Altered faecal and mucosal microbial composition in post-infectious irritable bowel syndrome patients correlates with mucosal lymphocyte phenotypes and psychological distress.

BACKGROUND: A subset of irritable bowel syndrome (IBS) patients, denoted post-infectious IBS (PI-IBS), develop symptoms after an enteric infection. Bacterial dysbiosis and mucosal inflammation have been proposed to be involved in the pathophysiology of this entity. AIM: To characterise the mucosal and faecal microbiota in PI-IBS, general IBS and healthy controls, and to investigate associations between the microbiota and the mucosal immune system. METHODS: Mucosal biopsies and faeces were collected from 13 PI-IBS patients, 19 general IBS patients and 16 healthy controls. Global bacterial composition was determined by generating 16S rRNA amplicons that were examined by phylogenetic microarray hybridisation, principal component and redundancy analysis. We correlated previously reported lymphocyte proportions with the microbiota. RESULTS: Faecal microbiota composition of PI-IBS patients differed significantly from both general IBS patients and healthy controls (P < 0.02). Both mucosal (P < 0.01) and faecal (P = 0.05) microbial diversity were reduced in PI-IBS compared to healthy controls. In the intraepithelial lymphocytes the previously published proportion of CD8(+) CD45RA(+) was negatively correlated with mucosal microbial diversity (P < 0.005). The previously published number of lamina propria lymphocytes was negatively correlated with mucosal microbial diversity (P < 0.05). Faecal microbial diversity was significantly negatively correlated with the Hospital Anxiety and Depression scale (P < 0.05). CONCLUSIONS: We present data that distinguishes the intestinal microbiota of PI-IBS patients from that of both general IBS patients and HC. The microbial composition is significantly associated with the HADs score and alterations in lymphocyte subsets proportions.

Rapid migration of thymic emigrants to the colonic mucosa in ulcerative colitis patients.

Inflammatory bowel disease (IBD) is associated with imbalances of the local intestinal immune responses, with dysregulated CD4(+) T cells contributing to the chronic inflammation. Having demonstrated altered T cell maturation in the thymus in two different mouse models of colitis, we set out to investigate whether abnormalities in T cell maturation is present in patients with ulcerative colitis (UC) or Crohn's disease (CD). Specimens were obtained from peripheral blood (CD; n = 14, UC; n = 22), colon and small intestinal specimens (CD; n = 6, UC; n = 13). As controls, peripheral blood specimens were obtained from healthy volunteers, patients with adenocarcinomas (n = 18) and colonic specimens from patients with adenocarcinomas (n = 14). Recent thymic emigrants were estimated by analysis of the normalized ratio of T cell receptor excision circles (TRECs) by real-time polymerase chain reaction (PCR). The frequency of naive- and proliferating T lymphocytes and markers of extrathymic T cell maturation in the mucosa was analyzed by flow cytometry and real time-PCR. TREC levels in peripheral blood T lymphocytes were similar between IBD patients and controls. In contrast, UC patients demonstrated significantly increased levels of TRECs both in intraepithelial and lamina propria lymphocytes from the colonic mucosa compared to patients with adenocarcinomas and CD. However, markers for extrathymic T cell maturation in the mucosa were not different between controls and IBD patients. The increased TREC levels in mucosal but not peripheral blood lymphocytes in UC patients in the absence of increased extrathymic maturation in situ in the mucosa together demonstrate that recent thymic emigrants are recruited rapidly to the inflamed mucosa of these patients.

Toll-like receptor cross-hyporesponsiveness is functional in interleukin-1-receptor-associated kinase-1 (IRAK-1)-deficient macrophages: differential role played by IRAK-1 in regulation of tumour necrosis factor and interleukin-10 production.

Signalling downstream Toll-like receptors (TLR) is regulated at several levels in order to activate the immune response and prevent excessive inflammation. Altered intracellular signalling may be one reason that repeated stimulation of various TLRs results in hyporesponsiveness and cross-tolerance. We report that TLR cross-tolerance is inducible in the absence of interleukin-1 receptor-associated kinase-1 (IRAK-1) in peritoneal macrophages. Similar to wild-type macrophages, IRAK-1-deficient macrophages respond with decreased tumour necrosis factor (TNF) production to a secondary TLR stimulation, but in opposite to IRAK-1(+/+), IRAK-1(-/-) macrophages display increased interleukin (IL)-10 production at TLR restimulation. IRAK-1-deficient peritoneal macrophages have a defective TNF and IL-10 production in response to lipoteichoic acid stimulation as well as a defective IL-10-but a normal TNF production in response to high concentration of lipopolysaccharide. Our results demonstrate that IRAK-1 is not necessary for induction of TLR cross-tolerance as judged by TNF production.

Dextran sulfate sodium-induced colitis generates a transient thymic involution--impact on thymocyte subsets.

One of the most widely used animal models for inflammatory bowel disease (IBD) is the dextran sulfate sodium (DSS)-induced colitis. We have previously reported that 5 days administration of DSS in C57Bl/6J mice induces a colonic inflammation that progresses into chronicity after DSS removal, whereas in BALB/cJ mice the inflammation resolves within 4 weeks post-DSS. Here we show that both thymic size and thymocyte numbers dramatically decreased in the acute phase of inflammation in C57Bl/6 mice, 7 days after DSS withdrawal. Mature, CD4(+) and CD8(+) single positive (SP) CD69(lo) CD62L(hi) thymocytes were enriched in these mice, accompanied by a major decrease in the number of immature double positive (DP) thymocytes. However, the different maturation stages within the DP thymocyte subset were unchanged between healthy and inflamed C57Bl/6J mice. Interestingly, as the inflammation progressed into the chronic phase, the thymus recovered and 2 weeks after the acute inflammatory phase all the thymic parameters investigated in this study were restored to normal. In contrast, BALB/cJ mice only develop mild thymic alterations. Nevertheless, we found that within the double negative (DN) thymocytes an increased frequency and also total numbers of CD44(+) CD25(-) (DN1) cells correlated with the severity of colitis, and that the frequency of CD44(-) CD25(-) (DN4) thymocytes decreased proportionally in the acute phase in BALB/cJ mice. Our observations suggest that the thymic effects are intimately connected to the intestinal inflammatory response in colitis regardless of the inflammatory stimuli.

Serum interleukin-1 receptor antagonist is an early indicator of colitis onset in Galphai2-deficient mice.

AIM: To study the serum concentration of IL-1beta, IL-1 receptor antagonist (IL-1Ra) and IL-18 in Galphai2-deficient mice at the age of 6 (healthy), 12 (pre-colitic) and 24 wk (colitic) and in healthy control mice. METHODS: At the time of killing, serum samples were collected and IL-1beta, IL-1Ra and IL-18 levels were measured using enzyme-linked immunosorbent assays. RESULTS: Serum concentration of IL-1Ra was significantly increased in pre-colitic (median: 524 ng/L; P=0.02) and colitic (450 ng/L; P=0.01), but not in healthy (196 ng/L) Galphai2-deficient mice as compared with controls (217 ng/L). Serum concentrations of IL-1beta did not differ between Galphai2-deficient mice and their controls, irrespective of age, IL-18 was significantly increased in colitic, but not in pre-colitic mice compared with controls (510 ng/L vs 190 ng/L; P=0.05). CONCLUSION: The increased serum concentrations of IL-18 and IL-1Ra in established diseases are suggested as markers of ongoing colitis. Interestingly, the significantly increased serum concentration of IL-1Ra in pre-colitic mice is found to be an early marker of disease progression.

Acellular Bordetella pertussis vaccine enhances mucosal interleukin-10 production, induces apoptosis of activated Th1 cells and attenuates colitis in Galphai2-deficient mice.

Mice deficient for the inhibitory G protein subunit alpha2 (Galphai2(-/-)) spontaneously develop a progressive inflammatory bowel disease resembling ulcerative colitis, and have a T helper 1 (Th1)-dominated immune response prior to onset of colitis, which is further augmented after the onset of disease. The present study was performed to investigate whether the Galphai2(-/-) mice were able to down-regulate the Th1-dominated inflammatory mucosal immune response and/or induce an anti-inflammatory Th2/T regulatory response and thereby diminish the severity of colitis following treatment with acellular Bordetella pertussis vaccine. The acellular vaccine against B. pertussis, the causative agent of whooping cough, has been demonstrated to induce a Th2-mediated response in both man and mice. We therefore treated Galphai2(-/-) mice intraperitoneally with a three-component acellular B. pertussis vaccine. The treated Galphai2(-/-) mice showed significantly increased interleukin (IL)-10 production in intestinal tissue, associated with significantly reduced colitis and decreased mortality, compared to untreated Galphai2(-/-) mice. The attenuation of colitis in Galphai2(-/-) mice was due, at least partly, to the B. pertussis surface antigen filamentous haemagglutinin (FHA), which almost completely inhibited proliferation of CD4(+) T cells and stimulated apoptosis of activated CD4(+) T helper 1 cells. In conclusion, the three-component acellular B. pertussis vaccine containing filamentous haemagglutinin increases the production of IL-10 in the intestinal mucosa, induces apoptosis of activated Th1 cells and attenuates colitis in Galphai2(-/-) mice.

Dietary antigen-specific T-cell responses: switch from an interleukin-10-dominated response in normal mice to a T-helper 1 cytokine profile in Galphai2-deficient mice prior to colitis.

We investigated dietary antigen-specific T-cell responses in mesenteric lymph nodes (MLN) and Peyer's patches (PP) in noncolitic control mice as well as in colitis-prone mice prior to onset of histological active colitis. T cells were restimulated in vitro with constituents isolated from the mouse diet. Interestingly, MLN T cells of littermate G(alpha)i2+/- control mice responded to soya with high production of interleukin (IL)-10, but did not produce proinflammatory T-helper 1 (Th1) cytokines. Recall dietary antigen stimulation of G(alpha)i2+/- PP T cells did not result in increased IL-10 production above the spontaneous production in the absence of antigenic stimulation. In strong contrast, MLN T cells from precolitic G(alpha)i2-/- mice produced high levels of interferon-gamma (IFN-gamma) upon restimulation with soya, which could be abolished using a major histocompatibility complex class II-blocking antibody. In conclusion, the present study demonstrates that MLN T lymphocytes in normal healthy mice respond with a significantly increased production of the regulatory cytokine IL-10 on re-encounter with dietary proteins in vitro. In marked contrast precolitic G(alpha)i2-/- mice respond to dietary antigens with a Th1-dominated cytokine response in the mucosa, prior to onset of colitis, with excessive IFN-gamma production. These results suggest that aberrant immune responses to dietary antigens could contribute as a potential pathogenic mechanism in the onset of colitis in G(alpha)i2-deficient mice.

Regression of Peyer's patches in G alpha i2 deficient mice prior to colitis is associated with reduced expression of Bcl-2 and increased apoptosis.

BACKGROUND: G protein deficient (G alpha i2-/-) mice spontaneously develop an inflammatory bowel disease (IBD) closely resembling ulcerative colitis. Previous studies have demonstrated that gut T cells are hyperreactive to the endogenous microflora in most IBD models. AIMS: The aim of this study was to analyse Peyer's patches (PP), the inductive sites for gut mucosal immune responses. SUBJECTS AND METHODS: G alpha i2-/- mice, an animal model for IBD, were analysed using immunological methods with regard to phenotype and function. RESULTS: We found significantly decreased numbers of PP in G alpha i2-/- mice. Even before the onset of colitis, G alpha i2 deficient animals exhibited diminished size of PP, as judged by histology. This involution of PP was associated with strongly increased levels of apoptotic lymphocytes, associated with decreased levels of antiapoptotic intracellular protein Bcl-2. PP T lymphocytes showed highly elevated production of interferon gamma in response to the enteric flora compared with PP T cells from wild-type mice, which produced predominantly interleukin 10. CONCLUSIONS: Thus even before the onset of colitis, the PP in G alpha i2 deficient mice is a Th1 dominated milieu associated with downregulated levels of Bcl-2, resulting in increased apoptosis of lymphocytes leading to regression of PP. We speculate that this Th1 dominated microenvironment in the inductive site for mucosal immune responses contributes to the development of colitis in G alpha i2 deficient mice.

Antibody response to dietary and autoantigens in G alpha i2-deficient mice.

BACKGROUND: Mice with a targeted mutation in the G protein subunit G alpha i2 gene develop a colonic mucosal inflammation, with a highly activated B-cell response. We wanted to investigate whether this increased B-cell activity was directed against dietary antigens and/or various self tissues. METHODS: The level of antibodies specific for dietary (gliadin, soya and fish meal) antigens was measured by ELISA. Reactivity against self antigens was measured by immunohistochemistry on cryo-sectioned mouse and rat tissue. Sera and intestinal lavages were analysed from G alpha i2-/- mice before and after development of colitis and in age-matched wild type litter mates. RESULTS: Titres of antibodies against dietary antigens were significantly enhanced both in serum and in large intestinal lavages from G alpha i2-/- mice with ongoing colitis but not prior to disease, as compared to wild type mice. The autoreactivity to self tissues was significantly increased in G alpha i2-/- mice both before and after development of colitis as compared to litter mate control animals. Self tissue reactivity was directed not only against epithelial cells of the colon, small intestine and gastric glands, but also against smooth muscle cells, hepatocytes, bile duct cells, renal tubule and collecting tubule cells of the kidney. In analogy to human ulcerative colitis, autoantibodies against epithelial cells, bile duct epithelium and neutrophil granulocytes were found. CONCLUSIONS: Earlier increase in levels of autoantibodies (before onset of colitis) than of food antibodies (after onset of colitis) suggests the latter response to be a secondary phenomenon to e.g. a destroyed barrier function.

Interaction of Bordetella pertussis with mast cells, modulation of cytokine secretion by pertussis toxin.

Together with macrophages and dendritic cells, mast cells have recently been shown to interact with certain pathogenic bacteria and present microbial antigens to the immune system. We show here that Bordetella pertussis can adhere to and be phagocytosed by mast cells. In addition, mast cells are able to process and present B. pertussis antigens to T lymphocytes. Furthermore, exposure of mast cells to B. pertussis induced the release of the proinflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). The release of IL-6 was strongly reduced by pertussis toxin expressed by B. pertussis. The production of IL-10, but not that of IL-4, by mast cells was also inhibited by pertussis toxin. Depletion of mast cells in vivo resulted in significant reduction of early TNF-alpha production in bronchoalveolar lavage (BAL) fluids of B. pertussis-infected mice. These data suggest that mast cells may play a role in the induction of immune responses against B. pertussis through the release of cytokines, especially TNF-alpha.

Immune activation in the intestinal mucosa before the onset of colitis in Galphai2-deficient mice.

G-protein subunit Galphai2-deficient mice spontaneously develop an inflammatory bowel disease that clinically and histopathologically resembles ulcerative colitis in humans. The aim of this study was to determine whether immunological changes precede the development of colitis in Galphai2-deficient mice. Therefore, Galphai2-deficient mice with no clinical or histopathological signs of colitis were compared with Galphai2-deficient mice with established colitis and wild-type animals, concerning immunological parameters. Healthy Galphai2-deficient mice displayed an increased frequency of CD4+ T cells and a decreased frequency of CD19+ B lymphocytes in the intestinal mucosa compared with control mice. The CD4+ population was characterized by a memory phenotype, i.e. increased expression of CD44 and decreased expression of CD45RB and CD62L, as well as increased expression of the mucosal homing receptors integrins alpha4beta7 and alphaEbeta7. Production of pro-inflammatory cytokines, interleukin (IL)-1beta and interferon (IFN)-gamma, were increased in Galphai2-deficient mice before clinical signs of disease were evident. In addition, total immunoglobulin (Ig)G and IgA levels in large intestinal secretions were increased significantly compared with wild-type mice, and antibodies specific for the normal intestinal flora in large intestinal secretions were present in Galphai2-deficient mice several weeks before the onset of colitis. In contrast, antibodies against tropomyosin, a putative autoantigen in human ulcerative colitis, were not found in Galphai2-deficient mice before the onset of colitis, although they were present in animals with established disease. In conclusion, activation of the intestinal immune system precedes histopathological and clinical signs of inflammation in Galphai2-deficient mice, suggesting that immune abnormalities play an important role in the induction of colitis.

The mucosal adjuvant effects of cholera toxin and immune-stimulating complexes differ in their requirement for IL-12, indicating different pathways of action.

Adjuvants that can improve mucosal vaccine efficacy are much warranted. In this comparative study between cholera toxin (CT) and immune-stimulating complexes (ISCOM) we found that, contrary to CT, ovalbumin (OVA)-ISCOM were poor inducers of mucosal anti-OVA IgA responses, but induced similar or better systemic immunity following oral immunizations. The addition of CT to the oral OVA-ISCOM protocol did not stimulate local anti-OVA IgA immunity, nor did it change the quality or magnitude of the systemic responses. Both vectors recruited strong innate immunity, but only OVA-ISCOM could directly induce IL-12, demonstrable at the protein and mRNA levels. CT had no inhibitory effects on lipopolysaccharide/IFN-gamma-induced IL-12 mRNA expression or IL-12 production. Furthermore, adjuvanticity of CT was unaffected in IL-12-deficient mice, while OVA-ISCOM showed partly impaired adjuvant effects by the lack of IL-12. CT abrogated the induction of oral tolerance stimulated by antigen feeding in these mice. In addition, CT did not alter TGF-beta levels, suggesting that the immunomodulating effect of CT was independent of IL-12 as well as TGF-beta production. Taken together, these findings indicate that mucosal adjuvanticity of CT and ISCOM are differently dependent on IL-12, suggesting that separate and distinct antigen-processing pathways are involved.

The influence of costimulation and regulatory CD4+ T cells on intestinal IgA immune responses.

It is thought that IgA B-cell differentiation is highly dependent on activated CD4+ T cells. In particular, cell-cell interactions in the Peyer's patches involving CD40 and/or CD80/CD86 have been implicated in germinal-center formation and IgA B-cell development. Also soluble factors, such as IL-4, IL-5, IL-6, and TGF beta may be critical for IgA B-cell differentiation in vivo. Here we report on some paradoxical findings with regard to IgA B-cell differentiation and specific mucosal immune responses that we have recently made using gene knockout mice. More specifically, we have investigated to what extent absence of CD4+ T cells, relevant cytokines, or T-cell-B-cell interactions would influence IgA B-cell differentiation in vivo. Using CD4- or IL-4-gene knockout mice or mice made transgenic for CTLA4Ig, we found that, although specific responses were impaired, total IgA production and IgA B-cell differentiation appeared to proceed normally. However, a poor correlation was found between, on the one hand, GC formation and IgA differentiation and, on the other hand, the ability to respond to T-cell-dependent soluble protein antigens in these mice. Thus, despite the various deficiencies in CD4+ T-cell functions seemingly intact IgA B-cell development was observed.

Lack of local suppression in orally tolerant CD8-deficient mice reveals a critical regulatory role of CD8+ T cells in the normal gut mucosa.

We found that feeding keyhole limpet hemocyanin (KLH) to CD8-deficient (CD8-/-) mice induced oral tolerance that was comparable in both magnitude and quality to that induced in wild-type (wt) mice. The tolerance was dose dependent, and only higher doses of KLH caused significant reduction in specific Ab and T cell responses. Both Th1 and Th2 CD4+ T cell functions were affected. Feeding KLH together with cholera toxin (CT) adjuvant, however, abrogated the induction of oral tolerance equally well in CD8-/- and wt mice. On the contrary, CT adjuvant was unable to abrogate already established oral tolerance in both CD8-/- and wt mice. Most importantly, whereas Ag feeding induced hyporesponsiveness in systemic as well as in local gut IgA responses in wt mice, a lack of local suppression was evident in orally tolerant CD8-/- mice following oral immunizations. Thus, contrary to the situation in wt mice, Ag feeding induces systemic, but not local, gut IgA hyporesponsiveness in CD8-/- mice, suggesting that CD8+ T cells in the normal gut mucosa exert an important down-regulatory function. In wt mice the local suppression extended to an unrelated Ag, OVA, given together with KLH and CT adjuvant, i.e., bystander suppression. Based on these results we propose that tolerance induced by feeding Ag is highly compartmentalized, requiring CD8+ T cells for local suppression of IgA responses, whereas systemic tolerance may affect CD4+ T cells of both Th1 and Th2 types independently of CD8+ T cells. Finally, the adjuvant effect of CT abrogates induction, but not established, oral tolerance through a mechanism that does not require CD8+ T cells.

CD8-deficient mice exhibit augmented mucosal immune responses and intact adjuvant effects to cholera toxin.

We used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4+ and CD8+ T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant. Phenotypic analysis of mucosa-associated tissues revealed normal CD3+ T-cell frequencies in CD4-/- and CD8-/- mice such that in CD4-/- mice the CD8+ and double-negative (DN) T cells were increased. In CD8-/- mice the CD4+ T cells were increased, with the exception that in the intraepithelial compartment the CD3+ T cells were predominantly DN gamma delta T-cell receptor (TCR)+ T cells. All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone. In the presence of CT adjuvant, however, CD8-/- mice consistently exhibited three- to fivefold stronger gut mucosal responses compared to normal C57B1/6 mice. By contrast, no difference was observed for systemic responses between CD8-/- and normal mice. Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8-/- mouse gut, the normal gut mucosa may host CD8+ T cells that exert a local suppressive effect on T- and B-cell responses. The magnitude of the enhancing effect of CT was comparable in CD8-/- and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8+ T cells. On the other hand, the adjuvant effect of CT required CD4+ T cells, because no or poor anti-KLH responses were observed in CD4-/- mice. In both normal and CD8-/- mice CT adjuvant promoted KLH-specific CD4+ T-cell printing without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance. Furthermore, CT adjuvant increased the frequency of CD4+ T cells expressing a memory phenotype, i.e. CD44high, LECAM-1low and CD45RBlow. We have shown, using gene-targeted mice, that CD8+ T cells are not required for the adjuvant effect of CT, and that CD8+ T cells may exert local mucosal down-regulation of intestinal immune responses.

Cholera toxin increases T lymphocyte responses to unrelated antigens.

We have found that CT adjuvant greatly augmented oral priming immunizations. Limiting dilution analysis revealed strong increases in the frequency of primed antigen specific T lymphocytes subsequent to the use of CT adjuvant. Moreover, several fold stronger lymphokine production accompanied the strong proliferative responses seen with CT-treated and KLH-primed T cells. The lymphokine pattern secreted by these T cells suggested that TH1 as well as TH2 types of cells were effectively primed by KLH, and that CT adjuvant did not exert a selective effect on any of these T-cell subsets. These effects of CT were truly due to immunomodulation and not simply the result of a more efficient uptake of gut luminal antigens in the presence of CT, because CT enhanced equally well T cell priming by peroral as well as parenteral immunization with KLH. Moreover, the CT-induced enhanced responsiveness to antigen was also observed with CD4(+)-enriched T cells suggesting that CT primarily affected T cell priming rather than the balance between CD4+ and CD8+ T cell subsets.

Cholera toxin adjuvant greatly promotes antigen priming of T cells.

Cholera toxin (CT) given perorally is a powerful mucosal immunogen and adjuvant. Information that explains the adjuvant effect of CT may be used for the development of more effective oral vaccines and might also contribute to our understanding of the mechanisms involved in regulating mucosal immunity. The present study was undertaken to investigate if CT administered together with keyhole limpet hemocyanin (KLH) would act to promote or inhibit priming of KLH-specific T cells and whether the adjuvant effect of CT is restricted to mucosal immune responses or is a generalized phenomenon due to direct immunomodulating effects of CT. We found that CT adjuvant greatly augmented the effectiveness of a single oral priming immunization with KLH: re-challenge with KLH in vitro 1 week following immunization gave several-fold stronger proliferation in KLH-specific spleen, mesenteric lymph node, Peyer's patch and gut lamina propria T cells from KLH + CT adjuvant as opposed to KLH only-treated mice. Moreover, several-fold stronger cytokine production, i.e. interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10 and interferon-gamma accompanied the enhanced proliferative response of T cells from CT adjuvant-treated mice. The adjuvant effect of CT was not restricted to mucosal immune responses but was evident also following a single parenteral immunization with KLH + CT. Limiting dilution analysis revealed that CT adjuvant promoted a 20- to 40-fold increase in the frequency of primed KLH-specific T cells. Phenotypic and functional analyses clearly demonstrated that CT adjuvant primarily enhanced priming of CD4+ rather than CD8+ T cells and the pattern of lymphokine secretion disclosed that CT most probably promoted antigen priming of both Th1 and Th2 type of CD4+ T precursor cells.