Hematopoietic cells are required to initiate a Chlamydia trachomatis-specific CD8+ T cell response

Chlamydia trachomatis is a global human pathogen causing diseases ranging from blinding trachoma to pelvic inflammatory disease. To explore how innate and adaptive immune responses cooperate to protect against systemic infection with C. trachomatis L2, we investigated the role of macrophages (Mphi) and dendritic cells (DCs) in the stimulation of C. trachomatis-specific CD8(+) T cells. We found that C. trachomatis infection of Mphi and DCs is far less productive than infection of nonprofessional APCs, the typical targets of infection. However, despite the limited replication of C. trachomatis within Mphi and DCs, infected Mphi and DCs process and present C. trachomatis CD8(+) T cell Ag in a proteasome-dependent manner. These findings suggest that although C. trachomatis is a vacuolar pathogen, some Ags expressed in infected Mphi and DCs are processed in the host cell cytosol for presentation to CD8(+) T cells. We also show that even though C. trachomatis replicates efficiently within nonprofessional APCs both in vitro and in vivo, Ag presentation by hematopoietic cells is essential for initial stimulation of C. trachomatis-specific CD8(+) T cells. However, when DCs infected with C. trachomatis ex vivo were adoptively transferred into naive mice, they failed to prime C. trachomatis-specific CD8(+) T cells. We propose a model for priming C. trachomatis-specific CD8(+) T cells whereby DCs acquire C. trachomatis Ag by engulfing productively infected nonprofessional APCs and then present the Ag to T cells via a mechanism of cross-presentation.     

N1L is an ectromelia virus virulence factor and essential for in vivo spread upon respiratory infection

The emergence of zoonotic orthopoxvirus infections and the threat of possible intentional release of pathogenic orthopoxviruses have stimulated renewed interest in understanding orthopoxvirus infections and the resulting diseases. Ectromelia virus (ECTV), the causative agent of mousepox, offers an excellent model system to study an orthopoxvirus infection in its natural host. Here, we investigated the role of the vaccinia virus ortholog N1L in ECTV infection. Respiratory infection of mice with an N1L deletion mutant virus (ECTVΔN1L) demonstrated profound attenuation of the mutant virus, confirming N1 as an orthopoxvirus virulence factor. Upon analysis of virus dissemination in vivo, we observed a striking deficiency of ECTVΔN1L spreading from the lungs to the livers or spleens of infected mice. Investigating the immunological mechanism controlling ECTVΔN1L infection, we found the attenuated phenotype to be unaltered in mice deficient in Toll-like receptor (TLR) or RIG-I-like RNA helicase (RLH) signaling as well as in those missing the type I interferon receptor or lacking B cells. However, in RAG-1(-/-) mice lacking mature B and T cells, ECTVΔN1L regained virulence, as shown by increasing morbidity and virus spread to the liver and spleen. Moreover, T cell depletion experiments revealed that ECTVΔN1L attenuation was reversed only by removing both CD4(+) and CD8(+) T cells, so the presence of either cell subset was still sufficient to control the infection. Thus, the orthopoxvirus virulence factor N1 may allow efficient ECTV infection in mice by interfering with host T cell function.     

Modulation of Innate Antigen-Presenting Cell Function by Pre-patent Schistosome Infection

Schistosomes are intravascular helminths that infect over 200 million people worldwide. Deposition of eggs by adult schistosomes stimulates Th2 responses to egg antigens and induces granulomatous pathology that is a hallmark of schistosome infection. Paradoxically, schistosomes require host immune function for their development and reproduction and for egress of parasite eggs from the host. To identify potential mechanisms by which immune cells might influence parasite development prior to the onset of egg production, we assessed immune function in mice infected with developing schistosomes. We found that pre-patent schistosome infection is associated with a loss of T cell responsiveness to other antigens and is due to a diminution in the ability of innate antigen-presenting cells to stimulate T cells. Diminution of stimulatory capacity by schistosome worms specifically affected CD11b⁺ cells and did not require concomitant adaptive responses. We could not find evidence for production of a diffusible inhibitor of T cells by innate cells from infected mice. Rather, inhibition of T cell responsiveness by accessory cells required cell contact and only occurred when cells from infected mice outnumbered competent APCs by more than 3∶1. Finally, we show that loss of T cell stimulatory capacity may in part be due to suppression of IL-12 expression during pre-patent schistosome infection. Modulation of CD4⁺ T cell and APC function may be an aspect of host immune exploitation by schistosomes, as both cell types influence parasite development during pre-patent schistosome infection.     

Correlates of protective immunity in poxvirus infection: where does antibody stand?

Even though smallpox has been eradicated, the threat of accidental or intentional release has highlighted the fact there is little consensus about correlates of protective immunity or immunity against re-infection with the causative poxvirus, variola virus (VARV). As the existing vaccine for smallpox has unacceptable rates of side effects and complications, new vaccines are urgently needed. Surrogate animal models of VARV infection in humans, including vaccinia virus (VACV) and ectromelia virus (ECTV) infection in mice, monkeypox virus (MPXV) infection in macaques have been used as tools to dissect the immune response to poxviruses. Mousepox, caused by ECTV, a natural mouse pathogen, is arguably the best surrogate small-animal model, as it shares many aspects of virus biology, pathology and clinical features with smallpox in humans. The requirements for recovery from a primary ECTV infection have been well characterized and include type I and II interferons, natural killer cells, CD4T cells, CD8T cell effector function and antibody. From a vaccine standpoint, it is imperative that the requirements for recovery from secondary infection are also identified. We have investigated host immune parameters in response to a secondary ECTV infection, and have identified that interferon and CD8T cell effector functions are not essential; however, T- and B-cell interaction and antibody are absolutely critical for recovery from a secondary challenge. The central role of antibody has been also been identified in the secondary response to other poxviruses. These findings have important clinical implications and would greatly assist the design of therapeutic interventions and new vaccines for smallpox.     

Direct CD28 costimulation is required for CD8+ T cell-mediated resistance to an acute viral disease in a natural host

Previous studies have suggested that, differing from model Ags, viruses that replicate extensively in the host still induce normal CD8+ T cell responses in the absence of CD28 costimulation. Because these studies were performed with viruses that do not normally cause acute disease, an important remaining question is whether CD28 costimulation is required for CD8+ T cell-mediated resistance to widely replicating but pathogenic viruses. To address this question, we studied the role of CD28 costimulation in CD8+ T cell-mediated resistance to mousepox, a disease of the mouse caused by the natural mouse pathogen, the ectromelia virus (ECTV). C57BL/6 (B6) mice are naturally resistant to mousepox, partly due to a fast and strong CD8+ T cell response. We found that B6 mice deficient in CD28 (CD28 knockout (KO)) are highly susceptible to lethal mousepox during the early stages of ECTV infection but can be protected by immunization with the antigenically related vaccinia virus (VACV) or by adoptive transfer of CD28 KO anti-VACV memory CD8+ cells. Of interest, a thorough comparison of the CD8+ T cell responses to ECTV and VACV suggests that the main reason for the susceptibility of CD28 KO mice to mousepox is a reduced response at the early stages of infection. Thus, while in the absence of CD28 costimulation the end point strength of the T cell responses to nonpathogenic viruses may appear normal, CD28 costimulation increases the speed of the T cell response and is essential for resistance to a life-threatening acute viral disease.     

Resistance to lethal ectromelia virus infection requires Type I interferon receptor in natural killer cells and monocytes but not in adaptive immune or parenchymal cells

Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.     

NOD2-deficient mice have impaired resistance to Mycobacterium tuberculosis infection through defective innate and adaptive immunity

NOD2/CARD15 mediates innate immune responses to mycobacterial infection. However, its role in the regulation of adaptive immunity has remained unknown. In this study, we examined host defense, T cell responses, and tissue pathology in two models of pulmonary mycobacterial infection, using wild-type and Nod2-deficient mice. During the early phase of aerosol infection with Mycobacterium tuberculosis, Nod2(-/-) mice had similar bacterial counts but reduced inflammatory response on histopathology at 4 and 8 wk postchallenge compared with wild-type animals. These findings were confirmed upon intratracheal infection of mice with attenuated Mycobacterium bovis bacillus Calmette-Guérin. Analysis of the lungs 4 wk after bacillus Calmette-Guérin infection demonstrated that Nod2(-/-) mice had decreased production of type 1 cytokines and reduced recruitment of CD8(+) and CD4(+) T cells. Ag-specific T cell responses in both the spleens and thoracic lymph nodes were diminished in Nod2(-/-) mice, indicating impaired adaptive antimycobacterial immunity. The immune regulatory role of NOD2 was not restricted to the lung since Nod2 disruption also led to reduced type 1 T cell activation following i.m. bacillus Calmette-Guérin infection. To determine the importance of diminished innate and adaptive immunity, we measured bacterial burden 6 mo after aerosol infection with M. tuberculosis and followed a second infected group for assessment of survival. Nod2(-/-) mice had a higher bacterial burden in the lungs 6 mo after infection and succumbed sooner than did wild-type controls. Taken together, these data indicate that NOD2 mediates resistance to mycobacterial infection via both innate and adaptive immunity.     

Inflammatory monocytes and neutrophils are licensed to kill during memory responses in vivo

Immunological memory is a hallmark of B and T lymphocytes that have undergone a previous encounter with a given antigen. It is assumed that memory cells mediate better protection of the host upon re-infection because of improved effector functions such as antibody production, cytotoxic activity and cytokine secretion. In contrast to cells of the adaptive immune system, innate immune cells are believed to exhibit a comparable functional effector response each time the same pathogen is encountered. Here, using mice infected by the intracellular bacterium Listeria monocytogenes, we show that during a recall bacterial infection, the chemokine CCL3 secreted by memory CD8+ T cells drives drastic modifications of the functional properties of several populations of phagocytes. We found that inflammatory ly6C+ monocytes and neutrophils largely mediated memory CD8+ T cell bacteriocidal activity by producing increased levels of reactive oxygen species (ROS), augmenting the pH of their phagosomes and inducing antimicrobial autophagy. These events allowed an extremely rapid control of bacterial growth in vivo and accounted for protective immunity. Therefore, our results provide evidence that cytotoxic memory CD8+ T cells can license distinct antimicrobial effector mechanisms of innate cells to efficiently clear pathogens.     

Intracellular Salmonella inhibit antigen presentation by dendritic cells

Dendritic cells (DC) are important APCs linking innate and adaptive immunity. During analysis of the intracellular activities of Salmonella enterica in DC, we observed that viable bacteria suppress Ag-dependent T cell proliferation. This effect was dependent on the induction of inducible NO synthase by DC and on the function of virulence genes in Salmonella pathogenicity island 2 (SPI2). Intracellular activities of Salmonella did not affect the viability, Ag uptake, or maturation of DC, but resulted in reduced presentation of antigenic peptides by MHC class II molecules. Increased resistance to reinfection was observed after vaccination of mice with SPI2-deficient Salmonella compared with mice vaccinated with SPI2-proficient Salmonella, and this correlated with an increased amount of CD4(+) as well as CD8(+) T cells. Our study is the first example of interference of an intracellular bacterial pathogen with Ag presentation by DC. The subversion of DC functions is a novel strategy deployed by this pathogen to escape immune defense, colonize host organs, and persist in the infected host.     

Defective early innate immune response to ectromelia virus in the draining lymph nodes of aged mice due to impaired dendritic cell accumulation

It is known that aging decreases natural resistance to viral diseases due to dysfunctional innate and adaptive immune responses, but the nature of these dysfunctions, particularly in regard to innate immunity, is not well understood. We have previously shown that C57BL/6J (B6) mice lose their natural resistance to footpad infection with ectromelia virus (ECTV) due to impaired maturation and recruitment of natural killer (NK) cells to the draining popliteal lymph node (dLN). More recently, we have also shown that in young B6 mice infected with ECTV, the recruitment of NK cells is dependent on a complex cascade whereby migratory dendritic cells (mDCs) traffic from the skin to the dLN, where they produce CCL2 and CCL7 to recruit inflammatory monocytes (iMOs). In the dLN, mDCs also upregulate NKG2D ligands to induce interferon gamma (IFN‐γ) expression by group 1 innate lymphoid cells (G1‐ILCs), mostly NK in cells but also some ILC1. In response to the IFN‐γ, the incoming uninfected iMOs secret CXCL9 to recruit the critical NK cells. Here, we show that in aged B6 mice, the trafficking of mDCs to the dLN in response to ECTV is decreased, resulting in impaired IFN‐γ expression by G1‐ILCs, reduced accumulation of iMOs, and attenuated CXCL9 production by iMOs, which likely contributes to decrease in NK cell recruitment. Together, these data indicate that defects in the mDC response to viral infection during aging result in a reduced innate immune response in the dLN and contribute to increased susceptibility to viral disease in the aged.     

Subdominant/cryptic CD8 T cell epitopes contribute to resistance against experimental infection with a human protozoan parasite

During adaptive immune response, pathogen-specific CD8(+) T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8(+) T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8(+) T cells of H-2(a) infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8(+) T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8(+) T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development.     

Poxvirus-encoded gamma interferon binding protein dampens the host immune response to infection

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-gamma) receptor 1. We generated an IFN-gamma binding protein (IFN-gammabp) deletion mutant of ECTV to study the role of viral IFN-gammabp (vIFN-gammabp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-gammabp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-gammabp from ECTV allowed the generation of an effective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-gammabp deletion mutant virus, designated ECTV-IFN-gammabp(Delta), produced increased levels of IFN-gamma and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-gamma and the virus' ability to dampen its effects.     

Differential effect of Listeria monocytogenes infection on cytokine production and cytotoxicity of CD8 T cells

Bacterial infection induces a shift to type 1 CD4 T cell subset in an infected host and this shift is important for protection of the host from disease development. Many researchers think that the shift is antigen-dependent, but we previously demonstrated an initial induction step for CD4 T cell subsets during Listeria monocytogenes (Lm) infection is antigen-independent. Although Listeria is a TLR2 ligand, the immune system of the Lm-infected host responded to the pathogen to induce expression of CD69 but not CD25 on CD4 T cells, CD8 T cells and B cells even in the absence of TLR2 or MyD88. The antigen-independent activation of type 1 CD4 T cells accelerate the clearance of pathogens by activating innate immune cells with type 1 cytokines. Type 1 CD4 T cells and CD8 T cells also collaborate to protect the host from intracellular Lm infection. Since CD8 T cells function mainly as cytotoxic T cells and CD69-positive CD8 T cells increase during Lm-infection, cytotoxic activity of CD8 T cells was evaluated during Lm-infection. Although CD8 T cells were activated to produce IFN-gamma, the cytotoxic function of CD8 T cells in Lymphocytic choriomeningitis virus (LCMV) p14 TCR-transgenic mouse was not augmented by Lm-infection. Therefore, Lm-infection differentially influences on cytokine production and cytotoxicity of CD8 T cells.     

Functional CD8+ T cell responses in lethal Ebola virus infection

Ebola virus (EBOV) causes highly lethal hemorrhagic fever that leads to death in up to 90% of infected humans. Like many other infections, EBOV induces massive lymphocyte apoptosis, which is thought to prevent the development of a functional adaptive immune response. In a lethal mouse model of EBOV infection, we show that there is an increase in expression of the activation/maturation marker CD44 in CD4(+) and CD8(+) T cells late in infection, preceding a dramatic rebound of lymphocyte numbers in the blood. Furthermore, we observed both lymphoblasts and apoptotic lymphocytes in spleen late in infection, suggesting that there is lymphocyte activation despite substantial bystander apoptosis. To test whether these activated lymphocytes were functional, we performed adoptive transfer studies. Whole splenocytes from moribund day 7 EBOV-infected animals protected naive animals from EBOV, but not Marburgvirus, challenge. In addition, we observed EBOV-specific CD8(+) T cell IFN-gamma responses in moribund day 7 EBOV-infected mice, and adoptive transfer of CD8(+) T cells alone from day 7 mice could confer protection to EBOV-challenged naive mice. Furthermore, CD8(+) cells from day 7, but not day 0, mice proliferated after transfer to infected recipients. Therefore, despite significant lymphocyte apoptosis, a functional and specific, albeit insufficient, adaptive immune response is made in lethal EBOV infection and is protective upon transfer to naive infected recipients. These findings should cause a change in the current view of the 'impaired' immune response to EBOV challenge and may help spark new therapeutic strategies to control lethal filovirus disease.     

Preferential infection of mature dendritic cells by mouse hepatitis virus strain JHM

Mouse hepatitis virus strain JHM (MHV-JHM) causes acute encephalitis and acute and chronic demyelinating diseases in mice. Dendritic cells (DCs) are key cells in the initiation of innate and adaptive immune responses, and infection of these cells could potentially contribute to a dysregulated immune response; consistent with this, recent results suggest that DCs are readily infected by another strain of mouse hepatitis virus, the A59 strain (MHV-A59). Herein, we show that the JHM strain also productively infected DCs. Moreover, mature DCs were at least 10 times more susceptible than immature DCs to infection with MHV-JHM. DC function was impaired after MHV-JHM infection, resulting in decreased stimulation of CD8 T cells in vitro. Preferential infection of mature DCs was not due to differential expression of the MHV-JHM receptor CEACAM-1a on mature or immature cells or to differences in apoptosis. Although we could not detect infected DCs in vivo, both CD8(+) and CD11b(+) splenic DCs were susceptible to infection with MHV-JHM directly ex vivo. This preferential infection of mature DCs may inhibit the development of an efficient immune response to the virus.     

Adaptive immune cells temper initial innate responses

Toll-like receptors (TLRs) recognize conserved microbial structures called pathogen-associated molecular patterns. Signaling from TLRs leads to upregulation of co-stimulatory molecules for better priming of T cells and secretion of inflammatory cytokines by innate immune cells. Lymphocyte-deficient hosts often die of acute infection, presumably owing to their lack of an adaptive immune response to effectively clear pathogens. However, we show here that an unleashed innate immune response due to the absence of residential T cells can also be a direct cause of death. Viral infection or administration of poly(I:C), a ligand for TLR3, led to cytokine storm in T-cell- or lymphocyte-deficient mice in a fashion dependent on NK cells and tumor necrosis factor. We have further shown, through the depletion of CD4+ and CD8+ cells in wild-type mice and the transfer of T lymphocytes into Rag-1-deficient mice, respectively, that T cells are both necessary and sufficient to temper the early innate response. In addition to the effects of natural regulatory T cells, close contact of resting CD4+CD25-Foxp3- or CD8+ T cells with innate cells could also suppress the cytokine surge by various innate cells in an antigen-independent fashion. Therefore, adaptive immune cells have an unexpected role in tempering initial innate responses.     

The ectromelia virus SPI-2 protein causes lethal mousepox by preventing NK cell responses

Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.     

The pathogenesis of schistosomiasis is controlled by cooperating IL-10-producing innate effector and regulatory T cells

IL-10 reduces immunopathology in many persistent infections, yet the contribution of IL-10 from distinct cellular sources remains poorly defined. We generated IL-10/recombination-activating gene (RAG)2-deficient mice and dissected the role of T cell- and non-T cell-derived IL-10 in schistosomiasis by performing adoptive transfers. In this study, we show that IL-10 is generated by both the innate and adaptive immune response following infection, with both sources regulating the development of type-2 immunity, immune-mediated pathology, and survival of the infected host. Importantly, most of the CD4(+) T cell-produced IL-10 was confined to a subset of T cells expressing CD25. These cells were isolated from egg-induced granulomas and exhibited potent suppressive activity in vitro. Nevertheless, when naive, naturally occurring CD4(+)CD25(+) cells were depleted in adoptive transfers, recipient IL-10/RAG2-deficient animals were more susceptible than RAG2-deficient mice, confirming an additional host-protective role for non-T cell-derived IL-10. Thus, innate effectors and regulatory T cells producing IL-10 cooperate to reduce morbidity and prolong survival in schistosomiasis.     

Mucosal immune responses are related to reduction of bacterial colonization in the stomach after therapeutic Helicobacter pylori immunization in mice

The aim of this study was to investigate the capacity of oral and parenteral therapeutic immunization to reduce the bacterial colonization in the stomach after experimental Helicobacter pylori infection, and to evaluate whether any specific immune responses are related to such reduction. C57BL/6 mice were infected with H. pylori and thereafter immunized with H. pylori lysate either orally together with cholera toxin or intraperitoneally (i.p.) together with alum using immunization protocols that previously have provided prophylactic protection. The effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori from the mouse stomach. Mucosal and systemic antibody responses were analyzed by ELISA in saponin extracted gastric tissue and serum, respectively, and mucosal CD4+ T cell responses by an antigen specific proliferation assay. Supernatants from the proliferating CD4+ T cells were analyzed for Th1 and Th2 cytokines. The oral, but not the parenteral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice. The oral immunization resulted in markedly elevated levels of serum IgG+M as well as gastric IgA antibodies against H. pylori antigen and also increased H. pylori specific mucosal CD4+ T cell proliferation with a Th1 cytokine profile. Although the parenteral immunization induced dramatic increases in H. pylori specific serum antibody titers, no increases in mucosal antibody or cellular immune responses were observed after the i.p. immunization compared to control infected mice. These findings suggest that H. pylori specific mucosal immune responses with a Th1 profile may provide therapeutic protection against H. pylori.