Depressed Type 1 Cytokine Synthesis by Superantigen-Activated CD4+ T Cells of Women with Human Papillomavirus-Related High-Grade Squamous Intraepithelial Lesions

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4⁺ T and CD8⁺ T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4⁺ T cells that produced IL-2 (P = 0.045), IFN-γ (P = 0.040), and TNF-α (P = 0.015) and a significantly lower percentage of activated CD8⁺ T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4⁺ T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8⁺ T cells.     

Regulatory T Cells Modulate Staphylococcal Enterotoxin B-Induced Effector T-Cell Activation and Acceleration of Colitis

Oral administration of bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) activates mucosal T cells but does not cause mucosal inflammation. We examined the effect of oral SEB on the development of mucosal inflammation in mice in the absence of regulatory T (Treg) cells. SCID mice were fed SEB 3 and 7 days after reconstitution with CD4⁺ CD45RB^high or CD4⁺ CD45RB^high plus CD4⁺ CD45RB^low T cells. Mice were sacrificed at different time points to examine changes in tissue damage and in T-cell phenotypes. Feeding SEB failed to produce any clinical effect on SCID mice reconstituted with CD4⁺ CD45RB^high and CD4⁺ CD45RB^low T cells, but feeding SEB accelerated the development of colitis in SCID mice reconstituted with CD4⁺ CD45RB^high T cells alone. The latter was associated with an increase in the number of CD4⁺ Vβ8⁺ T cells expressing CD69 and a significantly lower number of CD4⁺ CD25⁺ Foxp3⁺ T cells. These changes were not observed in SCID mice reconstituted with both CD45RB^high and CD45RB^low T cells. In addition, SEB impaired the development of Treg cells in the SCID mice reconstituted with CD4⁺ CD45RB^high T cells alone but had no direct effect on Treg cells. In the absence of Treg cells, feeding SEB induced activation of mucosal T cells and accelerated the development of colitis. This suggests that Treg cells prevent SEB-induced mucosal inflammation through modulation of SEB-induced T-cell activation.     

The role of antigen-presenting cells and interleukin-12 in the priming of antigen-specific CD4+ T cells by immune stimulating complexes

Immune stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A are vaccine adjuvants that promote a wide range of immune responses in vivo, including delayed-type hypersensitivity (DTH) and the secretion of both T helper 1 (Th1) and Th2 cytokines. However, the antigen-presenting cell (APC) responsible for the induction of these responses has not been characterized. Here we have investigated the role of dendritic cells (DC), macrophages (Mφ) and B cells in the priming of antigen-specific CD4⁺ T cells in vitro by ISCOMs containing ovalbumin (OVA). OVA ISCOMs pulsed bone marrow (BM)-derived DC but not BM Mφ, nor naïve B cells prime resting antigen-specific CD4⁺ T cells, and this response is greatly enhanced if DC are activated with lipopolysaccharide (LPS). Of the APC found in the spleen, only DC had the capacity to prime resting antigen specific CD4⁺ T cells following exposure to OVA ISCOMs in vitro, while Mφ and B cells were ineffective. DC, but not B cells purified from the draining lymph nodes of mice immunized with OVA ISCOMs also primed resting antigen-specific CD4⁺ T cells in vitro, suggesting that DC are also critical in vivo. Using DC and T cells from interleukin (IL)-12 p40^−/− mice, we also identified a crucial role for IL-12 in the priming of optimal CD4⁺ T cell responses by OVA ISCOMs. We suggest that DC are the principal APC responsible for the priming of CD4⁺ T cells by ISCOMs in vivo and that directed targeting of these vectors to DC may enhance their efficancy as vaccine adjuvants.     

Reciprocal Regulation of Th1- and Th2-Cytokine-Producing T Cells during Clearance of Parasitemia in Plasmodium falciparum Malaria

Flow cytometry for the intracellular detection of T-cell cytokines was performed for 15 Gabonese patients during acute uncomplicated Plasmodium falciparum malaria. A striking expansion of CD4⁺ and CD8⁺ T cells producing gamma interferon (IFN-γ) was found during drug-induced clearance of parasitemia, paralleled by a decrease of interleukin-2 (IL-2) production. The frequency of IL-4- and IL-13-producing CD4⁺ cells gradually decreased, whereas the frequency of T cells producing IL-2⁺–IFN-γ⁺, IL-4⁻–IL-5⁺, and IL-4⁺–IL-5⁺ cytokines as well as IL-4⁺–IFN-γ⁺ and IL-13⁺–IFN-γ⁺ cytokines was not significantly altered. The capacity for IL-10 production within the CD4⁺ subset increased due to an expansion of both IL-10⁺–IFN-γ⁻ and IL-10⁺–IFN-γ⁺ cytokine-expressing cells. Thus, a more pronounced Th2-driven immune response during acute untreated P. falciparum infection with a shift towards Th1 responsiveness induced by parasite clearance is suggested.     

Temporal regulation of interleukin-12p70 (IL-12p70) and IL-12-related cytokines in splenic dendritic cell subsets during Leishmania donovani infection

Dendritic cells (DC) play an essential role in initiating and directing T-cell responses, in part by production of interleukin-12p70 (IL-12p70), IL-23, and IL-27. However, comparative studies on the capacity for cytokine production of DC subsets are rare. Here, we compare splenic CD8α⁺, CD4⁺, and double-negative (DN) DC, isolated 5 h to 28 days after Leishmania donovani infection, for (i) production of IL-12p70, (ii) accumulation of IL-12/23p40, IL-12p35, IL-23p19, and IL-27p28 mRNAs, and (iii) their capacity to direct CD4⁺ T-cell differentiation. At 5 h, conventional DC (cDC) accumulated mRNA for IL-12/23p40 (CD8α>CD4>DN), IL-23p19 (CD4>CD8α>DN), and IL-27p28 (CD8α>CD4>DN), in an infection dose-dependent manner. IL-12p70 was restricted to CD8α⁺ cDC, reflecting the subset-specific accumulation of IL-12p35 mRNA. In contrast, cDC from mice infected for 14 to 28 days accumulated little mRNA for IL-12p40 and IL-12p19, though IL-27p28 mRNA remained detectable (CD8α>DN>CD4). IL-12p70 secretion by CD8α⁺ cDC was also absent, reflecting deficient IL-12/23p40, rather than IL-12p35, mRNA accumulation. The capacity of CD8α⁺ cDC isolated early after infection to direct Th1 cell differentiation was mediated through IL-12/23p40, whereas this ability in CD4⁺ and DN cDC was independent of IL-12/23p40 and did not result from overexpression of Delta 4 Notch-like ligand. However, DN cDC produced gamma interferon (IFN-γ) and also contained a rare population of CD11c^hi DX5⁺ IFN-γ-producing cells. Our data illustrate the extensive diversity in, and temporal regulation of, splenic cDC subsets during infection and suggest caution in interpreting data obtained with unfractionated or minimally purified DC.     

Expression of Regeneration and Tolerance Factor Correlates Directly with Human Immunodeficiency Virus Infection and Inversely with Hepatitis C Virus Infection

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause two of the most prevalent debilitating viral infections. HIV appears to induce a skewing toward a Th2 response, while in HCV infection a Th1 response appears to dominate. Regeneration and tolerance factor (RTF) may participate in driving or sustaining a Th2 cytokine response. The expression of RTF on CD3⁺ T cells of HIV-seropositive (HIV⁺) individuals is increased. The purpose of this study was to compare the expression of RTF during HIV infections with that during HCV infections. Three-color flow-cytometric analysis of peripheral blood collected from HIV⁺ HCV-seropositive (HCV⁺), HIV- and HCV-seropositive (HIV⁺ HCV⁺), and HIV- and HCV-seronegative (HIV⁻ HCV⁻) individuals was performed. Levels of RTF expression on T-lymphocyte subsets from these groups were compared, as were levels of RTF expression on activated T cells expressing CD38 and HLA-DR, to determine the relationship of RTF expression to these infections. We demonstrated that the expression of RTF on surfaces of T cells from HIV⁺ individuals is upregulated and that its expression on T cells from HCV⁺ individuals is downregulated. A twofold increase in the mean channel fluorescence of RTF on CD3⁺ T cells was seen in both HIV⁺ and HIV⁺ HCV⁺ individuals compared to HIV⁻ HCV⁻ individuals. HCV⁺ individuals had lower levels of RTF expression than HIV⁻ HCV⁻ individuals (P < 0.005 for CD4⁺; P < 0.0005 for CD8⁺). In terms of percentages of T cells expressing RTF, the groups were ranked as follows: HIV⁺ > HIV⁺ HCV⁺ > HIV⁻ HCV⁻ > HCV⁺. The results indicate that RTF expression correlates with HIV-associated immune activation and may be associated with Th2-type responses.     

Infection with Mycobacterium bovis BCG Diverts Traffic of Myelin Oligodendroglial Glycoprotein Autoantigen-Specific T Cells Away from the Central Nervous System and Ameliorates Experimental Autoimmune Encephalomyelitis

Infectious agents have been proposed to influence susceptibility to autoimmune diseases such as multiple sclerosis. We induced a Th1-mediated central nervous system (CNS) autoimmune disease, experimental autoimmune encephalomyelitis (EAE) in mice with an ongoing infection with Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) to study this possibility. C57BL/6 mice infected with live BCG for 6 weeks were immunized with myelin oligodendroglial glycoprotein peptide (MOG_35-55) to induce EAE. The clinical severity of EAE was reduced in BCG-infected mice in a BCG dose-dependent manner. Inflammatory-cell infiltration and demyelination of the spinal cord were significantly lessened in BCG-infected animals compared with uninfected EAE controls. ELISPOT and gamma interferon intracellular cytokine analysis of the frequency of antigen-specific CD4⁺ T cells in the CNS and in BCG-induced granulomas and adoptive transfer of MOG_35-55-specific green fluorescent protein-expressing cells into BCG-infected animals indicated that nervous tissue-specific (MOG_35-55) CD4⁺ T cells accumulate in the BCG-induced granuloma sites. These data suggest a novel mechanism for infection-mediated modulation of autoimmunity. We demonstrate that redirected trafficking of activated CNS antigen-specific CD4⁺ T cells to local inflammatory sites induced by BCG infection modulates the initiation and progression of a Th1-mediated CNS autoimmune disease.     

Primary CD8+ T-cell response to soluble ovalbumin is improved by chloroquine treatment in vivo

The efficiency of cross-presentation of exogenous antigens by dendritic cells (DCs) would seem to be related to the level of antigen escape from massive degradation mediated by lysosomal proteases in an acidic environment. Here, we demonstrate that a short course of treatment with chloroquine in mice during primary immunization with soluble antigens improved the cross-priming of naïve CD8⁺ T lymphocytes in vivo. More specifically, priming of chloroquine-treated mice with soluble ovalbumin (OVA), OVA associated with alum, or OVA pulsed on DCs was more effective in inducing OVA-specific CD8⁺ T lymphocytes than was priming of untreated mice. We conclude that chloroquine treatment improves the cross-presentation capacity of DCs and thus the size of effector and memory CD8⁺ T cells during vaccination.     

Antigen-Specific CD25−Foxp3−IFN-γhighCD4+ T Cells Restrain the Development of Experimental Allergic Encephalomyelitis by Suppressing Th17

The current study identifies within the Th1 subtype two distinct CD4⁺ populations: those capable of transferring inflammatory autoimmunity and others that regulate its development by suppressing Th17 in an interferon (IFN)-γ-dependent manner. These CD4⁺IFN-γ^highIL-4^lowIL-10^lowTGF-β^lowFOXp3⁻ cells in fact function as antigen-specific regulatory cells that restrain the development of autoimmunity by increasing the threshold of Th17 activation. We show that development of autoimmune conditions within the central nervous system is dependent on the Fas ligand-mediated apoptosis of these regulatory cells at early stages of disease. We also show that not only is the function of these cells IFN-γ dependent but also that stable over expression of IFN-γ in encephalitogenic CD4⁺ T cells redirects their biological function to become antigen-specific regulatory cells. This may also explain, in part, the pleiotropic role of IFN-γ in the regulation of autoimmunity, as previously observed by others.Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system (CNS) that serves as an experimental model for multiple sclerosis. It is believed that antigen-specific effector CD4⁺ T cells that produce interleukin (IL)-17 (ie, Th17) initiate the inflammatory process, which is later propagated by IFN-γ^highIL-4^low Th1 cells.^1,2,3,4,5 IFN-γ is a key cytokine that suppresses the selection and activity of Th17 cells.⁶ It is therefore thought that the dynamics of the autoimmune process includes the initiation of disease by Th17 and, later on, the propagation of the inflammatory process by Th1, which, via IFN-γ, suppresses and replaces Th17,⁷ thus making the Th17/Th1 ratio an important factor in the dynamics of disease.¹Thus far, much attention has been devoted to identifying regulatory T cells that restrain effector T cell functions. These include CD25⁺Foxp3⁺CD4⁺ T cells, which are likely to suppress effector cell functions nonspecifically,⁸ antigen-specific regulatory T cells that produce IL-10 (ie, Tr1),^9,10,11 and those producing transforming growth factor (TGF)-β (ie, Th3).¹² Antigen-specific regulatory T cells that selectively restrain Th17 have not been so far identified.We have previously shown that Fas ligand (FasL) plays a dual role in regulating EAE. Targeted neutralization of this tumor necrosis factor (TNF) family member at early stages of disease prevented the development of disease, whereas its neutralization at later stages, when the activity of Th1 drives the pathogenesis of disease,¹ aggravated its manifestation.¹³ The mechanistic basis of exacerbating disease most likely includes inhibition of apoptosis of Th1 cells at the autoimmune site.¹³ Nevertheless, understanding why early neutralization of FasL suppresses EAE remains elusive.The affinity of TCR to major histocompatibility complex II-peptide complex during CD4⁺ T cell selection affects their biological properties. Whereas higher affinity of biding enhances the selection of very high IFN-γ-producing Th1 cells, low affinity binding would results in lower IFN-γ-producing Th1 cells.¹⁴ Those CD4⁺ T cells that produce higher levels of IFN-γ were found to be more susceptible to FasL-mediated apoptosis, in an IFN-γ-dependent manner, probably as a part of the natural regulation of T cell homeostasis.¹⁵ The current study shows that these antigen-specific T cells emerge during early stages of an autoimmune condition and suppress Th17 cells in attempting to block the development of disease. Once they undergo FasL-induced apoptosis the autoimmune condition develops. Therefore, rescuing these cells by anti-FasL antibodies (Abs) at early stages of the disease suppresses its development.     

Primary activation of antigen-specific naive CD4+ and CD8+ T cells following intranasal vaccination with recombinant bacteria

The primary activation of T-helper and T-cytotoxic cells following mucosal immunization with recombinant Streptococcus gordonii was studied in vivo by adoptive transfer of ovalbumin (OVA)-specific transgenic CD8⁺ (OT-I) and CD4⁺ (OT-II) T cells. A recombinant strain, expressing on the surface the vaccine antigen Ag85B-ESAT-6 from Mycobacterium tuberculosis fused to OVA T-helper and T-cytotoxic epitopes (peptides 323 to 339 and 257 to 264), was constructed and used to immunize C57BL/6 mice by the intranasal route. Recombinant, but not wild-type, bacteria induced OVA-specific CD4⁺ and CD8⁺ T-cell clonal expansion in cervical lymph nodes, lung, and spleen. OVA-specific CD4⁺ and CD8⁺ T-cell proliferation appeared first in cervical lymph nodes and later in the spleen, suggesting a possible migration of activated cells from the inductive site to the systemic district. A significant correlation between the percentages of CD4⁺ and CD8⁺ proliferating T cells was observed for each animal. The expression of CD69, CD44, and CD45RB on proliferating T lymphocytes changed as a function of the cell division number, confirming T-cell activation following the antigen encounter. These data indicate that intranasal immunization with recombinant S. gordonii is capable of inducing primary activation of naive antigen-specific CD4⁺ and CD8⁺ T cells, both locally and systemically.     

Effects of CXCL10 on dendritic cell and CD4+ T-cell functions during Leishmania amazonensis infection

Leishmania amazonensis can cause progressive disease in most inbred strains of mice. We have previously reported that treatment with CXCL10 activates macrophage (MΦ) effector function(s) in parasite killing and significantly delays lesion development in susceptible C57BL/6 mice via enhanced gamma interferon (IFN-γ) and interleukin 12 (IL-12) secretion; however, the mechanism underlying this enhanced immunity against L. amazonensis infection remains largely unresolved. In this study, we utilized stationary promastigotes to infect bone marrow-derived dendritic cells (DCs) of C57BL/6 mice and assessed the activation of DC subsets and the capacity of these DC subsets to prime CD4⁺ T cells in vitro. We found that CXCL10 induced IL-12 p40 production but reduced IL-10 production in uninfected DCs. Yet L. amazonensis-infected DCs produced elevated levels of IL-10 despite CXCL10 treatment. Elimination of endogenous IL-10 led to increased IL-12 p40 production in DCs as well as increased proliferation and IFN-γ production by in vitro-primed CD4⁺ T cells. In addition, CXCL10-treated CD4⁺ T cells became more responsive to IL-12 via increased expression of the IL-12 receptor β₂ chain and produced elevated levels of IFN-γ. This report indicates the utility of CXCL10 in generating a Th1-favored, proinflammatory response, which is a prerequisite for controlling Leishmania infection.     

CD40-activated B cells are more potent than immature dendritic cells to induce and expand CD4+ regulatory T cells

CD4⁺ regulatory T cells (Tregs) play an important role in maintaining immune tolerance by suppressing pathologic immune responses. The generation of large numbers of antigen-specific Tregs ex vivo is critical for the development of clinical immunotherapy based on the adoptive transfer of Tregs. Both CD40-activated B cells (CD40-B) and immature dendritic cells (imDCs) have been used as professional antigen-presenting cells (APCs) to generate antigen-specific Tregs. However, the efficiencies of CD40-B and imDCs to generate CD4⁺ Tregs have not been compared directly and the mechanism driving the generation of these Tregs remains largely unknown. In this study, we found that CD40-B exhibited mature phenotypes and were more able to induce and expand CD4^highCD25⁺ Tregs than imDCs. Moreover, Tregs induced by CD40-B had greater suppressive capacity than those induced by imDCs. The generation of CD4^highCD25⁺ Tregs by CD40-B and imDCs is cell–cell contact dependent and partially relies on the expression of human leukocyte antigen (HLA)-DR and CD80/86. Differences in CD4^highCD25⁺ Treg generation efficiency were largely explained by the production of endogenous IL-2 by CD40-B. Our results suggest that CD40-B is better able to generate large numbers of antigen-specific Tregs than imDCs. Additionally, using CD40-B to generate Tregs may accelerate the clinical use of Treg-based immunotherapy in the treatment of allograft rejection, graft versus host disease (GVHD) and autoimmune diseases.     

Acquired pMHC I Complexes Greatly Enhance CD4＋ Th Cell＇s Stimulatory Effect on CD8＋T Cell-Mediated Diabetes in Transgenic RIP-mOVA Mice

CD4＋ helper T （Th） cells play pivotal roles in induction of CD8＋ CTL immunity. However, the mechanism of CD4＋ T cell help delivery to CD8＋ T cells in vivo is still elusive. In this study, we used ovalbumin （OVA）-pulsed dendritic cells （DCovA） to activate OT-II mouse CD4＋ T cells, and then studied the help effect of these CD4＋ T cells on CD8＋ cytotoxic T lymphocyte （CTL） responses＋ We also examined CTL mediated islet β cell destruction which leaded to diabetes in wild-type C57BL/6 mice and transgenic rat insulin promoter （RIP）-mOVA mice expressing β cell antigen OVA with self OVA-specific tolerance, respectively. In adoptive transfer experiments, we demonstrated that help, in the form of peptide/major histocompatibility complex （pMHC） I acquired from DCovA by DCovA activation, was required for induction of OVA-specific CTL responses in C57BL/6 mice. However, in combination ＋ ＋ ＋ with TCR transgenlc OT-I mouse CD8 T ceils, the tolerogenic dosage of CD4＋ Th cells with acquired pMHC I, but not CD4＋ （Kb-/-） Th cells without acquired pMHC I were able to cause diabetes in 8/10 （80%） RIP-mOVA mice. This study thus expands the current knowledge in T cell-mediated autoimmunity and provides insight into the nature of CD4＋ T cell-mediated help in CD8＋ CTL induction. Cellular ＆ Molecular Immunology. 2008;5（6）：407-415.     

Antrum- and Corpus Mucosa-Infiltrating CD4+ Lymphocytes in Helicobacter pylori Gastritis Display a Th1 Phenotype

In this study, cytokine patterns produced by CD4⁺ T cells isolated from antrum or corpus gastral biopsy specimens of 10 patients with Helicobacter pylori-positive gastritis were compared. To this end, expression of intracellular cytokines (interleukin-4 [IL-4] and gamma interferon) and of CD4 was assessed by flow cytometry. Ten to 60% of the isolated CD4⁺ T cells produced gamma interferon upon stimulation. With the exception of one patient, IL-4-positive CD4⁺ cells were not detected. Therefore, CD4⁺ cells infiltrating antrum and corpus stomach mucosa during H. pylori infection show a Th1 phenotype. This polarized Th1-type response may contribute to the inability of the immune system to eradicate H. pylori infection.     

Phase Variation of Hemoglobin Utilization in Neisseria gonorrhoeae

Most Neisseria gonorrhoeae isolates are unable to use human hemoglobin as the sole source of iron for growth (Hgb⁻), but a minor population is able to do so (Hgb⁺). This minor population grows luxuriously on hemoglobin, expresses two outer membrane proteins of 42 kDa (HpuA) and 89 kDa (HpuB), and binds hemoglobin under iron-stressed conditions. In addition to the previously reported HpuB, we identified and characterized HpuA, which is encoded by the gene hpuA, located immediately upstream of hpuB. Expression of both proteins was found to be controlled at the translational level by frameshift mutations in a run of guanine residues within the hpuA sequence encoding the mature HpuA protein. The “on-phase” hemoglobin-utilizing variants contained 10 G’s, while the “off-phase” variants contained 9 G’s. Insertional hpuB mutants of FA19 Hgb⁺ and FA1090 Hgb⁺ no longer expressed HpuB but still produced HpuA. A polar insertional mutation of the upstream hpuA gene in FA1090 Hgb⁺ eliminated production of both HpuA and HpuB, whereas a nonpolar insertional mutant expressed HpuB only. Insertional mutagenesis of either hpuA or hpuB or both substantially decreased the hemoglobin binding ability of the FA1090 Hgb⁺ variant and prevented growth on hemoglobin plates. Therefore, both HpuA and HpuB were required for the utilization of hemoglobin for growth.     

Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-Stage Plasmodium chabaudi AS Infection in Mice

Dendritic cells (DCs) are important accessory cells for promoting NK cell gamma interferon (IFN-γ) production in vitro in response to Plasmodium falciparum-infected red blood cells (iRBC). We investigated the requirements for reciprocal activation of DCs and NK cells leading to Th1-type innate and adaptive immunity to P. chabaudi AS infection. During the first week of infection, the uptake of iRBC by splenic CD11c⁺ DCs in resistant wild-type (WT) C57BL/6 mice was similar to that in interleukin 15^−/− (IL-15^−/−) and IL-12p40^−/− mice, which differ in the severity of P. chabaudi AS infection. DCs from infected IL-15^−/− mice expressed costimulatory molecules, produced IL-12, and promoted IFN-γ secretion by WT NK cells in vitro as efficiently as WT DCs. In contrast, DCs from infected IL-12p40^−/− mice exhibited alterations in maturation and cytokine production and were unable to induce NK cell IFN-γ production. Coculture of DCs and NK cells demonstrated that DC-mediated NK cell activation required IL-12 and, to a lesser extent, IL-2, as well as cell-cell contact. In turn, NK cells from infected WT mice enhanced DC maturation, IL-12 production, and priming of CD4⁺ T-cell proliferation and IFN-γ secretion. Infected WT mice depleted of NK cells, which exhibit increased parasitemia, had impaired DC maturation and DC-induced CD4⁺ Th1 cell priming. These findings indicate that DC-NK cell reciprocal cross talk is critical for control and rapid resolution of P. chabaudi AS infection and provide in vivo evidence for the importance of this interaction in IFN-γ-dependent immunity to malaria.     

Lack of Interleukin-12 in p40-Deficient Mice Leads to Poor CD8+ T-Cell Immunity against Encephalitozoon cuniculi Infection

A CD8⁺ T-cell response is critical for protection against Encephalitozoon cuniculi infection. However, the factors responsible for the generation of CD8⁺ T-cell immunity during E. cuniculi infection and the cytokines involved in this process have not been identified. In the present study, we demonstrated that p40-deficient animals, which are unable to produce interleukin-12 (IL-12), have a serious defect in expansion of the CD8⁺ T-cell response which compromises the survival of an infected host. Adoptive transfer of CD8⁺ T cells from immunocompetent donors protected SCID mice infected with E. cuniculi, whereas administration of CD8⁺ T cells from p40^−/− mice failed to protect infected SCID mice. In vitro dendritic cell (DC) cultures from knockout mice pulsed with E. cuniculi spores were unable to develop a robust CD8⁺ T-cell immune response. Addition of exogenous IL-12 or transfer of CD8⁺ T cells that were initially primed with DC from p40^−/− animals to DC cultures from immunocompetent mice (directly or via transwells) led to optimal expansion of these cells. This IL-12-mediated reinstatement of CD8⁺ T-effector immunity was independent of gamma interferon (IFN-γ) as addition of antibody to the cultures failed to have an effect. These studies demonstrated that IL-12 plays a predominant role in the expansion of effector CD8⁺ T-cell immunity against E. cuniculi, which is critical for host survival. These findings are very important for understanding the protective immune mechanisms needed to protect an immunocompromised host against an opportunistic infection and can be extended to other microsporidial pathogens.Encephalitozoon cuniculi is an opportunistic pathogen recently associated with HIV infection, organ transplants, travelers, and the elderly (8, 37). Cell-mediated immunity has been shown to be critical for host resistance against E. cuniculi infection (32), and previous studies in our laboratory have suggested that CD8⁺ T cells play a predominant role during this type of infection (16). Mice lacking CD8⁺ T cells are unable to survive E. cuniculi challenge, and a CD8⁺ T-cell cytotoxic response is essential for protective immunity against this parasite.Priming of the CD8⁺ T-cell response against several intracellular pathogens has been shown to be largely dependent on interleukin-12 (IL-12) (25, 40), a heterodimeric proinflammatory cytokine that has been postulated to serve as a bridge between the innate and adaptive immune responses (35). Formed by a light-chain p35 and a heavy-chain p40, this cytokine has multiple functions. It stimulates the development of Th1 lymphocytes (18), induces the production of gamma interferon (IFN-γ) by mouse CD4⁺ Th1 clones (12), and induces the production of cytotoxic CD8⁺ T lymphocytes (34). In the present study, we demonstrate that the absence of IL-12 causes a severe defect in the expansion of the effector CD8⁺ T-cell response against E. cuniculi infection. In vitro studies demonstrate that addition of exogenous IL-12 even 48 to 72 h postculture can reverse the defect in a CD8⁺ T-cell population primed with p40^−/− dendritic cells (DC).     

Relative importance of T-cell subsets in monocytotropic ehrlichiosis: a novel effector mechanism involved in Ehrlichia-induced immunopathology in murine ehrlichiosis

Infection with gram-negative monocytotropic Ehrlichia strains results in a fatal toxic shock-like syndrome characterized by a decreased number of Ehrlichia-specific CD4⁺ Th1 cells, the expansion of tumor necrosis factor alpha (TNF-α)-producing CD8⁺ T cells, and the systemic overproduction of interleukin-10 (IL-10) and TNF-α. Here, we investigated the role of CD4⁺ and CD8⁺ T cells in immunity to Ehrlichia and the pathogenesis of fatal ehrlichiosis caused by infection with low- and high-dose (10³ and 10⁵ bacterial genomes/mouse, respectively) ehrlichial inocula. The CD4⁺ T-cell-deficient mice showed exacerbated susceptibility to a lethal high- or low-dose infection and harbored higher bacterial numbers than did wild-type (WT) mice. Interestingly, the CD8⁺ T-cell-deficient mice were resistant to a low dose but succumbed to a high dose of Ehrlichia. The absence of CD8⁺ T cells abrogated TNF-α and IL-10 production, reduced tissue injury and bacterial burden, restored splenic CD4⁺ T-cell numbers, and increased the frequency of Ehrlichia-specific CD4⁺ Th1 cells in comparison to infected WT mice. Although fatal disease is perforin independent, our data suggested that perforin played a critical role in controlling bacterial burden and mediating liver injury. Similar to WT mice, mortality of infected perforin-deficient mice was associated with CD4⁺ T-cell apoptosis and a high serum concentration of IL-10. Depletion of IL-10 restored the number of CD4⁺ and CD8⁺ T cells in infected WT mice. Our data demonstrate a novel mechanism of immunopathology in which CD8⁺ T cells mediate Ehrlichia-induced toxic shock, which is associated with IL-10 overproduction and CD4⁺ T-cell apoptosis.