Th2-type Cytokine Responses Research Articles

The phenotype and survival of antigen-stimulated transgenic CD4 T cells in vivo: the influence of persisting antigen

Naive and primed/memory CD4 T cells are distinguished by changes in the expression of activation/adhesion molecules that correspond with an altered function. Adoptively transferred TCR transgenic (tg) CD4 T cells specific for ovalbumin peptide (OVA-pep) were analysed for changing phenotype and the speed of change in vivo following antigen challenge with alum-precipitated (ap) OVA-pep, a conjugate that stimulated a Th2-type cytokine response. The change of CD45RB in relation to number of divisions showed that the transition from CD45RB(hi) (naive) to CD45RB(low) (primed/memory) was incremental; with each cell cycle the number of CD45RB(hi) molecules on the cell surface was diluted by approximately half and replaced by the low-weight isoform. Similarly, the change to CD44(hi) expression increased gradually during four rounds of proliferation. The loss of CD62L expression occurred early and was independent of cell division. CD69 was up-regulated quickly within 1-2 cycles, but down-regulated after about seven divisions. The expression of CD49d was not altered during the early rounds of division, although it was up-regulated on 30-60% of tg T cells dividing repeatedly (>or=8 cycles). When analysed on day 3 following stimulation, CD25 was no longer up-regulated. The intra-peritoneal injection of ap-OVA-pep stimulated tg T cells in the spleen and mesenteric lymph node one day in advance of those in more distant peripheral lymph nodes. Evidence indicated that residual antigen persisted for at least 4 weeks and was able to stimulate naive tg T cells. However, residual antigen had no net effect on extending or reducing survival of the transferred population.

Combination of Nasal Lymphotactin and Macrophage Chemoattractant Protein-1 Expressing Plasmids Elicits Antigen-Specific Salivary S-IgA Antibody Immunity

It has been shown that mucosal application of chemokines with protein antigen (Ag) successfully induces mucosal and systemic antibody (Ab) responses, as well as CD4+ T cell responses. We examined whether plasmids expressing lymphotaction (LTN) and/or macrophage chemoattractant proteine-1 (MCP-1) cDNA (pLTN and pMCP-1) exhibit nasal adjuvanticity when administered with protein Ag. When either pLTN or pMCP-1 was employed as a nasal adjuvant in mice, OVA-specific IgG Ab responses were seen in plasma ; however, both failed to induce OVA-specific secretory IgA (S-IgA) Abs in external secretions except anti-OVA IgG and IgA Abs in nasal washes of mice given pLTN. However, mice nasally immunized with a combination of pLTN and pMCP-1 showed OVA-specific S-IgA Ab responses in both saliva and fecal extracts. Further, that combination of chemokines used as a nasal adjuvant induced the highest levels of CD4+ T cell proliferative and Th2-type cytokine responses in cervical lymph nodes (CLN), as compared with a single chemokine nasal administration. In addition, a significantly increased number of CD11c+ dendritic cells (DCs) which expressed co-stimulatory molecules such as MHC II, CD40 and CD86 noted in the nasopharyngeal-associated lymphoreticular tissue (NALT) of mice given pLTN and pMCP-1. These results show that a combination of pLTN and pMCP-1 given as a nasal adjuvant induces optimal mucosal S-IgA Ab responses in submandibular glands, which are mediated by increased number of DCs in the NALT and Th2-type CD4+ T cells in the CLN.

Glutamine supplementation influences immune development in the newly weaned piglet

A study was conducted to determine changes that occur in immune function during the early post-weaning period and the effect of supplementing glutamine (gln, 4% w/w) to the weaning diet of piglets. Dutch-Landrace piglets ( n=10/group) were killed prior to weaning (21 d) or randomized to one of two nutritionally complete weaning diets with or without gln. With age there was an increased ability of peripheral blood mononuclear cells (PBMC) and mesenteric lymph nodes (MLN) cells to proliferate (rate of 3H-thymidine uptake) to T cell mitogens ( P < 0.05 ). PBMC from older piglets produced less of a Th-1 type response after stimulation ( P < 0.05 ). Adding gln to the weaning diet significantly ( P < 0.05 ) modified immune cells in the MLN, in a potentially beneficial manner (with respect to mucosal infections) by preventing an increase in antigen naïve CD4+ cells, increasing the proliferative response to pokeweed mitogen and supporting a Th-1 type cytokine response after T cell (phytohemagglutinin) stimulation.

Predominant T helper type 2‐inflammatory responses promote murine colon cancers

Colon cancer is one of the most serious complications of inflammatory bowel diseases, especially ulcerative colitis (UC). Previous studies have shown that characteristic immunological event during inflammation in UC is the expression of T helper-type 2 (Th2) cell-derived cytokines. In this study, we investigated the influence of a predominant Th2-type cytokine response in colitis on carcinogen-induced colon tumors. Wild type (WT), interferon gamma (IFN-gamma) gene deficient (-/-) [Th2 dominant] or interleukin (IL)-4(-/-) [Th1-dominant] mice of BALB/c background were used in this study. To compare tumor formation, mice were given the carcinogen azoxymethane (AOM) and intrarectal administration of trinitrobenzene sulfonic acid (TNBS), to induce colitis. Thirty-three weeks after initial treatment, the total colon was examined. When IFN-gamma(-/-) mice were treated with AOM and TNBS, significantly higher number of tumors were seen (8.4 +/- 1.7) than in WT (3.3 +/- 2.9) or IL-4(-/-) (3.1 +/- 3.4) mice, which received identical treatments. A separate set of experiment, using less doses of AOM and TNBS also showed the higher frequency of tumor formation in IFN-gamma(-/-) mice than in IL-4(-/-) mice. Histologically, the tumors were well- or moderately-differentiated adenocarcinomas. No invasion into the submucosal or serosal layers of the intestine was seen. In immunohistological staining, some tumors in IFN-gamma(-/-) mice showed distinct nuclear expression of beta-catenin, in contrast to the strong membrane staining seen in tumors of IL-4(-/-) mice. In conclusion, colonic inflammation associated with Th2-dominant cytokine responses enhanced the formation of malignant neoplasms.

Downregulation of Interleukin-13 by PUVA Treatment in a Human Model of Graft-Versus-Host Disease May Highlight the Importance of This Cytokine in the Mechanism of Extracorporeal Phototherapy Treatment.

Graft-versus-Host disease (GvHD) remains the most serious complication following haemopoietic stem cell transplantation, with an incidence of 40–60% and can be fatal in up to 50% of cases. Extracorporeal phototherapy (ECP) is a novel treatment of both acute and chronic GvHD involving psoralen and UVA (PUVA) treatment of peripheral blood cells with high reported success even in those resistant to conventional immunosuppressive treatments. ECP appears to induce selective immune suppression without increased rates of infection or disease relapse. It is well tolerated by patients with minimal side effects and therefore is emerging as a highly desirable therapy. However, its mechanism of action remains poorly understood. An in vitro human skin explant model for GvHD has been used to study the role of cytokine changes induced by ECP. The model involves sensitising donor lymphocytes with recipient lymphocytes in vitro in a primary mixed lymphocyte reaction and then evaluating the secondary response on recipient skin biopsies by grading the graft-versus-host reactivity (grades I-IV) histopathologically using the Lerner grading system for GvHD. The assay has been proved to be superior to other assays used to predict clinical GvHD and has been used to investigate the pathophysiology of GvHD. Skin explant supernatants were collected and stored at −80 C and measured in batches using a multiplex Th1/Th2 cytokine assay. In 20 experiments where PUVA treatment of responder cells caused down regulation of GvHR in the skin explant model, statistically significant downregulation of IL-13, IL-5 and IFNgamma (p=<0.05) was demonstrated. No changes of IL-6, IL-1b, IL-10, IL-2, IL-4 or TNFalpha levels were demonstrated. Further evidence for the importance of IL-13 was highlighted in 3 skin explant assays where no downregulation of GvHR was seen with PUVA treatment and IL-13 levels remained high. Jordon et Al (Blood Jan2004) have documented the association between IL-13 production and the incidence of acute GvHD. Our data adds further evidence to challenge the notion that acute GvHD is purely a Th1-type cytokine response and that there may be a significant link between the Th2-type cytokine IL-13 with acute GvHD. Furthermore, downregulation of IL-13 and by PUVA may be important in the control of GvHD. More skin explant assays will be performed to consolidate these provisional findings.

Partially Assembled K99 Fimbriae Are Required for Protection

Antibodies to K99 fimbriae afford protection to F5+ bovine enterotoxigenic Escherichia coli (ETEC). Previous studies show that murine dams immunized with Salmonella vaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion of fanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion of fanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion of fanGH is sufficient to confer protection.

Comparison of immune responses after intra-typic heterologous and homologous vaccination against foot-and-mouth disease virus infection in pigs

This study compares the immune responses and protection induced by intra-typic heterologous vaccination with that induced by homologous vaccination against challenge with foot-and-mouth disease virus (FMDV). Humoral and cell-mediated immune responses and protection against challenge with FMDV O Taiwan were examined in a non-vaccinated group, a group vaccinated with O Taiwan FMD vaccine and a group vaccinated with O Manisa FMD vaccine. Five pigs from each group were challenged with FMDV type O Taiwan 14 days after vaccination and five other pigs were contact-exposed to the inoculated pigs. Both homologous and heterologous vaccination protected against challenge with FMDV O Taiwan at 2 weeks after vaccination. In the heterologous vaccinated group, cross-neutralizing antibody titres against O Taiwan could be detected although the ratio ‘ r 1’ was 0.4, which was significantly smaller than the critical r-value. Cell-mediated immune responses were detected after both homologous and heterologous vaccination. Virus-induced in vitro lymphocyte (cross-) proliferation and production of both a Th1-type (IFN-γ) and a Th2-type (IL-10) cytokine response were demonstrated in cultures of peripheral blood mononuclear cells (PBMC). The findings show that heterologous (emergency) vaccination can prevent clinical disease and shedding of virus. The induction of the cell-mediated immune responses after (heterologous) vaccination needs more research but data on these responses might provide additional tools for both vaccine choice and vaccine development.

Nickel Elicits Concomitant and Correlated in vitro Production of Th1‐, Th2‐Type and Regulatory Cytokines in Subjects with Contact Allergy to Nickel

Nickel (Ni2+) elicits production of functionally distinct cytokines in vitro, but the relation between the cytokine profile and the degree of the allergic reaction in vivo needs to be better defined in order to improve the understanding of the immunological mechanisms involved in contact allergy and to facilitate development of in vitro diagnostics. The aim of the study was to define Th1-type [interferon-gamma (IFN-gamma)], Th2-type [interleukin-4 (IL-4), IL-5 and IL-13] and regulatory (IL-10) cytokine responses to Ni2+ in peripheral blood mononuclear cells (PBMC) from subjects with varying patch test reactivity to Ni2+. The study included subjects with strong (+3), moderate (+2), weak (+1) or negative (controls) patch test reactivity to Ni2+ (n = 10 per group). All +3 and +2 subjects but only three +1 subjects had a clinical history of contact allergy to Ni(2+). Cytokine production of PBMC stimulated with Ni(2+) was determined by enzyme-linked immunospot and/or enzyme-linked immunosorbent assay. Ni2+ elicited significant production of all cytokines in PBMC from patch-test-positive subjects versus controls with a positive correlation between each cytokine and the patch test reactivity as well as with other cytokines. More subjects responded to Ni2+ above cut-off values with Th2-type cytokines as compared with IFN-gamma or IL-10; 100% of +3, 80% of +2, 50% of +1 and 0% of control subjects displayed reactivity to Ni2+ based on IL-4 and IL-13 assays. Despite the prevailing view of Ni2+ allergy as a type-1-mediated condition, the in vivo reactivity to Ni2+ correlated with a mixed Th1-type, Th2-type and regulatory cytokine response to Ni2+in vitro. The results accentuate the importance of type 2 responses in contact allergy and also demonstrate that IL-4 and IL-13 are reliable markers for Ni2+ allergy.

Evaluation of the immunogenicity of pBudCE4.1 plasmids encoding mycolyl-transferase Ag85A and phosphate transport receptor PstS-3 from Mycobacterium tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis ( Mtb), remains a major health problem. The only currently available vaccine, BCG, confers only variable protection and an improved vaccine is urgently needed. Administration of DNA vaccines encoding the secreted mycolyl-transferase Ag85A and the surface-exposed phosphate transport receptor PstS-3 elicit an immune response capable of protecting mice challenged with Mtb. In order to combine the protection against Mtb infection induced by these two DNA vaccines, we have cloned Ag85A and PstS-3 in pBudCE4.1 vector, in which antigenic expression is controlled by two independent promoters. (BALB/cxC57BL/6)F1 mice were vaccinated with this combination vaccine and immune responses were compared to those induced by vaccination with plasmids encoding the single antigens on pBudCE4.1 or pV1J.ns-tPA backbone. Antibody and Th1 type cytokine responses against Ag85A were comparable, whereas responses against PstS-3 were clearly lower in mice vaccinated with the combination plasmid, suggesting antigenic competition with the mycolyl-transferase being the dominant antigen.

Host control of Mycobacterium tuberculosis is regulated by 5-lipoxygenase–dependent lipoxin production

Th1 type cytokine responses are critical in the control of Mycobacterium tuberculosis infection. Recent findings indicate that 5-lipoxygenase-dependent (5-LO-dependent) lipoxins regulate host IL-12 production in vivo. Here, we establish lipoxins as key chemical mediators in resistance to M. tuberculosis infection. High levels of lipoxin A4 (LXA4) were detected in sera from infected WT but not infected 5-LO-deficient mice. Moreover, lungs from M. tuberculosis-infected 5-lo-/- animals showed increased IL-12, IFN-gamma, and NO synthase 2 (NOS2) mRNA levels compared with the same tissues in WT mice. Similarly, splenocyte recall responses were enhanced in mycobacteria-infected 5-lo-/- versus WT mice. Importantly, bacterial burdens in 5-lo-/- lungs were significantly lower than those from WT mice, and this enhancement in the resistance of the 5-lo-/- animals to M. tuberculosis was completely prevented by administration of a stable LXA4 analog. Together our results demonstrate that lipoxins negatively regulate protective Th1 responses against mycobacterial infection in vivo and suggest that the inhibition of lipoxin biosynthesis could serve as a strategy for enhancing host resistance to M. tuberculosis.

The Immune Response to Human Metapneumovirus Is Associated with Aberrant Immunity and Impaired Virus Clearance in BALB/c Mice

Human metapneumovirus (HMPV), recently identified in isolates from children hospitalized with acute respiratory tract illness, is associated with clinical diagnosis of pneumonia, asthma exacerbation, and acute bronchiolitis in young children. HMPV has been shown to cocirculate with respiratory syncytial virus (RSV) and mediate clinical disease features similarly to RSV. Little is known regarding the pathophysiology or immune response associated with HMPV infection; thus, animal models are needed to better understand the mechanisms of immunity and disease pathogenesis associated with infection. In this study, we examine features of the innate and adaptive immune response to HMPV infection in a BALB/c mouse model. Primary HMPV infection elicits weak innate and aberrant adaptive immune responses characterized by induction of a Th2-type cytokine response at later stages of infection that coincides with increased interleukin-10 expression and persistent virus replication in the lung. Examination of the cytotoxic T lymphocyte and antibody response to HMPV infection revealed a delayed response, but passive transfer of HMPV-specific antibodies provided considerable protection. These features are consistent with virus persistence and indicate that the immune response to HMPV is unique compared to the immune response to RSV.

Generation of a Monoclonal Antibody Agonist to Toll-Like Receptor 4

Toll-Like Receptor 4 (TLR4) has recently been identified as the lipopolysaccharide (LPS) receptor. Upon stimulation of TLR4 with LPS, a Th1 type cytokine response is observed. This immune modulation is thought to have a potential benefit in anti-tumor therapy. We have generated a TLR4 antibody agonist (5D24.D4) that mimics the action of the natural ligand. 5D24.D4 and LPS stimulation of TLR4 leads to a similar response, including IL-8 secretion, activation of NF-kB-mediated transcription, and the induced expression of an overlapping set of target genes. 5D24.D4 may be beneficial as an adjuvant anti-tumor therapy.

Evaluation of host immune responses to pulmonary cryptococcosis using a temperature-sensitive C. neoformans calcineurin A mutant strain

Cryptococcus neoformans is an opportunistic fungal pathogen that threatens individuals with impaired cell-mediated immunity (CMI). Presently, there are no standardized vaccines available to prevent cryptococcal infections and conventional anti-fungal drug therapy does not induce host immune reactivity and thus cannot efficiently resolve C. neoformans infections in immunocompromised individuals. The present study was designed to characterize pulmonary immune responses following infection with an avirulent temperature-sensitive (ts) mutant, calcineurin A1 ( cna1) compared to the pathogenic C. neoformans strain H99 and its potential to induce protective anti-cryptococcal immunity. Host CMI responses in cna1-inoculated mice were observed to be dose-dependent, and comprise increases in pulmonary macrophages and CD4 + T lymphocytes. However, cytokine analysis demonstrated a mixed pulmonary cytokine response (increases in IL-4, and MCP-1) with no induction of IFN-γ. Also, pre-immunization with the ts cna1 mutant did not result in protection from a subsequent secondary pulmonary infection with the pathogenic C. neoformans strain H99. Taken together, these results suggest that host pulmonary CMI responses to the ts cna1 mutant that is eventually eliminated from the host without the induction of IFN-γ appear to be dose-dependent, diverse, and require further stimulation to induce C. neoformans-specific Th1-type cytokine responses to resolve subsequent experimental pulmonary cryptococcal infections.

HLA class II alleles and measles virus-specific cytokine immune response following two doses of measles vaccine

Measles virus-specific T cells and the production of cytokines play a critical role in the immune response following measles immunization. To understand the genetic factors that influence variation in IFN-gamma and IL-4 responses following measles immunization and to provide insight into the factors influencing both cellular and humoral immunity to measles, we assessed associations between human leukocyte antigen (HLA) class II genes and measles-specific Th1 and Th2-type cytokine responses in peripheral blood lymphocytes from 339 children previously vaccinated with two doses of measles-mumps-rubella vaccine (MMR-II). Median values for measles-specific IFN-gamma and IL-4 secretion levels were 40.73 and 9.71 pg/ml, respectively. The global tests suggested associations between measles-specific IFN-gamma response and alleles of the DRB1 and DQB1 loci (P=0.07 and P=0.02, respectively). Specifically, DRB1*0301, *0901, and *1501 alleles were significantly associated with IFN-gamma secretion. The alleles that suggested evidence of an HLA association with IL-4 secretion were DRB1*0103, *0701, and *1101. Th1 cytokine responses and DQB1 allele associations revealed that the alleles with the strongest association with IFN-gamma secretion were DQB1*0201, *0303, *0402, and *0602. Specific alleles with a suggestive association with low measles-specific Th2 cytokine responses were DQB1*0202 and *0503. In addition, DPB1*0101, *0201, and *0601 alleles provided suggestive evidence of an HLA association with measles-induced IFN-gamma response, while DPB1*0501 was associated with an IL-4 response. These data suggest that IFN-gamma and IL-4 cytokine responses to measles may be genetically restricted in part by HLA class II genes, which in turn can restrict the cellular immune response to measles vaccine.

The role of IFN-γ in cattle infected with Theirelia sergenti

The spleen plays an important role in cellular immunity leading to parasite elimination, especially in hemo-protozoan parasite infection. In the present study, we quantified the serum IFN-γ in splenectomized (SP) and non-splenectomized (NSP) cows infected with Theirelia sergenti (TS) to investigate whether the spleen plays a role in the Th1-type cytokine responses in cows following parasite infection. A transient increase in IFN-γ was observed in the early stages of infection in the NSP cows, and the cows did not develop parasitemia. In contrast, the SP cows showed no IFN-γ response at the early stage of infection, and the cows developed parasitemia following anemia. The NSP cows produced IgG1 and IgG2 antibodies to TS-specific, whereas the SP cows showed increases only in IgG1. Increased PBMC proliferation by cocanavalin A stimulation was observed concomitant with the IFN-γ response. Pretreatment with IFN-γ suppressed the propagation of TS in the SP cattle. These results suggest that the spleen plays an important role in the resistance to TS infection, leading to a Th1-type immune response. IFN-γ might contribute to the activation of immunocytes and play an important role in the immune response to resist TS proliferation.

IL-13 - Predictor of or Protector from Acute Graft Versus Host Disease?.

Experimental and clinical data suggest that acute graft vs. host disease (aGVHD) after allogeneic (allo-) stem cell transplantation (SCT) predominantly involves a Th1-type cytokine response. Paradoxically, the production of Interleukin 13 (IL-13), a typical Th2 cytokine, by donor T cells responding to host antigen in vitro was recently shown to correlate with the severity of clinical aGVHD. We used a well-established murine SCT model (BALB/c → B6) and mice deficient in IL-13 to more clearly investigate the role of IL-13 produced by donor cells in the pathogenesis of GVHD. In our first set of experiments, B6, BALB/c IL-13+/+ and BALB/c IL-13−/− T cells were stimulated with B6 splenic dendritic cells. BALB/c IL-13+/+ and IL-13−/− T cells proliferated equally to alloantigens (123718±9038 vs.100565±3083 cpm) compared to syngeneic controls (2411±349 cpm). In addition, IL-13+/+ T cells produced significant amounts of IL-13 (1882.5±301.9 pg/ml), whereas IL-13 levels in wells with IL-13−/− T cells were very low (37.7±2.1 pg/ml) and did not differ from syngeneic controls (52.9±7.4 pg/ml). Next, lethally irradiated B6 mice received bone marrow and T cells from either syngeneic B6, or allogeneic BALB/c IL-13+/+ or BALB/c IL-13−/− donors and serum cytokine levels were determined on day +7. As shown in table 1, serum levels of IL-13, IL-5, IFNγ and TNFa were all elevated in mice receiving allo-SCT from IL-13+/+ donors compared to syngeneic controls. By contrast, analysis of sera from IL-13−/− SCT recipients revealed significant reductions in IL-13 and IL-5 levels, no change in IFNγ and a significant increase in TNFa levels compared to allo controls. Subsequent experiments revealed that the altered cytokine milieu observed early after allo-SCT correlated with enhanced GVHD; day 100 survival in allo-recipients of IL13−/− SCT was significantly decreased when compared to mice transplanted with IL-13+/+ donor cells (table 1) although GVHD of the gut (table 1) and liver (data not shown) did not differ in animals surviving at day +42. In summary our data demonstrate that donor T cell production of IL-13 does not predict the severity of GVHD and may instead have a protective effect on mortality from GVHD in this system. The decrease in survival observed in recipients of IL-13−/− donor cells may be explained by 1) the significant decrease in Th2 cytokines early after SCT, 2) loss of the strong immuno-modulatory effects of IL-13 on macrophage function, and 3) the associated increase in TNFa levels observed in these animals. The previously described correlation between the in vitro production of IL-13 by donor T cells and clinical aGVHD may therefore reflect the overall activation status of these T cells rather than a direct functional link to GVHD pathophysiology.Table 1: Serum cytokines (pg/ml) 7 days after SCTIL-13IL-5IFN γα TNFsurvival (day 100)gut pathology (day 42)syngeneic28.0±16.932.1±7.9ND2.2±1.5100%15.3±3.2allogeneic wt161.2±29.0115.0±21.32059±32038.5±5.140%26.0±1.1allogeneic IL-13−/−13.7±4.8**53.5±9.4*2042±31370.5±11.0*0%26.2±1.2(** p< 0.01; * p < 0.05; ND = not detectable)

Characterization of the T‐cell epitopes of a major peanut allergen, Ara h 2

The development of safe and effective immunotherapy for peanut allergy has been complicated by the high anaphylactic potential of native peanut extracts. We sought to map the T-cell epitopes of the major peanut allergen, Ara h 2 in order to develop T-cell targeted vaccines. A panel of eight peanut-specific CD4+ T-cell lines (TCL) was derived from eight peanut-allergic subjects and proliferative and cytokine responses to stimulation with a set of overlapping 20-mer peptides representing the entire sequence of Ara h 2 determined. Proliferation was assessed in 72 h assays via tritiated thymidine incorporation, while interleukin (IL)-5 and interferon (IFN)-gamma production were assessed via sandwich enzyme-linked immunosorbent assay (ELISA) of cell culture supernatants. Eight of the 17 Ara h 2 peptides were recognized by one or more subjects, with the two peptides showing highest reactivity [Ara h 2 (19-38) and Ara h 2 (73-92)] being recognized by three subjects each. Adjoining peptides Ara h 2 (28-47) and Ara h 2 (100-119) induced proliferative responses in two subjects. Each of these peptides was associated with a Th2-type cytokine response. Two highly immunogenic T-cell reactive regions of Ara h 2 have been identified, Ara h 2 (19-47) and Ara h 2 (73-119), providing scope for the development of safe forms of immunotherapy for peanut allergy.

Effect of renal cell carcinomas on the development of type 1 T-cell responses.

We reported that in renal cell carcinoma patients with active disease, T-cell reactions to the tumor-associated antigens MAGE-6 and EphA2 are highly skewed toward TH2-type cytokine responses [interleukin (IL) 5]. Herein, we determined whether tumor-derived products, including gangliosides isolated from renal cell carcinoma patients, participate in the down-regulation of type 1 T-cell responses. T cells from healthy volunteers or renal cell carcinoma patients were cultured in the presence and absence of supernatants derived from renal cell carcinoma explants or with gangliosides isolated from those tumor supernatants. T cells were stimulated or not with either autologous dendritic cells pulsed with superantigen (Staphylococcus enterotoxin B) or with phorbol 12-myristate 13-acetate and ionomycin and then were assessed for type 1 or type 2 responses (cytokine production and gene expression) and apoptosis. Tumor supernatants efficiently inhibited the TH1-type responses [interferon (IFN) gamma] of T cells stimulated with either S. enterotoxin B or phorbol 12-myristate 13-acetate and ionomycin but had no inhibitory effect on activated T-cell production of type 2 cytokines (IL-4, IL-5, and IL-10). Likewise, IFN-gamma mRNA and protein production were inhibited when T cells were cocultured with either renal cell carcinoma supernatant-derived gangliosides or a commercial source of purified GD1a. It was also determined that gangliosides impair type 1 responses by inducing apoptosis of activated T cells. We propose that renal cell carcinoma-derived tumor products such as gangliosides can induce a type 2 bias in antitumor immunity by initiating apoptosis in the IFN-gamma-producing type 1 effector cells. This represents a relevant mechanism by which renal cell carcinoma can inhibit protective antitumor immunity.

CARBON MONOXIDE INHALATION PROTECTS HEART ALLOGRAFTS FROM CHRONIC REJECTION

O69 Aims: Chronic rejection (CR) remains the major obstacle in successful organ transplantation. Heme oxygenase system is shown to provide protection against several inflammatory reactions including CR. Since carbon monoxide (CO), a by-product of heme catalysis, has also been shown to exert similar cytoprotective effects against a variety of stimuli, this study evaluated the efficacy of CO in preventing CR in rat heterotopic heart transplantation (HHT) model. Methods: Fully allogeneic HHT was performed in LEW to BN rat strain combination under short course FK506 (0.5 mg/kg, d0-6). Recipients were either kept in air or exposed to CO (20 ppm) for 28 days continuously after HHT. Cytokine mRNA levels, flowcytometry, mixed lymphocyte reaction (MLR), graft histopathology, and graft survival were analyzed. Results (Table): In CO-treated recipients, blood carboxyhemoglobin (COHb) levels were maintained at 10 % during CO exposure, compared to 1.4 % in air controls. CO treatment was remarkably effective in prolonging heart graft survival to a median of >100 days from 45 days in air control group. Severe histopathological changes associating with CR seen in control allografts were markedly reduced in CO-treated allografts with less numbers of infiltrating ED1+ macrophages. Th1 type cytokine (IL-2, IFNγ) upregulation in the spleen of air group at d28 was significantly downregulated with CO, associating with a considerable reduction of splenic CD25 (IL-2R) expression. Levels of anti-donor IgG antibodies at d50 were also notably reduced in CO-treated recipients. In addition, early short CO exposure (d0-13) was more effective in prolonging graft survival than later exposure (d14-28) (74 vs 57 days), suggesting that CO exposure during the early acute phase was more effective. As CO was shown to exert potent protection against oxidative stress, CO inhibited an early upregulation (3 hours post reperfusion) of inflammatory mediators (TNFα, endothelin-1, IL-1β, IL-6) in this model with significantly lower serum CPK levels. These results suggested that the efficacy of CO in preventing CR in this study was in part mediated via the anti-inflammatory effect of CO on I/R injury, which was known to have strong impacts on subsequent alloimmune responses and CR development. Additionally, direct inhibitory effect of CO on T cell activation was confirmed in vitro assays. When C3H/He mouse T cells were stimulated with anti-CD3 mAb for 48 hours under 20 ppm CO, T cell proliferation was markedly reduced to 78% without affecting T cell viability. Inhibition of T cell proliferation with CO associated with decreased production of IL-2 and IFNγ in the culture supernatant.Table: . (*p<0.05 vs air, mRNA levels; fold-increase vs normal samples).Conclusion: The study for the first time shows that low dose CO inhalation to recipients effectively improves whole organ allograft function and survival by inhibiting the development of CR. CO appears to downregulate alloimmune responses via the direct inhibition of Th1 type cytokine responses and via the indirect regulation of anti-inflammatory responses.

Genetic approaches to assessing evidence for a T helper type 1 cytokine defect in adult asthma.

Recent evidence suggests that deficiency in the Th1 cytokine pathway may underlie the susceptibility to allergic asthma. This study examined whether (1) single-nucleotide polymorphisms exist in the promoter region of the two interleukin (IL)-12 subunit genes in patients with asthma; (2) messenger RNA and protein expressions of signal transducers and activators of transcription, IL-12, IFN-gamma, and their receptors are altered in asthma; and (3) linkage to genes in the Th1 pathway is present in families with asthma in Iceland. The promoter regions of the IL-12 subunit genes were sequenced in 94 patients with asthma and 94 control subjects without asthma. Linkage was examined in 169 families that included over 570 patients with asthma and 950 of their unaffected relatives. The results demonstrate no evidence of linkage to microsatellite markers in close association with genes within the Th1 pathway, and no polymorphism was detected in the promoter regions of the two IL-12 subunit genes in the cohort with asthma patients. Moreover, we found no differences in the messenger RNA or protein expression signals of genes in the IL-12 pathway between the patients and control subjects. We conclude that decrease in Th1 type cytokine response is unlikely to present a primary event in asthma.