Central role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium

Citrobacter rodentium, an attaching-effacing bacterial pathogen, establishes an acute infection of the murine colonic epithelium and induces a mild colitis in immunocompetent mice. This study describes the role of T-cell subsets and B lymphocytes in immunity to C. rodentium. C57Bl/6 mice orally infected with C. rodentium resolved infection within 3 to 4 weeks. Conversely, systemic and colonic tissues of RAG1(-/-) mice orally infected with C. rodentium contained high and sustained pathogen loads, and in the colon this resulted in a severe colitis. C57Bl/6 mice depleted of CD4(+) T cells, but not CD8(+) T cells, were highly susceptible to infection and also developed severe colitis. Mice depleted of CD4(+) T cells also had diminished immunoglobulin G (IgG) and IgA antibody responses to two C. rodentium virulence-associated determinants, i.e., EspA and intimin, despite having a massively increased pathogen burden. Mice with an intact T-cell compartment, but lacking B cells ( micro MT mice), were highly susceptible to C. rodentium infection. Systemic immunity, but not mucosal immunity, could be restored by adoptive transfer of convalescent immune sera to infected micro MT mice. Adoptive transfer of immune B cells, but not na?ve B cells, provided highly variable immunity to recipient micro MT mice. The results suggest that B-cell-mediated immune responses are central to resolution of a C. rodentium infection but that the mechanism through which this occurs requires further investigation. These data are relevant to understanding immunity to enteric attaching and effacing bacterial pathogens of humans.     

Tumor necrosis factor receptor p55-deficient mice respond to acute Yersinia enterocolitica infection with less apoptosis and more effective host resistance

Tumor necrosis factor (TNF) has generally been regarded as a protective cytokine in host defense against bacterial infections. In the present study, we evaluated the role of TNF in the acute phase of infection by Yersinia enterocolitica by using mice rendered genetically deficient in TNF receptor p55 (TNFRp55(-/-)). Unexpectedly, TNFRp55(-/-) mice showed more effective resistance to the bacteria, reflected in enhanced bacterial clearance and less tissue damage, than did control C57BL/6 mice. C57BL/6 mice showed evidence of extensive apoptosis in the spleen accompanied by a selective decrease in the CD4(+)-T-cell population of splenocytes, whereas TNFRp55(-/-) mice were spared these changes. The splenocytes from TNFRp55(-/-) mice also maintained a robust gamma interferon IFN-gamma response to mitogenic stimulation, while the comparable response in C57BL/6 mice was impaired. In addition, splenocytes harvested from infected mice demonstrated lower production of interleukin-10 IL-10 in TNFRp55(-/-) mice than in C57BL/6 mice. These findings suggest that Yersinia can induce TNFRp55-mediated apoptosis of splenocytes in the acute phase of the infection and that alteration of T-cell-generated cytokines can dramatically alter the early events in host defense against this pathogen.     

Toll-like receptor 4 contributes to colitis development but not to host defense during Citrobacter rodentium infection in mice

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are noninvasive bacterial pathogens that infect their hosts' intestinal epithelium, causing severe diarrheal disease. These infections also cause intestinal inflammation, although the mechanisms underlying the inflammatory response, as well as its potential role in host defense, are unclear. Since these bacteria are gram-negative, Toll-like receptor 4 (TLR4), the innate receptor for bacterial lipopolysaccharide may contribute to the host response; however, the role of TLR4 in the gastrointestinal tract is poorly understood, and its impact has yet to be tested against this family of enteric bacterial pathogens. Since EPEC and EHEC are human specific, we infected mice with Citrobacter rodentium, a mouse-adapted attaching and effacing (A/E) bacterium that infects colonic epithelial cells, causing colitis and epithelial hyperplasia, using a similar array of virulence proteins as EPEC and EHEC. We demonstrated that C. rodentium activates TLR4 and rapidly induced NF-kappaB nuclear translocation in host cells in a partially TLR4-dependent manner. Infection of TLR4-deficient mice revealed that TLR4-dependent responses mediate much of the inflammation and tissue pathology seen during infection, including the induction of the chemokines MIP-2 and MCP-1, as well as the recruitment of macrophages and neutrophils into the infected intestine. Surprisingly, spread of C. rodentium through the colon was delayed in TLR4-deficient mice, whereas the duration of the infection was unaffected, indicating that TLR4-mediated responses against this A/E pathogen are not host protective and are ultimately maladaptive to the host, contributing to both the morbidity and the pathology seen during infection.     

The sphingosine-1-phosphate analogue FTY720 impairs mucosal immunity and clearance of the enteric pathogen Citrobacter rodentium

The sphingosine-1-phosphate (S1P) analogue FTY720 is therapeutically efficacious in multiple sclerosis and in the prevention of transplant rejection. It prevents the migration of lymphocytes to sites of pathology by trapping them within the peripheral lymph nodes, mesenteric lymph nodes (MLNs), and Peyer's patches. However, evidence suggests that its clinical use may increase the risk of mucosal infections. We investigated the impact of FTY720 treatment on susceptibility to gastrointestinal infection with the mouse enteric pathogen Citrobacter rodentium. This attaching and effacing bacterium induces a transient bacterial colitis in immunocompetent mice that resembles human infection with pathogenic Escherichia coli. FTY720 treatment induced peripheral blood lymphopenia, trapped lymphocytes in the MLNs, and prevented the clearance of bacteria when mice were infected with luciferase-tagged C. rodentium. FTY720-treated C. rodentium-infected mice had enhanced colonic inflammation, with significantly higher colon mass, colon histopathology, and neutrophil infiltration than vehicle-infected animals. In addition, FTY720-treated infected mice had significantly lower numbers of colonic dendritic cells, macrophages, and T cells. Gene expression analysis demonstrated that FTY720-treated infected mice had an impaired innate immune response and a blunted mucosal adaptive immune response, including Th1 cytokines. The data demonstrate that the S1P analogue FTY720 adversely affects the immune response to and clearance of C. rodentium.     

IL-36α expression is elevated in ulcerative colitis and promotes colonic inflammation

A role for the IL-36 family of cytokines has been identified in the pathogenesis of psoriasis. Although significant mechanistic overlap can exist between psoriasis and inflammatory bowel disease (IBD), to date there have been no reports investigating the IL-36 family in gastrointestinal inflammation. Here we demonstrate that expression levels of IL-36α are specifically elevated in the colonic mucosa of ulcerative colitis patients. This elevated expression is mirrored in the inflamed colonic mucosa of mice, wherein IL-36 receptor deficiency confirmed this pathway as a mediator of mucosal inflammation. Il36r−/− mice exhibited reduced disease severity in an acute DSS-induced model of colitis in association with decreased innate inflammatory cell infiltration to the colon lamina propria. Consistent with these data, infection with the enteropathogenic bacteria Citrobacter rodentium, resulted in reduced innate inflammatory cell recruitment and increased bacterial colonization in the colons of il36r−/− mice. Il36r−/− mice also exhibited altered T helper cell responses in this model, with enhanced Th17 and reduced Th1 responses, demonstrating that IL-36R signaling also regulates intestinal mucosal T-cell responses. These data identify a novel role for IL-36 signaling in colonic inflammation and indicate that the IL-36R pathway may represent a novel target for therapeutic intervention in IBD.     

The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes

The intracellular sensor Nod2 is activated in response to bacteria, and the impairment of this response is linked to Crohn's disease. However, the function of Nod2 in host defense remains poorly understood. We found that Nod2-/- mice exhibited impaired intestinal clearance of Citrobacter rodentium, an enteric bacterium that models human infection by pathogenic Escherichia coli. The increased bacterial burden was preceded by reduced CCL2 chemokine production, inflammatory monocyte recruitment, and Th1 cell responses in the intestine. Colonic stromal cells, but not epithelial cells or resident CD11b+ phagocytic cells, produced CCL2 in response to C. rodentium in a Nod2-dependent manner. Unlike resident phagocytic cells, inflammatory monocytes produced IL-12, a cytokine that induces adaptive immunity required for pathogen clearance. Adoptive transfer of Ly6C(hi) monocytes restored the clearance of the pathogen in infected Ccr2-/- mice. Thus, Nod2 mediates CCL2-CCR2-dependent recruitment of inflammatory monocytes, which is important in promoting bacterial eradication in the intestine.     

Pathogenic roles of tumor necrosis factor receptor p55-mediated signals in dimethylnitrosamine-induced murine liver fibrosis

TNF-alpha has pleiotropic functions, but its role in liver fibrosis has not yet been clarified. To understand the pathophysiologic role of the TNF-alpha/TNF receptor (TNFR) p55 signals in liver fibrosis, 10 mg/kg of dimethylnitrosamine, a specific hepatotoxicant, was administered twice a week into the peritoneal cavity of both TNFRp55 knock-out (KO) and wild-type mice, and the severity of fibrosis was monitored histologically and biochemically. In wild-type mice, histologic analysis demonstrated evident fibrotic changes 1 week after the initiation of dimethylnitrosamine administration, consistent with increased liver collagen contents. Concomitantly, the numbers of Kupffer cells and activated hepatic stellate cells (HSCs) were increased in liver tissue. On the contrary, fibrotic changes were attenuated and the numbers of Kupffer cells and HSCs were decreased in TNFRp55-KO mice. Moreover, gene expression of TNF-alpha and monocyte chemoattractant protein-1, which are involved in Kupffer cell activation or migration, was decreased in the liver of TNFRp55-KO mice. Collectively, TNFRp55-mediated signals may regulate activation of Kupffer cells and HSCs and eventually enhance fibrotic process.     

Interleukin-1 and tumor necrosis factor receptor signaling is not required for bacteria-induced osteoclastogenesis and bone loss but is essential for protecting the host from a mixed anaerobic infection

Bacterial infection causes significant morbidity, mediated in part by the up-regulation of inflammatory cytokines. Cytokine induction is thought to stimulate osteolysis in conditions such as periodontal disease and otitis media. To establish the relative importance of interleukin-1 (IL-1) and tumor necrosis factor (TNF) in mediating the response to a mixed anaerobic infection, we used an in vivo model in which the dental pulp was inoculated with six anaerobic pathogens, in mice with functional deletions of receptors to IL-1 (IL-1RI(-/-)), TNF (TNFRp55(-/-)-p75(-/-)), or both (TNFRp55(-/-)-IL-1RI(-/-)). Polymorphonuclear and mononuclear phagocyte recruitment occurred to the greatest extent in TNFRp55(-/-)-IL-1RI(-/-) mice, and to a lesser extent in IL-1RI(-/-) or TNFRp55(-/-)-p75(-/-) mice, and the least in wild-type mice, demonstrating that recruitment of these phagocytes is not dependent on IL-1 or TNF receptor signaling. A similar pattern was observed for bacterial penetration into host tissue. Because it had recently been reported that TNF played a critical role in mediating lipopolysaccharide-induced bone loss, we anticipated that mice with targeted deletions of TNFRp55(-/-) would have reduced osteoclastogenesis. Surprisingly, osteolytic lesion formation was greatest in animals lacking TNF and/or IL-1 receptors. These results indicate that IL-1 or TNF receptor signaling is not required for bacteria-induced osteoclastogenesis and bone loss, but does play a critical role in protecting the host against mixed anaerobic infections.     

Helicobacter hepaticus-induced colitis in interleukin-10-deficient mice: cytokine requirements for the induction and maintenance of intestinal inflammation

We have previously shown that specific-pathogen-free interleukin-10 (IL-10)-deficient (IL-10 KO) mice reconstituted with Helicobacter hepaticus develop severe colitis associated with a Th1-type cytokine response. In the present study, we formally demonstrate that IL-12 is crucial for disease induction, because mice deficient for both IL-10 and IL-12 p40 show no intestinal pathology following H. hepaticus infection. By using monoclonal antibodies (MAbs) to IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), we have further analyzed the role of these cytokines in the maintenance of the Th1 response and inflammation in IL-10 KO mice with established H. hepaticus-induced colitis. Treatment of infected colitic IL-10 KO mice with anti-IL-12 p40 resulted in markedly reduced intestinal inflammation, colonic IFN-gamma, TNF-alpha, and inducible nitric oxide synthase (iNOS) mRNA levels, and H. hepaticus-specific IFN-gamma secretion by mesenteric lymph node (MLN) cells compared to the findings in control MAb-treated mice. Moreover, the diminished pathology was associated with decreased numbers of colonic CD3(+) T cells and significantly reduced frequencies of Helicobacter-reactive CD4(+) Th1 cells in MLN. In contrast, anti-IFN-gamma and/or anti-TNF-alpha had no effect on intestinal inflammation in IL-10 KO mice with established colitis. Using IL-10/IFN-gamma double-deficient mice, we further show that IFN-gamma is not required for the development of colitis following H. hepaticus infection. MLN cells from infected IL-10/IFN-gamma KO animals secreted elevated amounts of IL-12 and TNF-alpha following bacterial antigen stimulation, indicating alternative pathways of disease induction. Taken together, our results demonstrate a crucial role for IL-12 in both inducing and sustaining intestinal inflammation through recruitment and maintenance of a pool of pathogenic Th1 cells.     

The resistance against Listeria monocytogenes and the formation of germinal centers depend on a functional death domain of the 55 kDa tumor necrosis factor receptor

The biological functions mediated by the death domain of the 55-kDa tumor necrosis factor receptor (TNFRp55) in vivo are still elusive. TNFRp55 mutants lacking a functional death domain were expressed in TNFRp55-/- and in TNFRp55+/- mice as transgenes under control of the H-2Kb promoter. Analysis of these animals revealed that signals originating from the TNFRp55 death domain are indispensable for the protection against Listeria monocytogenes, the expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the spleen and the development of splenic germinal centers. Furthermore, the transgenic coexpression of the TNFRp55 mutants in TNFRp55+/- mice exerts a dominant negative effect on the signaling of the endogenous receptor chains in vivo.     

Absence of TNFRp55 influences virus-induced autoimmunity despite efficient lymphocytic infiltration

Tumor necrosis factor (TNF)-alpha is a multipotent cytokine associated with many cellular functions, including inflammation and anti-viral defense. Many studies have implicated TNF-alpha in the pathogenesis of autoimmune diseases. TNF-alpha responses are mediated through binding to specific cell surface receptors, TNFRp55 and TNFRp75. The objective of the present study was to investigate the contribution of the TNFRp55 in the inflammatory response associated with autoimmune diabetes development in a viral transgenic model. In this model, the animals express lymphocytic choriomeningitis virus (LCMV)-glycoprotein (gp) in the beta cells of the pancreas under the control of the rat insulin promoter (RIP-gp). Diabetes is induced following LCMV infection due to beta cell destruction by LCMV-specific CD8+ cytotoxic T lymphocytes. TNFRp55-deficient RIP-gp animals were examined to assess the importance of the TNFRp55. The kinetics and onset of lymphocytic infiltration into the pancreatic islets and hyperglycemia was not altered in the absence of TNFRp55 after LCMV infection. Animals were evaluated following recombinant LCMV-gp vaccinia virus infection to test whether properties of the infectious agent influence autoimmunity. Interestingly, the kinetics were accelerated and the frequency of diabetes was increased in TNFRp55-deficient mice compared with control animals. This accelerated onset of diabetes is likely a result of increased viral replication in the TNFRp55-deficient host. Thus, these data demonstrate that TNFRp55 is not essential for producing the local inflammatory effects which contribute to organ-specific autoimmunity in this transgenic model. However, the absence of TNFRp55 altered the kinetics and incidence of the disease in a pathogen-dependent fashion.      

Vaccine-induced protection against gastrointestinal bacterial infections in the absence of secretory antibodies

Secretory IgA (SIgA) is widely held to be responsible for the defense of the mucosae against pathogenics and other potentially harmful agents. In this study, polymeric Ig receptor (pIgR) knockout mice, which lack secretory antibodies (SAb), were used to investigate the role of vaccine-elicited SAb in protection against gastrointestinal bacterial infections. An essential role for specific SAb in protection against Vibrio cholerae was evident from experiments showing that vaccinated pIgR(-/-) mice, but not vaccinated C57BL/6 mice, were susceptible to cholera toxin challenge. Vaccination of C57BL/6 mice with Salmonella typhimurium elicited strong antigen-specific, mucosal responses, which blocked in vitro invasion of epithelia. However, vaccinated C57BL/6 and pIgR(-/-) mice were equally resistant to challenge infection with virulent S. typhimurium. Finally, we investigated the importance of SIgA in protection against recurrent infections with Citrobacter rodentium. Although higher numbers of bacteria were detected early after challenge infection in feces of vaccinated pIgR(-/-) mice compared with vaccinated C57BL/6 mice, both mouse strains showed complete clearance after 9 days. These results suggested that, in immune animals, SIgA is crucial for the protection of gastrointestinal surfaces against secreted bacterial toxins, may inhibit early colonization by C. rodentium, but is not essential for protection against re-infection with S. typhimurium or C. rodentium.     

Interleukin-1 receptor signaling rather than that of tumor necrosis factor is critical in protecting the host from the severe consequences of a polymicrobe anaerobic infection

Infection of the dental pulp leads to an osteolytic lesion that results from a polymicrobial infection consisting largely of pathogenic anaerobes. Infection causes significant morbidity and mortality mediated by bacterial factors and in some cases by the up-regulation of inflammatory cytokines. The inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF), in particular, play a complex and central role in the responses to microbial pathogens. However, relatively little is known about the significance of these cytokines in protecting the host from focal polymicrobial anaerobic infections. To establish the relative importance of IL-1 and TNF in mediating the response to a mixed anaerobic infection, we inoculated the dental pulp of mice with six anaerobic pathogens containing functional deletions of receptors to IL-1 (IL-1R1(-/-)), TNF (TNFRp55(-/-)-p75(-/-)), or both (TNFRp55(-/-)-IL-1RI(-/-)). The results indicate that IL-1 receptor signaling and TNF receptor signaling both play similarly important roles in protecting the host from local tissue damage. However, IL-1 receptor signaling is considerably more important than TNF receptor signaling in preventing the spread of infection into surrounding fascial planes, since IL-1R1(-/-) but not TNFRp55(-/-)-p75(-/-) mice exhibited significantly higher morbidity and mortality. Moreover, all of the fatal infections occurred in male mice, suggesting the importance of gender differences in limiting the impact of these infections.     

Mast cells limit systemic bacterial dissemination but not colitis in response to Citrobacter rodentium

Enteropathogenic Escherichia coli and enterohemorrhagic E. coli cause an inflammatory colitis in human patients characterized by neutrophil infiltration, proinflammatory cytokine expression, and crypt hyperplasia. Citrobacter rodentium causes a similar colitis in mice and serves as a model for enteropathogenic E. coli infection in humans. C. rodentium induces systemic T-cell-dependent antibody production that facilitates clearance of the bacteria and protects the host from reinfection. The role of innate immune cells in infectious colitis, however, is less well understood. In this study, we have determined the role of mast cells in the inflammatory response and disease induced by C. rodentium. Mice deficient in mast cells exhibit more severe colonic histopathology and have a higher mortality rate following infection with C. rodentium than do wild-type animals. Despite unimpaired neutrophil recruitment and lymphocyte activation, mast cell-deficient mice have a disseminated infection evident in crucial organ systems that contributes to sepsis. Importantly, mast cells also have the capacity to directly kill C. rodentium. Together, these results suggest that mast cells protect the host from systemic infection by reducing the bacterial load and preventing dissemination of the bacterium from the colon.     

Mice lacking T and B lymphocytes develop transient colitis and crypt hyperplasia yet suffer impaired bacterial clearance during Citrobacter rodentium infection

The bacterial pathogen Citrobacter rodentium belongs to a family of gastrointestinal pathogens that includes enteropathogenic and enterohemorrhagic Escherichia coli and is the causative agent of transmissible colonic hyperplasia in mice. The molecular mechanisms used by these pathogens to colonize host epithelial surfaces and form attaching and effacing (A/E) lesions have undergone intense study. In contrast, little is known about the host's immune response to these infections and its importance in tissue pathology and bacterial clearance. To address these issues, wild-type mice and mice lacking T and B lymphocytes (RAG1 knockout [KO]) were infected with C. rodentium. By day 10 postinfection (p.i.), both wild-type and RAG1 KO mice developed colitis and crypt hyperplasia, and these responses became more exaggerated in wild-type mice over the next 2 weeks, as they cleared the infection. By day 24 p.i., bacterial clearance was complete, and the colitis had subsided; however, crypt heights remained increased. In contrast, inflammatory and crypt hyperplastic responses in the RAG1 KO mice were transient, subsiding after 2 weeks. By day 24 p.i., RAG1 KO mice showed no signs of bacterial clearance and infection was often fatal. Surprisingly, despite remaining heavily infected, tissues from RAG1 KO mice surviving the acute colitis showed few signs of disease. These results thus emphasize the important contribution of the host immune response during infection by A/E bacterial pathogens. While T and/or B lymphocytes are essential for host defense against C. rodentium, they also mediate much of the tissue pathology and disease symptoms that occur during infection.     

Interleukin-17 contributes to generation of Th1 immunity and neutrophil recruitment during Chlamydia muridarum genital tract infection but is not required for macrophage influx or normal resolution of infection

Interleukin 17 (IL-17) contributes to development of Th1 immunity and neutrophil influx during Chlamydia muridarum pulmonary infection, but its role during C. muridarum genital tract infection has not been described. We detected similar numbers of Chlamydia-specific Th17 and Th1 cells in iliac nodes of wild-type mice early during genital C. muridarum infection, while Th1 cells predominated later. il17ra(-/-) mice exhibited a reduced chlamydia-specific Th1 response in draining iliac nodes and decreased local IFN-γ production. Neutrophil influx into the genital tract was also decreased. However, il17ra(-/-) mice resolved infection normally, and no difference in pathology was observed compared to the wild type. Macrophage influx and tumor necrosis factor alpha (TNF-α) production were increased in il17ra(-/-) mice, providing a compensatory mechanism to effectively control chlamydial genital tract infection despite a reduced Th1 response. In ifnγ(-/-) mice, a marked increase in cellular infiltrates and chronic pathology was associated with an increased Th17 response. Although neutralization of IL-17 in ifnγ(-/-) mice decreased neutrophil influx, macrophage infiltration remained intact and the bacterial burden was not increased. Collectively, these results indicate that IL-17 contributes to the generation of Th1 immunity and neutrophil recruitment but is not required for macrophage influx or normal resolution of C. muridarum genital infection. These data highlight the redundant immune mechanisms operative at this mucosal site and the importance of examining site-specific responses to mucosal pathogens.     

Regulatory T cells promote a protective Th17-associated immune response to intestinal bacterial infection with C. rodentium

Intestinal infection with the mouse pathogen Citrobacter rodentium induces a strong local Th17 response in the colon. Although this inflammatory immune response helps to clear the pathogen, it also induces inflammation-associated pathology in the gut and thus, has to be tightly controlled. In this project, we therefore studied the impact of Foxp3⁺ regulatory T cells (Treg) on the infectious and inflammatory processes elicited by the bacterial pathogen C. rodentium. Surprisingly, we found that depletion of Treg by diphtheria toxin in the Foxp3^DTR (DEREG) mouse model resulted in impaired bacterial clearance in the colon, exacerbated body weight loss, and increased systemic dissemination of bacteria. Consistent with the enhanced susceptibility to infection, we found that the colonic Th17-associated T-cell response was impaired in Treg-depleted mice, suggesting that the presence of Treg is crucial for the establishment of a functional Th17 response after the infection in the gut. As a consequence of the impaired Th17 response, we also observed less inflammation-associated pathology in the colons of Treg-depleted mice. Interestingly, anti-interleukin (IL)-2 treatment of infected Treg-depleted mice restored Th17 induction, indicating that Treg support the induction of a protective Th17 response during intestinal bacterial infection by consumption of local IL-2.     

Differentiation, regulation, and death of T helper cell subsets during infection with leishmania major

The development and regulation of specific types of immune responses are dependent on understanding the biology of T helper cell (Th cells) subsets. We primarily use murine infections with the intracellular protozoan parasite,Leishmania major, as our model to investigate Th1/2 cell biology, where following infection, some inbred strains of mice develop a Th1 response and heal, but others develop a Th2 response and fail to control parasite replication. We focus on three major questions:

1. What factors are involved in Th cell development?
2. How can we switch an established immune response from one type to another?
3. How are immune responses downregulated once the parasites are eliminated?

We demonstrated that interleukin (IL) 12 promotes Thl cell development and that IL 12 is an effective adjuvant for cell-mediated immunity. We are now defining factors regulating the expression of the IL 12 receptor and the importance of CD28-B7 interactions for the development of vaccine-induced immunity. We also found that IL 12, in combination with chemotherapy, abrogates a Th2 response. These results have implications for treatment of infectious diseases, autoimmunity, and allergy. Currently, we are studying how such switching occurs. Finally, we found that TNFRp55^-/- mice are unable to heal leishmanial lesions in spite of eliminating the parasites. This result suggests that the tumor necrosis factor receptor (TNFR) p55 plays a critical, and previously unrecognized, role in downregulating pathogen-induced inflammatory responses. Our current hypothesis is that the TNFRp55 is required for induction of cell death in these lesions, and that in its absence, lymphocytes accumulate at the site of inflammation.