants with mAbs to CD3 and TCR-alphabeta but not TCR-gammadelta induced IL-5 secretion. Allergen-induced IL-5 and IL-13 production by the asthmatic tissue was inhibited by anti-CD80 and, to a lesser extent, by anti-CD86 mAbs. In contrast, the production of these cytokines by PBMCs was not affected by mAbs to CD80, was inhibited by anti-CD86, and was strongly attenuated in the presence of both Abs. FACS analysis revealed that stimulated asthmatic bronchial tissue was comprised of CD4+ T cells that expressed surface CD28 (75. 3%) but little CTLA-4 (4.0%). Neutralizing mAbs to CD40 ligand had no effect on the cytokine levels produced by asthmatic tissue or PBMCs. Collectively, these findings suggest that allergen-specific alphabeta T cells are resident in asthmatic bronchial tissue and demonstrate that costimulation by both CD80 and CD86 is essential for allergen-induced cytokine production. In contrast, CD86 appears to be the principal costimulatory molecule required in PBMC responses. Attenuation of type 2 alphabeta T cell responses in the bronchial mucosa by blocking these costimulatory molecules may be of therapeutic potential in asthma.     

Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.     

IL-4 increases type 2, but not type 1, cytokine production in CD8+ T cells from mild atopic asthmatics

Background

Virus infections are the major cause of asthma exacerbations. CD8⁺ T cells have an important role in antiviral immune responses and animal studies suggest a role for CD8⁺ T cells in the pathogenesis of virus-induced asthma exacerbations. We have previously shown that the presence of IL-4 during stimulation increases the frequency of IL-5-positive cells and CD30 surface staining in CD8⁺ T cells from healthy, normal subjects. In this study, we investigated whether excess IL-4 during repeated TCR/CD3 stimulation of CD8⁺ T cells from atopic asthmatic subjects alters the balance of type 1/type 2 cytokine production in favour of the latter.

Methods

Peripheral blood CD8⁺ T cells from mild atopic asthmatic subjects were stimulated in vitro with anti-CD3 and IL-2 ± excess IL-4 and the expression of activation and adhesion molecules and type 1 and type 2 cytokine production were assessed.

Results

Surface expression of very late antigen-4 [VLA-4] and LFA-1 was decreased and the production of the type 2 cytokines IL-5 and IL-13 was augmented by the presence of IL-4 during stimulation of CD8⁺ T cells from mild atopic asthmatics.

Conclusion

These data suggest that during a respiratory virus infection activated CD8⁺ T cells from asthmatic subjects may produce excess type 2 cytokines and may contribute to asthma exacerbation by augmenting allergic inflammation.     

Donor lymphocyte infusion induces long-term donor-specific cardiac xenograft survival through activation of recipient double-negative regulatory T cells

Previous studies have shown that pretransplant donor lymphocyte infusion (DLI) can enhance xenograft survival. However, the mechanism by which DLI induces xenograft survival remains obscure. Using T cell subset-deficient mice as recipients we show that CD4+, but not CD8+, T cells are necessary to mediate the rejection of concordant cardiac xenografts. Adoptive transfer of naive CD4+ T cells induces rejection of accepted cardiac xenografts in CD4-/- mice. This rejection can be prevented by pretransplant DLI in the absence of any other treatment. Furthermore, we demonstrate that DLI activates alphabeta-TCR+CD3+CD4-CD8- double-negative (DN) regulatory T (Treg) cells in xenograft recipients, and that DLI-activated DN Treg cells can inhibit the proliferation of donor-specific xenoreactive CD4+ T cells in vitro. More importantly, adoptive transfer of DLI-activated DN Treg cells from xenograft recipients can suppress the proliferation of xenoreactive CD4+ T cells and their ability to produce IL-2 and IFN-gamma in vivo. Adoptive transfer of DLI-activated DN Treg cells also prevents CD4+ T cell-mediated cardiac xenograft rejection in an Ag-specific fashion. These data provide direct evidence that DLI can activate recipient DN Treg cells, which can induce donor-specific long-term cardiac xenograft survival by suppressing the proliferation and function of donor-specific CD4+ T cells in vivo.     

IL-7 is required for the development of the intrinsic function of marginal zone B cells and the marginal zone microenvironment

The characteristic microarchitecture of the marginal zone (MZ), formed by locally interacting MZ-specific B cells, macrophages, and endothelial cells, is critical for productive marginal zone B cell (MZB cell) Ab responses. Reportedly, IL-7-deficient mice, although severely lymphopenic, retain small numbers of CD21(high)CD23(low) B cells consistent with MZB cell phenotype, suggesting that IL-7 signaling is not exclusively required for MZB cell lymphopoiesis. In this study, we investigated the function of IL-7(-/-) MZB cells and the IL-7(-/-) microenvironment using a model of hamster heart xenograft rejection, which depends exclusively on MZB cell-mediated production of T cell-independent IgM xenoantibodies (IgMXAb). C57BL/6-IL-7(-/-) mice accepted xenografts indefinitely and failed to produce IgMXAb, even after transfer of additional IL-7(-/-) or wild-type C57BL/6 MZB cells. Transfer of wild-type but not IL-7(-/-) B cells enabled SCID mice to produce IgMXAb. When transferred to SCID mice, wild-type but not IL-7(-/-) B cells formed B cell follicles with clearly defined IgM(+), MOMA-1(+), and MAdCAM-1(+) MZ structures. Conversely, adoptively transferred GFP(+) C57BL/6 B cells homed to the MZ area in a SCID but not an IL-7(-/-) environment. Naive IL-7(-/-) mice showed absent or aberrant splenic B cell structures. We provide evidence that IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against T cell-independent type II Ags, for their homing potential, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.     

Asthma and gender impact accumulation of T cell subtypes

Background

The "Th2 hypothesis for asthma" asserts that an increased ratio of Th2:Th1 cytokine production plays an important pathogenic role in asthma. Although widely embraced, the hypothesis has been challenged by various empirical observations and has been described as overly simplistic. We sought to establish whether CD3+CD28-mediated and antigen-independent accumulation of type 1 and type 2 T cells differs significantly between nonasthmatic and asthmatic populations.

Methods

An ex vivo system was used to characterize the regulation of IFN-γ-producing (type 1) and IL-13-producing (type 2) T cell accumulation in response to CD3+CD28 and IL-2 stimulation by flow cytometry.

Results

IL-13-producing T cells increased in greater numbers in response to antigen-independent stimulation in peripheral blood lymphocytes from female atopic asthmatic subjects compared with male asthmatics and both male and female atopic non-asthmatic subjects. IFN-γ⁺ T cells increased in greater numbers in response to either antigen-independent or CD3+CD28-mediated stimulation in peripheral blood lymphocytes from atopic asthmatic subjects compared to non-asthmatic subjects, regardless of gender.

Conclusions

We demonstrate that T cells from asthmatics are programmed for increased accumulation of both type 2 and type 1 T cells. Gender had a profound effect on the regulation of type 2 T cells, thus providing a mechanism for the higher frequency of adult asthma in females.     

V alpha 24-invariant NKT cells from patients with allergic asthma express CCR9 at high frequency and induce Th2 bias of CD3+ T cells upon CD226 engagement

We have demonstrated that Valpha24(+)Vbeta11(+) invariant (Valpha24(+)i) NKT cells from patients with allergic asthma express CCR9 at high frequency. CCR9 ligand CCL25 induces chemotaxis of asthmatic Valpha24(+)i NKT cells but not the normal cells. A large number of CCR9-positive Valpha24(+)i NKT cells are found in asthmatic bronchi mucosa, where high levels of Th2 cytokines are detected. Asthmatic Valpha24(+)i NKT cells, themselves Th1 biased, induce CD3(+) T cells into an expression of Th2 cytokines (IL-4 and IL-13) in cell-cell contact manner in vitro. CD226 are overexpressed on asthmatic Valpha24(+)i NKT cells. CCL25/CCR9 ligation causes directly phosphorylation of CD226, indicating that CCL25/CCR9 signals can cross-talk with CD226 signals to activate Valpha24(+)i NKT cells. Prestimulation with immobilized CD226 mAb does not change ability of asthmatic Valpha24(+)i NKT cells to induce Th2-cytokine production, whereas soluble CD226 mAb or short hairpin RNA of CD226 inhibits Valpha24(+)i NKT cells to induce Th2-cytokine production by CD3(+) T cells, indicating that CD226 engagement is necessary for Valpha24(+)i NKT cells to induce Th2 bias of CD3(+) T cells. Our results are providing with direct evidence that aberration of CCR9 expression on asthmatic Valpha24(+)i NKT cells. CCL25 is first time shown promoting the recruitment of CCR9-expressing Valpha24(+)i NKT cells into the lung to promote other T cells to produce Th2 cytokines to establish and develop allergic asthma. Our findings provide evidence that abnormal asthmatic Valpha24(+)i NKT cells induce systemically and locally a Th2 bias in T cells that is at least partially critical for the pathogenesis of allergic asthma.     

The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells

Susceptibility and development of Th2 cells in BALB/c mice infected with Leishmania major result from early IL-4 production by Vbeta4Valpha8 CD4+ T cells in response to the Leishmania homolog of mammalian RACK1 Ag. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. We further showed that transfer of 10(7) BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 10(7) control BALB/c spleen cells were resistant to infection with L. major and developed a Th1 response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.     

Colitis-inducing potency of CD4+ T cells in immunodeficient, adoptive hosts depends on their state of activation, IL-12 responsiveness, and CD45RB surface phenotype

We studied the induction, severity and rate of progression of inflammatory bowel disease (IBD) induced in SCID mice by the adoptive transfer of low numbers of the following purified BALB/c CD4+ T cell subsets: 1) unfractionated, peripheral, small (resting), or large (activated) CD4+ T cells; 2) fractionated, peripheral, small, or large, CD45RBhigh or CD45RBlow CD4+ T cells; and 3) peripheral IL-12-unresponsive CD4+ T cells from STAT-4-deficient mice. The adoptive transfer into SCID host of comparable numbers of CD4+ T cells was used to assess the colitis-inducing potency of these subsets. Small CD45RBhigh CD4+ T lymphocytes and activated CD4+ T blasts induced early (6-12 wk posttransfer) and severe disease, while small resting and unfractionated CD4+ T cells or CD45RBlow T lymphocytes induced a late-onset disease 12-16 wk posttransfer. SCID mice transplanted with STAT-4-/- CD4+ T cells showed a late-onset IBD manifest > 20 wk posttransfer. In SCID mice with IBD transplanted with IL-12-responsive CD4+ T cells, the colonic lamina propria CD4+ T cells showed a mucosa-seeking memory/effector CD45RBlow Th1 phenotype abundantly producing IFN-gamma and TNF-alpha. In SCID mice transplanted with IL-12-unresponsive STAT-4-/- CD4+ T cells, the colonic lamina propria, mesenteric lymph node, and splenic CD4+ T cells produced very little IFN-gamma but abundant levels of TNF-alpha. The histopathologic appearance of colitis in all transplanted SCID mice was similar. These data indicate that CD45RBhigh and CD45RBlow, IL-12-responsive and IL-12-unresponsive CD4+ T lymphocytes and lymphoblasts have IBD-inducing potential though of varying potency.     

Monocyte-derived dendritic cells generated after a short-term culture with IFN-alpha and granulocyte-macrophage colony-stimulating factor stimulate a potent Epstein-Barr virus-specific CD8+ T cell response

Cellular immune responses are crucial for the control of EBV-associated lymphoproliferative diseases. To induce an anti-EBV cell-mediated immunity, we have used dendritic cells (DCs) generated by a 3-day culture of human CD14(+) monocytes in the presence of GM-CSF and type I IFN (IFN-DCs) and pulsed with peptides corresponding to CTL EBV epitopes. The functional activity of IFN-DCs was compared with that of APCs differentiated by culturing monocytes for 3 days with GM-CSF and IL-4 and indicated as IL-4-DCs. Stimulation of PBLs from EBV-seropositive donors with EBV peptide-pulsed autologous IFN-DCs resulted in a stronger expansion of specific T lymphocytes producing IFN-gamma with respect to stimulation with peptide-loaded IL-4-DCs, as assessed by ELISPOT assays. When purified CD8(+) T cells were cocultured with EBV peptide-pulsed IFN-DCs or IL-4-DCs, significantly higher levels of specific cytotoxic activity were observed in CD8(+) T cell cultures stimulated with IFN-DCs. Injection of peptide-pulsed IFN-DCs into SCID mice transplanted with autologous PBLs led to the recovery of a significantly greater number of EBV-specific human CD8(+) T cells from the spleen and the peritoneal cavity with respect to that recovered from mice injected with peptide-pulsed IL-4-DCs. Moreover, a significant delay in lymphoma development was observed when peptide-pulsed IFN-DCs were injected into SCID mice reconstituted with PBMCs endowed with a high capability of lymphoma induction, whereas injection of unpulsed IFN-DCs was ineffective. Our results indicate that IFN-DCs efficiently promote in vitro and in vivo the expansion of CD8(+) T lymphocytes acting as cytotoxic effectors against EBV-transformed cells.     

Effects of beta-arrestin 2 on cytokine production of CD4+ T lymphocytes of mice with allergic asthma

Beta-arrestin 2 has been shown to participate in the pathogenesis of asthma by inducing Th2 cell migration to the lungs. Whether beta-arrestin 2 regulates cytokine production of CD4+ T cells is still unknown. The aim of the present study was to investigate the effect of beta-arrestin 2 on the cytokine production of CD4+ T lymphocytes and the mechanism involved in a mouse model for asthma. After silencing beta-arrestin 2 expression in CD4+ T lymphocytes from asthmatic mice by RNA interference (RNAi), the interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) levels in CD4+ T lymphocyte culture supernatants with or without terbutaline stimulation were determined. Cell-surface beta2 adrenergic receptor (beta2AR) as well as GATA3 expression of CD4+ T lymphocytes were also measured. CD4+ T lymphocytes of mice with allergic asthma expressed higher levels of beta-arrestin 2 on both mRNA and protein levels. beta-arrestin 2 RNAi decreased IL-4 (43.16%) and GATA3 (protein 77.21%, mRNA 62.98%) expression after terbutaline stimulation. Cell-surface beta2AR of CD4+ T lymphocytes decreased (15.27%) after terbutaline treatment, but recovered after beta-arrestin 2 RNAi down-modulation. These findings demonstrate that beta-arrestin 2 regulates IL-4 production and GATA3 expression of CD4+ T lymphocytes partly through the beta2AR signaling pathway in an allergic asthma model.     

Role of innate and adaptive immunity in the outcome of primary infection with Chlamydia pneumoniae, as analyzed in genetically modified mice

Infection with Chlamydia pneumoniae is a common cause of acute respiratory disease in man and is also associated with atherosclerotic cardiovascular disorder. Herein, we have compared bacterial load and immune parameters of C. pneumoniae-infected mice genomically lacking T cell coreceptors, cytokine receptors, or cytotoxic effector molecules. A protective role for CD8+ cells is shown by the enhanced severity of infection of CD8-/- or TAP-1-/-/beta2-microglobulin -/- mice. CD8+ cells hindered a parasite growth-promoting role of CD4+ T cells, as indicated by the higher sensitivity to early infection of CD8-/- than CD4-/-/CD8-/- mice, which was further confirmed in experiments in which SCID mice were reconstituted with either CD4+ or CD4+ plus CD8+ T cells. Interestingly, CD4+ T cells played a dual role, detrimental early (14 and 24 days) after infection but protective at later time points (60 days after infection). The CD8+ T cell protection was perforin independent. The early deleterious role of CD4+ in the absence of CD8+ T cells was associated with enhanced IL-4 and IL-10 mRNA levels and delayed IFN-gamma mRNA accumulation in lungs. In line with this, IFN-gammaR-/- (but not TNFRp55 -/-) mice showed dramatically increased susceptibility to C. pneumoniae, linked to reduced inducible nitric oxide synthase (iNOS) mRNA accumulation, but not to diminished levels of specific Abs. The increased susceptibility of iNOS-/- mice indicates a protective role for iNOS activity during infection with C. pneumoniae. The higher sensitivity of IFN-gammaR-/- mice to C. pneumoniae compared with that of SCID or recombination-activating gene-1-/- mice suggested a relevant protective role of IFN-gamma-dependent innate mechanisms of protection.     

T cell subsets and their soluble products regulate eosinophilia in allergic and nonallergic asthma.

Lymphokines derived from activated T cells regulate the proliferation and postmitotic differentiation of eosinophils in vitro. We investigated whether peripheral blood eosinophilia, which is a characteristic feature of both allergic and nonallergic asthma, correlates with T cell activation and lymphokine production in asthmatic patients. Flow cytometric analysis of T cell activation markers revealed that asthmatic individuals are characterized by increased numbers of IL-2R (CD25)-bearing T cell subsets. The absolute number of IL-2R+ T cells correlated with the eosinophilia observed in the asthmatic patients. Purified CD4+ and CD8+ T cells from allergic and nonallergic asthmatic individuals spontaneously secreted factors that extend the lifespan of eosinophils in vitro. T cells from normal donors displayed this effect only after polyclonal stimulation with anti-CD3 antibody. The eosinophil lifespan-extending factors were also found in sera of asthmatic patients. Identification of these factors was performed by using neutralizing antibodies against IL-3, IL-5, and granulocyte-macrophage CSF. In sera, mainly IL-5 and granulocyte-macrophage CSF were responsible for prolonged eosinophil survival, whereas granulocyte-macrophage CSF was dominant in T cell supernatants. These results indicate that T cells and secretion of lymphokines play an important regulatory function toward eosinophils, which are thought to represent major proinflammatory effector cells in certain types of asthma.     

The mechanism of a defective IFN-gamma response to bacterial toxins in an atopic dermatitis model, NC/Nga mice, and the therapeutic effect of IFN-gamma, IL-12, or IL-18 on dermatitis

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-gamma levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-gamma response to staphylococcal enterotoxin B was correlated to the lack of regular Vbeta8(+) T cells and Vbeta8(+) NK T cells, and the low IFN-gamma response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-gamma, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.     

Blocking IL-15 prevents the induction of allergen-specific T cells and allergic inflammation in vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.     

Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-gamma. Serum levels of human IFN-gamma in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-gamma abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-gamma and IFN-gamma-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-gamma production, and the associated changes in gene expression are all dependent upon CD4+ T cells.     

Human CD4+ T cells mediate rejection of porcine xenografts

It has previously been demonstrated that xenograft rejection in rodents is dependent on CD4+ T cells. However, because of the lack of an appropriate in vivo model, little is known about the cellular basis of human T cell-mediated rejection of xenografts. In this study, we have evaluated the ability of human T cells to mediate rejection of porcine skin grafts in a novel in vivo experimental system using immunodeficient mice as recipients. Recombinase-activating gene-1-deficient mice (R-) lacking mature B and T cells were grafted with porcine skin and received human lymphocytes stimulated in vitro with irradiated porcine PBMC. Skin grafts on mice given either unseparated, activated human lymphocytes, or NK cell-depleted lymphocyte populations were rejected within 18 days after adoptive cell transfer. In contrast, skin grafts on mice given T cell-depleted human lymphocytes or saline showed no gross or histologic evidence of rejection up to 100 days after adoptive transfer. Purified CD4+ T cells were also able to mediate rejection of porcine skin grafts. These data suggest that human CD4+ T cells are sufficient to induce rejection of porcine xenografts. Thus, strategies directed toward CD4+ T cells may effectively prevent cellular rejection of porcine xenografts in humans.     

Anti-inflammatory non-steroidal drug able to modulate IL-10 in allergic asthma

Our studies target alternative/adjuvant therapies in allergic diseases, able to qualitatively/quantitatively modify cytokine profiles produced by both CD4+ T-cell subsets (mainly Th1 and Th2) and B-cells, macrophages, etc. Current investigations aim to identify compounds capable to down-regulate IL-10 as an exponent of Th2 cell function and, consequently, to up-regulate Th1 cytokine levels. Experiments on ten allergic asthmatic patients and ten healthy subjects as control were performed. Cytokine production, triggered in PBMCs culture systems by PHA, was modulated with Indomethacin, a non-steroidal anti-inflammatory drug and IL-10 was measured in 24 hours culture supernatants. According to our experimental data, IL-10 level of asthmatic patients' PBMCs in the resting state is not significantly different from control. PHA-activated PBMCs from asthmatic patients do not display significantly higher IL-10 levels than the normal subjects. The results obtained up-to-date reveal the fact that Indomethacin strongly down-regulates IL-10 levels in PBMCs cultures, in both asthmatic allergic patients and healthy subjects. It is obvious that the inhibitory effect of Indomethacin on IL-10 released by PBMCs is higher in the case of allergic asthmatic patients. The results obtained in this study demonstrate that Indomethacin is a possible therapeutic candidate in allergic asthma.     

Pulmonary CCL18 recruits human regulatory T cells

CCL18 is both a constitutively expressed and an inducible chemokine, whose role in the inflammatory reaction is poorly known. The aim of this study was to evaluate whether CCL18 has the capacity to attract human T cells with a regulatory function (regulatory T cells [Treg]). Results from chemotaxis assays performed on different types of Treg showed that CD4(+)CD25(+)CD127(low) cells, but neither T regulatory type 1 clones nor Treg differentiated in vitro with anti-CD3/CD46 mAbs, were recruited by CCL18 in a dose-dependent manner. CCL18-recruited memory CD4(+) T cells were enriched in CD25(high), CD25(+)CD127(low), latency-associated peptide/TGF-β1, and CCR4-expressing T cells, whereas there was no enrichment in Foxp3(+) cells as compared with controls. Stimulated CCL18-recruited memory T cells produced significantly increased amounts of the regulatory cytokines IL-10 and TGF-β1, as well as IL-4, but not IFN-γ and IL-17. Cell surface CCL18 binding was found predominantly on IL-10(+) (26.3 ± 5.8%) and on a few latency-associated peptide/TGF-β1(+) (18.1 ± 1.9%) and IL-4(+) (14.5 ± 2.9%) memory T cells. In an in vivo model of SCID mice grafted with human skin and reconstituted with autologous PBMCs, the intradermal injection of CCL18 led to the cutaneous recruitment of CD4(+), CD25(+), and IL-10(+) cells, but not Foxp3(+) cells. Furthermore, CCL18-recruited memory T cells inhibited the proliferation of CD4(+)CD25(-) effector T cells through an IL-10-dependent mechanism. These data suggest that CCL18 may contribute to maintaining tolerance and/or suppressing deleterious inflammation by attracting memory Tregs into tissues, particularly in the lung, where it is highly and constitutively expressed.