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Central memory CD4(+) T cells provide a pool of lymph node-homing, Ag-experienced cells that are capable of responding rapidly after a secondary infection. We have previously described a population of central memory CD4(+) T cells in Leishmania major-infected mice that were capable of mediating immunity to a secondary infection. In this study, we show that the Leishmania-specific central memory CD4(+) T cells require IL-12 to produce IFN-gamma, demonstrating that this population needs additional signals to develop into Th1 cells. In contrast, effector cells isolated from immune mice produced IFN-gamma in vitro or in vivo in the absence of IL-12. In addition, we found that when central memory CD4(+) T cells were adoptively transferred into IL-12-deficient hosts, many of the cells became IL-4 producers. These studies indicate that the central memory CD4(+) T cell population generated during L. major infection is capable of developing into either Th1 or Th2 effectors. Thus, continued IL-12 production may be required to ensure the development of Th1 cells from this central memory T cell pool, a finding that has direct relevance to the design of vaccines dependent upon central memory CD4(+) T cells.

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNgamma, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+ CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFbeta, TGFbetaRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFbeta and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

Cryptosporydium parvum is an intracellular parasite that infects gastrointestinal epithelium and produces diarrhea that is self-limited in immunocompetent persons but potentially life-threatening in immunocompromised, especially those with the acquired immunodeficiency syndrome (AIDS). C. parvum enteric infection's incidence in a pediatric HIV/AIDS cohort, during a 6 years period, was studied. Clinical and immunologic characteristics of the dual infection were also recorded. Highly active antiretroviral therapy (HAART) was started or continued by all the patients during follow-up. Azithromicyn was used as antiparasitic drug. Cryptosporidiosis incidence was 13.7%; 33 out 240 children showed chronic diarrhea lasting 14 days at least, or recurrent, without dehydration and electrolytic disturbance. Peripheral blood T CD4+ percentage levels of the patients were variable and without relationship with C. parvum presence. Viral load levels in 31 out 33 patients were over cut-off at the enteric episode time. Mild or moderate eosinophilia were recorded in 23% of the patients and other intestinal parasites were present in 11 children. When the number of enteric episodes were compared with the clinical and immunological patient's status, not significant differences were recorded. HAART is the best treatment to improve immune function in HIV patients avoiding potentially fatal complications that accompany acute diarrhea during concomitant infection with C. parvum.

Background: Interleukin (IL)-10 plays an important role in down-regulating the immune response to filarial parasites. The goal of this study was to characterize the predominant cellular source of IL-10 in human filarial infections., Methods: Multicolor flow cytometry was used to determine the frequencies of IL-10 production from various lymphocyte populations in the circulation of 23 patients with filarial infections and 8 uninfected control subjects., Results: The frequencies of cells spontaneously producing IL-10 was significantly greater in filaria-infected patients than in uninfected control subjects (geometric mean, 93 vs. 18 IL-10-producing cells/100,000 peripheral blood mononuclear cells; P = .03). Most IL-10-producing cells in filaria-infected patients were T cells, with CD4(+) and CD8(+) cells accounting for 48% and 27%, respectively, of all IL-10-producing cells; CD19(+) B cells, CD14(+) monocytes, and CD56(+) NK cells accounted for 10%, 8%, and 7%, respectively. Surprisingly, only 12% of the IL-10-producing CD3(+)CD4(+) cells were CD25(+). Seventy-seven percent of IL-10-producing CD4(+) T cells stained negatively for both IL-4 and interferon (IFN)-gamma, 22% were positive for IL-4, and <1% were positive for IFN-gamma., Conclusions: These experiments demonstrate that the most frequent producers of IL-10 in human filarial infections are CD4(+) T cells, many of which are skewed toward a type 2 phenotype and most of which are not CD25(+).

Schistosome infection induces profound Th-biasing and immune suppression. Although much has been examined in mice, few studies have examined responses of naïve humans to schistosome antigens. In this study, we examined the response of naïve human peripheral blood mononuclear cells (nPBMC) to stimulation with Schistosoma mansoni soluble egg antigen (SEA) using a priming in vitro (PIV) assay. We found that SEA induced a pronounced CD4+ T-helper cell response based on cytokine secretion and phenotyping markers. SEA-stimulated nPBMC (SEA cells) at day 7 post-priming and after the first recall consisted predominantly of Th0-like CD4+ T cells. Following the second recall, the majority of donor (10/12) responses were Th2-like. The cell population consisted of approximately 64% CD4+, 17% CD8(+high), 12% CD19+, and 7% CD23+ cells. The CD4+ population also expressed HLA-DR+, CD54+, CD45RO+ and CD25+ whereas the CD19+ cells expressed CD80 and CD86. Following priming, we detected high levels of IL-6, IFN-gamma, IL-12p40, IL-10 and IL-5. Upon restimulation, SEA cells secreted IL-5 and high levels of IL-10, typical of a Th2-like response. The data presented herein shows that the majority of naïve donor dendritic cells, following stimulation with SEA, prime and clonally expand SEA-specific T cells towards a Th2-type response. However, two donors responded with an atypical response, producing IFN-gamma coincident with low levels of IL-10. Whether this differential response was due to HLA or other genes was not determined but is currently under investigation.

Alternatively activated macrophages (AAMPhi) are found in abundance during chronic Th2 inflammatory responses to metazoan parasites. Important roles for these macrophages are being defined, particularly in the context of Th2-mediated pathology and fibrosis. However, a full understanding of the requirements for alternative activation, particularly at the innate level, is lacking. We present evidence that alternative activation by the Th2 cytokines IL-4 and IL-13 is an innate and rapid response to tissue injury that takes place even in the absence of an infectious agent. This early response does not require CD4+ Th2 cells because it occurred in RAG-deficient mice. However, class II-restricted CD4+ T cell help is essential to maintain AAMPhi in response to infection, because AAMPhi were absent in RAG-deficient and MHC class II-deficient, but not B cell-deficient mice after chronic exposure to the nematode parasite, Brugia malayi. The absence of AAMPhi was associated with increased neutrophilia and reduced eosinophilia, suggesting that AAMPhi are involved in the clearance of neutrophils as well as the recruitment of eosinophils. Consistent with this hypothesis, AAMPhi show enhanced phagocytosis of apoptotic neutrophils, but not latex beads. Our data demonstrate that alternative activation by type 2 cytokines is an innate response to injury that can occur in the absence of an adaptive response. However, analogous to classical activation by microbial pathogens, Th2 cells are required for maintenance and full activation during the ongoing response to metazoan parasites.

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.

Schistosoma mansoni infection of mice increases the frequency of cells that are CD4+ CD25+ in the acute (4 and 8 weeks) and chronic (16 week) stages of infection. Depletion of > 85% of CD25+ cells in the acute or chronic stages of schistosome infection caused no overt changes in morbidity or immunological responses. The absence of effect in mice with CD25+ cells depleted may be due to the preferential expression of IL-4 and IL-10, two cytokines that are protective in schistosome infection, on CD25- CD4+ cells. We also assessed infection-induced changes of other regulatory markers, GITR, CD103 and CTLA-4 on CD4+ cells. We identified a marked expansion of CTLA-4+ population on CD25- CD4+ cells in acute and chronic infection. Blocking of CTLA-4 during acute, but not chronic infection, caused significant weight loss and altered the type 2 cytokine response of mice, with increased IL-4 and IL-5 production associated with significantly more Th2 cells and eosinophils in the liver granuloma. This study illustrates the complexity of regulation of T cells in schistosome infection and highlights a specific role for CTLA-4+, but not CD25+ cells, in the regulation of Th2 responses in helminth infection.

Activation of Th2 CD4(+) T cells is necessary and sufficient to elicit allergic airway disease, a mouse model with many features of human allergic asthma. Effectively controlling the activities of these cells could be a panacea for asthma therapy. Blood-feeding parasites have devised remarkable strategies to effectively evade the immune response. For example, ticks such as Ixodes scapularis, which must remain on the host for up to 7 days to feed to repletion, secrete immunosuppressive proteins. Included among these proteins is the 15-kDa salivary protein Salp15, which inhibits T cell activation and IL-2 production. Our objective for these studies was to evaluate the T cell inhibitory properties of Salp15 in a mouse model of allergic asthma. BALB/cJ mice were Ag sensitized by i.p. injection of OVA in aluminum hydroxide, with or without 50 mug of Salp15, on days 0 and 7. All mice were challenged with aerosolized OVA on days 14-16 and were studied on day 18. Compared with control mice sensitized with Ag, mice sensitized with Ag and Salp15 displayed significantly reduced airway hyperresponsiveness, eosinophilia, Ag-specific IgG1 and IgE, mucus cell metaplasia, and Th2 cytokine secretion in vivo and by CD4(+) T cells restimulated with Ag in vitro. Our results demonstrate that Salp15 can effectively prevent the generation of a Th2 immune response and the development of experimental asthma. These studies, and those of others, support the notion that a lack of ectoparasitism may contribute to the increasing prevalence of allergic asthma.

Objective: To observe the changes of CD4+CD25+ regulatory T cells in the spleen of mice infected with T.gondii., Methods: Twenty-eight female C57BL/6 mice were randomly divided into four groups. Three groups of mice were inoculated intraperitoneally with 10(4) tachyzoites in 200 microl sterile PBS. At 2, 4 and 6 days post-infection, the spleens were removed. The expression level of Foxp3 mRNA in splenic CD4+ T cells was quantitated by real-time PCR. The percentage of CD4+CD25+ regulatory T cells in CD4+ T cells was determined by flow cytometry, and the absolute numbers of splenic CD4+CD25 - regulatory T cells and CD4+ T cells were assessed. The fourth group was injected intraperitoneally with 200 microl sterile PBS as control., Results: The relative mRNA level of Foxp3 in splenic CD4+ T cells at day 4 (1.89+/-0.23) and day 6 (1.79+/-0.24) post-infection was significantly higher than control (1.00+/-0.12) (P< 0.01). After an initial up-regulation at 2 days post-infection (15.07%+/-2.73%) (P<0.05), the proportion of CD4+CD25+ regulatory T cells in CD4+ T cells at day 4 (24.29%+/-3.19%) and day 6 (19.80%+/-2.66%) post-infection was significantly higher than control (11.58%+/-2.04%) (P<0.01). At day 6 post-infection, both the percentage of splenic CD4+ T cells in splenocytes(5.49%+/-l.71%) and absolute number of CD4+ T cells (1.71+/-0.44)x106 greatly decreased(P<0.01)., Conclusion: The proportion of splenic CD4+CD25+ regulatory T cells in CD4+ T cells has been up-regulated following T. gondii infection, which is mainly due to a great reduction of CD4+ T cells in the spleen.

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection, causing significant morbidity and mortality among young children and nonimmune adults in the developing world. Although previous work on experimental CM has identified T cells as key mediators of pathology, the APCs and subsets therein required to initiate immunopathology remain unknown. In this study, we show that conventional dendritic cells but not plasmacytoid dendritic cells are required for the induction of malaria parasite-specific CD4+ T cell responses and subsequent experimental CM. These data have important implications for the development of malaria vaccines and the therapeutic management of CM.

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.

Mice from most inbred strains are resistant to infection with Leishmania major whereas mice from BALB strains are highly susceptible. Resistance and susceptibility result from the development of Th1 or Th2 cells, respectively. In this report, we document an IL-2 mRNA burst, preceding the reported early IL-4 response, in draining lymph nodes of susceptible mice infected with L. major. Neutralization of IL-2 during the first days of infection redirected Th1 cell maturation and resistance to L. major, through interference with the rapid IL-4 transcription in Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) cells. A burst of IL-2 transcripts also occurred in infected C57BL/6 mice that do not mount an early IL-4 response. However, although the LACK protein induced IL-2 transcripts in susceptible mice, it failed to trigger this response in resistant C57BL/6 mice. Reconstitution experiments using C.B.-17 SCID mice and LACK-reactive CD4(+) T cells from IL-2(-/-) BALB/c mice showed that triggering of the early IL-4 response required autocrine IL-2. Thus, in C57BL/6 mice, the inability of LACK-reactive CD4(+) T cells to express early IL-4 mRNA transcription, important for disease progression, appears due to an incapacity of these cells to produce IL-2.

Both T cells and APC have been strongly implicated in the immune suppression observed during filarial nematode infections, but their relative roles are poorly understood, particularly in regard to timing and locality of action. Using Litomosoides sigmodontis infection of susceptible BALB/c mice, we have studied the progression of filarial immunosuppression leading to patent infection with blood microfilaremia. Patent infection is associated with decreased immune responsiveness in the draining thoracic lymph nodes (tLN) and intrinsically hyporesponsive CD4+ T cells at the infection site. We now show that we are able to separate, both in time and space, different suppressive mechanisms and cell populations that contribute to filarial hyporesponsiveness. L. sigmodontis infection recruited a F4/80+ population of alternatively activated macrophages that potently inhibited Ag-specific CD4+ T cell proliferative responses even in the presence of competent naive APC. T cell responsiveness was partially restored by neutralizing TGF-beta, but not by blocking IL-10 or CTLA-4 signaling. During prepatent infection, the macrophage population was restricted to the infection site. However, once infection became patent with systemic release of microfilariae, the suppressive macrophage activity extended peripherally into the tLN. In contrast, the hyporesponsive CD4+ T cell phenotype remained localized at the infection site, and the tLN CD4+ T cell population recovered full Ag responsiveness in the absence of suppressive macrophages. Filarial immunosuppression, therefore, evolves over time at sites increasingly distal to infection, and the mechanisms of filarial down-regulation are dependent on proximity to the infection site.

Natural killer (NK) cells promote dendritic cell (DC) maturation and influence T cell differentiation in vitro. To better understand the nature of the putative interactions among these cells in vivo during the early phases of an adaptive immune response, we have used immunohistochemical analysis and dynamic intravital imaging to study NK cell localization and behavior in lymph nodes (LNs) in the steady state and shortly after infection with Leishmania major. In the LNs of naive mice, NK cells reside in the medulla and the paracortex, where they closely associate with DCs. In contrast to T cells, intravital microscopy revealed that NK cells in the superficial regions of LNs were slowly motile and maintained their interactions with DCs over extended times in the presence or absence of immune-activating signals. L. major induced NK cells to secrete interferon-gamma and to be recruited to the paracortex, where concomitant CD4 T cell activation occurred. Therefore, NK cells form a reactive but low mobile network in a strategic area of the LN where they can receive inflammatory signals, interact with DCs, and regulate colocalized T cell responses.

Background & Objective: The risk of human immunodeficiency virus (HIV) co-infection in patients with visceral leishmaniasis (VL) or kala-azar in endemic areas has posed a major challenge in control programmes. We undertook this study to identify the high risk groups vulnerable to Leishmania-HIV co-infection in VL endemic State of Bihar, India. Further, immunological responses were also evaluated in these patients before and after treatment for VL to see the immune impairment associated with CD4 T cell count., Methods: A total of 1511 subjects attending Voluntary Counselling and Testing Centre (VCTC) at Patna, Bihar were included in this study. VL was confirmed by splenic or bone marrow aspirates testing for parasite. HIV states was confirmed by two kits. Immunological parameters (CD4, CD8, IFN-gamma, IL-4) were studied in co-infection patients., Results: Of the 280 (18.53%) HIV-positive individuals, eight were diagnosed serologically and pathologically as VL patients co-infected with HIV. The humoral and cellular immune responses were evaluated in 18 Indian VL patients with (n = 8) or without HIV (n = 10) and 10 HIV seropositive subjects. Among the eight confirmed cases of VL, false negative direct agglutination test (DAT) result was observed in two who had HIV co-infection (sensitivity 80%), while none in 10 other VL cases who were HIV negative (sensitivity 100%). A very low CD4 cell count was observed in VL cases that had HIV co-infection compared to HIV negative VL or controls. All VL cases with or without HIV infection had lower Th1/Th2 ratio compared to controls. VL patients with or without HIV infection responded well to anti-leishmanial/anti-retroviral therapy with considerable degree of immunological reconstitution., Interpretation & Conclusion: A different immune response was noticed in patients with co-infection of HIV and Leishmania. Anti-leishmanial drug treatment led to improvement in immunological response in co-infected patients. Further studies need to be done to see the effect of combined therapy for VL and HIV on immunological parameters in these patients.

Pneumocystis infection leads to a life threatening pneumonia in susceptible individuals. While depletion or dysfunction of CD4+T cells is a key determinant of susceptibility to Pneumocystis, the host response that leads to resolution of infection or lung injury is less well understood. We had previously shown that mice deficient in the T cell costimulatory molecule CD28 are susceptible to infection with Pneumocystis. A detailed analysis revealed that they clear Pneumocystis with delayed kinetics. This is associated with an influx of naïve CD8+ T cells. Depletion of CD8+ T cells did not alter organism burden, suggesting these cells are not responsible for clearance. Analysis of the cytokine milieu demonstrated a consistent increase in mRNA for IL-10 and IFN-gamma in the CD28-deficient mice. These data suggest that CD28 function in important in the efficiency of the host response to Pneumocystis pneumonia.

Parasites are designed by evolution to invade the host and survive in its organism until they are ready to reproduce. Parasites release a variety of molecules that help them to penetrate the defensive barriers and avoid the immune attack of the host. In this respect, particularly interesting are enzymes and their inhibitors secreted by the parasites. Serine-, aspartic-, cysteine-, and metalloproteinases are involved in tissue invasion and extracellular protein digestion. Helminths secrete inhibitors of these enzymes (serpins, aspins, and cystatins) to inhibit proteinases, both of the host and their own. Proteinases and their inhibitors, as well as helminth homologues of cytokines and molecules containing phosphorylcholine, influence the immune response of the host biasing it towards the anti-inflammatory Th2 type. Nucleotide-metabolizing enzymes and cholinesterase are secreted by worms to reduce inflammation and expel the parasites from the gastrointestinal tract. An intracellular metazoan parasite, Trichinella spiralis, secretes, among others, protein kinases and phosphatases, endonucleases, and DNA-binding proteins, which are all thought to interfere with the host cellular signals for muscle cell differentiation. Secretion of antioxidant enzymes is believed to protect the parasite from reactive oxygen species which arise from the infection-stimulated host phagocytes. Aside from superoxide dismutase, catalase (rarely found in helminths), and glutathione peroxidase (selenium-independent, thus having a poor activity with H(2)O(2)), peroxiredoxins are probably the major H(2)O(2)-detoxifying enzymes in helminths. Secretion of antioxidant enzymes is stage-specific and there are examples of regulation of their expression by the concentration of reactive oxygen species surrounding the parasite. The majority of parasite-secreted molecules are commonly found in free-living organisms, thus parasites have only adapted them to use in their way of life.

CD4 T cells play a central role in the immune response to malaria. They are required to help B cells produce the antibody that is essential for parasite clearance. They also produce cytokines that amplify the phagocytic and parasitocidal response of the innate immune system, as well as dampening this response later on to limit immunopathology. Therefore, understanding the mechanisms by which T helper cells are activated and the requirements for development of specific, and effective, T cell memory and immunity is essential in the quest for a malaria vaccine. In this paper on the CD4 session of the Immunology of Malaria Infections meeting, we summarize discussions of CD4 cell priming and memory in malaria and in vaccination and outline critical future lines of investigation. B. Stockinger and M.K. Jenkins proposed cutting edge experimental systems to study basic T cell biology in malaria. Critical parameters in T cell activation include the cell types involved, the route of infection and the timing and location and cell types involved in antigen presentation. A new generation of vaccines that induce CD4 T cell activation and memory are being developed with new adjuvants. Studies of T cell memory focus on differentiation and factors involved in maintenance of antigen specific T cells and control of the size of that population. To improve detection of T cell memory in the field, efforts will have to be made to distinguish antigen-specific responses from cytokine driven responses.

DM functions as a peptide editor for MHC class II-bound peptides. We examined the hypothesis that DM peptide editing plays a key role in focusing the in vivo CD4 T cell responses against complex pathogens and protein Ags to only one, or at most a few, immunodominant peptides. Most CD4 T cells elicited in the wild-type BALB/c (H-2d) mice infected with Leishmania major predominantly recognize a single epitope 158-173 within Leishmania homologue of activated receptor for c-kinase (LACK), as is the case when these mice are immunized with rLACK. Using DM-deficient (DM-/-) H-2d mice, we now show that in the absence of DM, the in vivo CD4 T cell responses to rLACK are skewed away from the immunodominant epitopes and are diversified to include two novel epitopes (LACK 33-48 and 261-276). DM-/- B10.BR (H-2k) mice showed similar results. These results constitute the first demonstration of the role of DM peptide editing in sculpting the specificity and immunodominance in in vivo CD4 T cell responses.

The hepatic immunopathology in schistosomiasis mansoni is mediated by CD4 T cells specific for egg antigens and varies considerably among mouse strains. Previous studies in high pathology C3H mice suggested that a strong T cell response was due to the recognition of an immunodominant epitope within the major egg antigen Sm-p40 (Sm-p40(234-246)). Using a panel of T cell hybridomas, we have now examined the egg antigen-specific TCR repertoire in two high pathology strains, C3H and CBA. We found that nearly half of the hybridomas responded to the Sm-p40(234-246 )epitope and, of these, nearly all expressed Valpha11.3 associated with Vbeta8. Furthermore, in response to egg antigen stimulation, transcript levels of Valpha11.3J36 (the most prevalent rearrangement expressed by Sm-p40(234-246)-specific hybridomas), increased in high pathology (CBA) but not in low pathology BALB/c strains. Our findings suggest that exacerbated schistosome egg-induced immunopathology can be driven by T cells expressing a highly restricted TCR structure specific for a single parasite epitope.

Recent publications by Zaph et al. have highlighted the distinct requirements for generating and maintaining different subpopulations of CD4(+) memory T cells after infection with Leishmania major in mice. These studies have advanced the understanding of the nature of long-lasting immunity to Leishmania and, when considered within the context of previous work on both murine and human leishmaniasis, will aid the design of effective vaccines.

IL-10 plays a vital role in controlling the inflammatory response during acute Toxoplasma gondii infection, however the production of IL-10 during the chronic phase of toxoplasmosis has been associated with parasite persistence. To address this paradox, the production and effect of IL-10 in the brain during toxoplasmic encephalitis (TE) was investigated. Analysis of brain mononuclear cells (BMNC) from chronically infected mice revealed that infiltrating macrophages and CD4(+) T cells were the major sources of IL-10. Endogenous levels of IL-10 inhibited the production of IL-12, IFN-gamma, TNF-alpha, and IL-6 from both hematopoetic and non-hematopoetic cells in the brain, as well as anti-microbial activity of astrocytes. Furthermore, IL-10-/- mice that progressed to the chronic phase of infection had equivalent parasite burden to WT mice but developed a lethal inflammatory response within the brain characterized by increased numbers of CD4(+) T cells and macrophages, and elevated production of inflammatory cytokines. Finally, partial depletion of CD4(+) T cells decreased the severity of the inflammation in the brain and allowed IL-10-/- mice to survive infection. Together these results point to a vital role for IL-10 in the control of CD4(+) T cell mediated inflammation in the brain during TE.

Lymphatic filariasis is a disease characterized by immune dysregulation involving APC and T cell populations. To assess the contribution of TLR in mediating this dysregulation, we examined the expression of TLR1, TLR2, TLR4, and TLR9 on B cells and monocytes of filaria-infected and uninfected individuals. Baseline expression of TLR was significantly lower in B cells but not in monocytes of the filaria-infected group compared with the uninfected group. Upon stimulation with filarial Ag, a diminished up-regulation of TLR was observed in both B cells and monocytes of infected individuals. Finally, stimulation of B cells and monocytes with TLR ligands resulted in decreased B cell and monocyte activation/cytokine production, indicating a state of immune tolerance. This dysregulation is associated with diminished CD4(+) T cell production of IFN-gamma and IL-5. The diminished expression and function of TLR is thus a likely consequence of chronic Ag stimulation and could serve as a novel mechanism underlying the dysfunctional immune response in filariasis.

Canine leishmaniasis caused by Leishmania infantum is endemic in the foxhound population in North America. Studies of canine leishmaniasis in the Mediterranean basin indicate a role for both CD4+ and CD8+ lymphocytes with clinical illness and in asymptomatic dogs. Limited information is available on the strain of L. infantum infecting foxhounds in North America. The present study investigated changes in cellular immune responses in dogs experimentally infected with 1x10(7) (low dose, LD; N=4) or 2x10(8) (high dose, HD; N=4) promastigotes of a United States isolate of L. infantum and control dogs (N=2) for 72 weeks. Density gradient separation was used to enrich for peripheral blood lymphocytes from canine blood. Lymphocyte subsets (CD4+ and CD8+) were quantified by flow cytometric analysis. Lymphocyte population expression levels over the course of the present study were compared to clinical status of the dog and antibody responses in infected and control dogs. No significant differences (P>0.05) were observed in either CD4+ or CD8+ lymphocyte expression in of the groups over the experimental period. This study suggests that the cellular immune responses to North American L. infantum in experimentally infected dogs may differ from other strains of L. infantum.

Mice lacking functional CD1d genes were used to study mechanisms of resistance to the protozoan parasite Toxoplasma gondii. Wild-type (WT) BALB/c mice, CD1d-deficient BALB/c mice, and WT C57BL/6 mice all survived an acute oral infection with a low dose of mildly virulent strain ME49 T. gondii cysts. In contrast, most CD1d-deficient C57BL/6 mice died within 2 wk of infection. Despite having parasite burdens that were only slightly higher than WT mice, CD1d-deficient C57BL/6 mice displayed greater weight loss and intestinal pathology. In C57BL/6 mice, CD4(+) cells can cause intestinal pathology during T. gondii infection. Compared with WT mice, infected CD1d-deficient C57BL/6 mice had higher frequencies and numbers of activated (CD44(high)) CD4(+) cells in mesenteric lymph nodes. Depletion of CD4(+) cells from CD1d-deficient mice reduced weight loss and prolonged survival, demonstrating a functional role for CD4(+) cells in their increased susceptibility to T. gondii infection. CD1d-deficient mice are deficient in Valpha14(+) T cells, a major population of NKT cells. Involvement of these cells in resistance to T. gondii was investigated using gene-targeted Jalpha18-deficient C57BL/6 mice, which are deficient in Valpha14(+) T cells. These mice did not succumb to acute infection, but experienced greater weight loss and more deaths than B6 mice during chronic infection, indicating that Valpha14(+) cells contribute to resistance to T. gondii. The data identify CD4(+) cells as a significant component of the marked susceptibility to T. gondii infection observed in CD1d-deficient C57BL/6 mice, and establish T. gondii as a valuable tool for deciphering CD1d-dependent protective mechanisms.

Although activation of CD4(+) T cells mediates pathogenesis in Leishmania amazonensis (La)-infected mice, these susceptible mice do not develop a polarized Th2 response, suggesting a unique mechanism of disease susceptibility. To understand how Th cell activities are regulated, we examined the frequency and phenotypes of regulatory T (Treg) cells. At 1-3 wk of infection, relatively high percentages of CD4(+)CD25(+)CD86(+) T cells, as well as high levels of FoxP3, TGF-beta1, and IL-10RI transcripts, were detected in the skin and draining lymph nodes, indicating local accumulation of Treg cells. Lesion-derived, IL-10-producing CD4(+)CD25(+) cells effectively suppressed proliferation and cytokine (IL-2 and IFN-gamma) production of CD4(+)CD25(-) effector cells. Adoptive transfer of lesion-derived CD4(+)CD25(+) cells to syngeneic, naive C57BL/6 mice before infection significantly reduced disease development. To further validate the beneficial role of Treg cells in La infection, we adoptively transferred CD25(+) T cell-depleted splenocytes (derived from naive mice) into RAG1(-/-) mice. This transfer rendered RAG1(-/-) mice more susceptible to La infection than the mice receiving control splenocytes. The beneficial effect of Treg cells was transitory and correlated with decreased activation of IFN-gamma-producing effector T cells. This study uncovers an intriguing role of Treg cells in restraining pathogenic responses during nonhealing Leishmania infection and emphasizes a balance between Treg and Th1-like effector cells in determining the outcome of New World cutaneous leishmaniasis.

Leishmaniasis is a complex of diseases caused by protozoan parasites belonging to the genus Leishmania. The development of specific resistance against re-infection after cure suggests that a vaccine approach is feasible. Various studies in humans and experimental animals strongly suggest that Th1 type of cell-mediated immune response is important for protection against the disease. A defined antigen that could elicit a specific T-cell-mediated immune response in the host would be an ideal candidate for the vaccine against this parasite. In order to select a candidate antigen, we established a screening system to identify the recombinant clone, expressing antigen having T-cell epitopes from a cDNA library. We screened the library using an established Leishmania specific cell line (LSCL) from a naive healthy human subject. The cell line with predominantly CD4+ cells behaved in a Leishmania specific manner. Fifty-two immuno-reactive clones were screened against the LSCL in vitro and we identified three cDNA clones expressing recombinant antigens that could induce proliferation of these cells to produce INFgamma. The protective efficacy of one of these recombinant proteins was investigated in a hamster model of experimental visceral leishmaniasis and showed protection against a virulent challenge. The identified antigens might be potential candidates for vaccine against Leishmania.

In the BALB/c mouse model, primary infection with Toxoplasma gondii during the second third of gestation leads to a high percentage of infected foetuses. However, immunity induced by infection contracted before pregnancy prevents parasites from crossing the placenta and completely protects the foetuses, as well as the pregnant women. In order to clarify the roles of CD4+, CD8+ T lymphocytes and IFN-gamma in this protection, pregnant BALB/c mice were treated with depleting monoclonal antibodies against CD4, CD8, IFN-gamma, or control antibody. Only the foetuses of the groups treated with anti-CD8 and anti-IFN-gamma antibodies developed congenital toxoplasmosis. The maternal production of IFN-gamma was depressed in the mice depleted of CD4 and CD8 cells (P < 0.001). Determination of the blood parasite load demonstrated that materno-foetal transmission of T. gondii correlates with maternal parasitaemia. Together, these results show that CD8+ T lymphocytes and IFN-gamma play an important role in protection against congenital toxoplasmosis during reinfection.

Reactivation of dormant infections causes an immense burden of morbidity and mortality in the world at large. Reactivation can occur as a result of immunosuppression, environmental insult, or aging; however, the cause of reactivation of such infections is often not clear. We have previously shown that persistence of the parasite Leishmania major is controlled by endogenous CD4(+) CD25(+) regulatory T (T reg) cells. In this report, we show that despite efficient parasite clearance at secondary sites of infection, Leishmania superinfection can cause disease reactivation at the primary site. Our results strongly suggest that T reg cells, whose numbers increase in sites of reactivation, are directly responsible for such reactivation. Depletion of CD25(+) cells at the time of secondary challenge prevented disease reactivation at the site of persistent infection while strengthening the expression of immunity at the site of secondary challenge. Finally, transfer of T reg cells purified from infected mice into chronically infected mice was sufficient to trigger disease reactivation and prevent the expression of an effector memory response. Our results demonstrate that after persistence is achieved, an equilibrium between T reg cells and effector lymphocytes, which can be disturbed by superinfection, controls the efficiency of recall immune responses and disease reactivation.

In this study we present the first systematic analysis of the immunity induced by normal Plasmodium yoelii sporozoites in mice. Immunization with sporozoites, which was conducted under chloroquine treatment to minimize the influence of blood stage parasites, induced a strong protection against a subsequent sporozoite and, to a lesser extent, against infected RBC challenges. The protection induced by this immunization protocol proved to be very effective. Induction of this protective immunity depended on the presence of liver stage parasites, as primaquine treatment concurrent with sporozoite immunization abrogated protection. Protection was not found to be mediated by the Abs elicited against pre-erythrocytic and blood stage parasites, as demonstrated by inhibition assays of sporozoite penetration or development in vitro and in vivo assays of sporozoite infectivity or blood stage parasite development. CD4(+) and CD8(+) T cells were, however, responsible for the protection through the induction of IFN-gamma and NO.

Memory peripheral Th2 immune responses to infectious pathogens are not well studied due to the lack of suitable models and the difficulty of assessing Th2 cytokine expression at sites of inflammation. We have examined the localized immune response to a nematode parasite that encysts in the small intestine. An unexpected architecture was observed on day 4 of the memory response, with granulocytes and macrophages infiltrating the cyst and CD4(+), TCR-alphabeta(+) T cells surrounding the cyst. Laser capture microdissection analysis showed a pronounced CD4-dependent Th2 cytokine pattern at the cyst region only during the memory response, demonstrating that the Th2 memory response is readily distinguished from the primary response by the rapid accumulation of Th2 effector cells at the host:parasite interface.

Basophil contribution to the IL-4 pool in filarial infections was assessed using PBMC from 20 patients with active filarial infections and from 9 uninfected subjects. Patient basophils released histamine in response to Brugia malayi Ag (BmAg). They also released IL-4 within 2 h after exposure to BmAg, as assessed by intracellular cytokine flow cytometry. This IL-4 induction was Ag specific, as IL-4 was not detected in BmAg-exposed basophils obtained from uninfected subjects. Although there were, on average, 64 times more CD4(+) T cells than basophils in the peripheral circulation of filaria-infected patients, the absolute numbers of basophils and CD4(+) T cells producing IL-4 per 100000 PBMC were equivalent (geometric mean: 16 IL-4-producing basophils/100000 PBMC vs 22 IL-4-producing CD4(+) T cells/100000 PBMC). Basophils also released IL-4 in response to both low and high concentrations of BmAg, whereas CD4(+) T cells released IL-4 only after incubation with a high concentration of BmAg, raising the possibility that basophils, due to their lower threshold for activation, may actually release IL-4 more frequently than CD4(+) T cells in vivo. Furthermore, IL-4 production in vitro by Ag-stimulated purified basophils or CD4(+) T cells provided evidence that basophils release greater quantities of IL-4 per cell than CD4(+) T cells in response to BmAg. These results suggest that, when Ag-specific IgE is present in a filaria-infected individual, basophils function to amplify the ongoing Th2 response by releasing IL-4 in greater amounts and possibly more frequently than CD4(+) T cells in response to filarial Ag.

Background/aims: Hepatitis C virus (HCV) remains the most common cause of chronic liver disease in Egypt. Despite the high prevalence of HCV and Schistosoma mansoni (S. mansoni) in Egypt, the effect of co-infection on the immune response against HCV genotype 4a has not been extensively examined., Methods: We evaluated the HCV 4a-specific responses against the core and non-structural 5B proteins in chronic HCV with or without S. mansoni co-infection in 38 volunteers from Egypt., Results: HCV 4a-specific responses were detected in 8/15 and 13/23 individuals with HCV alone or with concomitant schistosomiasis, respectively. Despite the alteration in the Th1 cytokine profile caused by schistosomiasis, the overall immune response rate against HCV was not affected (P=0.11). Seven individuals demonstrated HCV-specific responses against conserved regions of the Core that were previously identified for genotypes 1, 2 and 3 despite differences in HLA class I distribution., Conclusions: Egyptian patients infected with HCV genotype 4 can mount HCV-specific T cell responses, both CD4+ and CD8+ T cell-mediated, despite the prevalence of concomitant schistosomiasis. These findings suggest that S. mansoni co-infection may not represent a major obstacle to developing an HCV vaccine in this population.

Here we investigated whether the depletion of CD4+ lymphocytes, observed in mononuclear cells incubated with Taenia solium metacestode E/S products or with living cysts was due to apoptosis. Using the deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), electron microscopy and DNA gel electrophoresis, we found signs of apoptosis in these cells. Results showed that cysteine protease activity was responsible for this effect, since E-64 prevented cell death in all cases. Electron microscopy studies showed that lymphocytes exhibited features of apoptosis such as cellular membrane integrity, strangling and fragmentation of nuclei, chromatin condensation, apoptotic bodies and loss of microvilli. In contrast, lymphocytes co-cultured with living metacestodes plus E-64 exhibited integrity of their structures. DNA fragmentation was detected by TUNEL assays and DNA gel electrophoresis. The results suggested that cell death induced by the cysteine protease from the T. solium metacestode may be involved in down-regulation of cell-mediated responses in infected hosts.

Although the early human immune response to the infective-stage larvae (L3) of Brugia malayi has not been well-characterized in vivo (because of the inability to determine the precise time of infection), the consensus has been that it must involve a predominant Th2 environment. We have set up an in vitro system to study this early immune response by culturing PBMC from unexposed individuals with live L3 of B. malayi. After 24 h of culture, T cell responses were examined by flow cytometry and by quantitative real-time RT-PCR for multiple cytokines. T cells were activated early following exposure to L3 as indicated by up-regulation of surface markers CD69 and CD71. The frequency of T cells expressing proinflammatory Th1 cytokines (IFN-gamma, TNF-alpha, GM-CSF, IL-1alpha, and IL-8) but not Th2 cytokines (IL-4, IL-5, IL-6, IL-10, and IL-13) was significantly increased in response to L3. This T cell response occurred in both the CD4 and CD8 T cell compartment and was restricted to the effector/memory pool (CD45RO(+)). This T cell response was not due to LPS activity from the parasite or from its endosymbiont, Wolbachia; moreover, it required the presence of APC as well as direct contact with live L3. Real-time RT-PCR analysis of multiple cytokines in the T cells confirmed the increased expression of proinflammatory Th1 cytokines. Up-regulation of these cytokines suggests that the primary immune response to the live infective stage of the parasite is not predominantly Th2 in nature but rather dominated by a proinflammatory response.

Amoebic liver abscesses (ALA) are the most frequent and severe extraintestinal clinical presentations of amoebiasis. During the early establishment of amoebae in the liver parenchyma, as well as during the extension of the tissue necrosis, parasites interact with the parenchymal liver cells and, as a consequence of these interactions, hepatocytes can be destroyed and host immune cells can become activated. However, little is known about the nature of these interactions in the liver or about the factors involved in the local immune response. In this investigation we studied the localization of Entamoeba histolytica trophozoites, TCD4+, TCD8+ cells, CD68+ macrophages and CD15+ neutrophils in human ALA using immunohistochemical techniques. Trophozoites were found close to undamaged hepatocytes in both lysed and non-lysed areas with either sparse or abundant inflammatory infiltrate. CD8+ cells were more abundant than CD4+ T cells. CD 68+ macrophages and CD15+ neutrophils were also detected, suggesting that neutrophils, macrophages and T cells might be related to the local host immune mechanisms in ALA. We also found that E. histolytica possesses proteins recognized by antibodies raised against inducible nitric oxide synthase.

In infection with Schistosoma mansoni, parasite eggs precipitate an intrahepatic granulomatous and fibrosing inflammation that is mediated by CD4(+) Th cells. Compared with CBA mice, C57BL/6 mice develop smaller granulomas composed of cells that exhibit reduced proliferative responses to schistosome egg Ags. In the present study, we investigated CD4(+) T cell apoptosis as a possible mechanism that could account for this subdued response. We found throughout the course of several infection weeks a markedly higher proportion of apoptotic CD4(+) T cells in granulomas from C57BL/6 mice than in those from CBA mice ex vivo; the apoptosis further increased upon cell cultivation in vitro. Activation-induced cell death or CD8(+) T cells failed to account for the enhanced apoptosis as infected Fas-, Fas ligand,- and CD8-deficient mice exhibited similar apoptosis to that seen in wild-type counterparts. However, a strikingly lower IL-2 production by schistosome egg Ag-stimulated C57BL/6 granuloma and mesenteric lymph node cells suggested the possibility of apoptosis due to growth factor deprivation. Indeed, the CD4(+) T cell apoptosis was significantly reversed by addition of rIL-2 in vitro, or by injection of rIL-2 in vivo, which also resulted in significant exacerbation of granulomatous inflammation. These findings indicate that apoptosis by neglect can represent a significant means of controlling CD4(+) T cells that mediate the immunopathology in schistosomiasis.

Parasite Ag-specific T cell unresponsiveness and diminished IFN-gamma production are immunologic hallmarks of patent infection with lymph-dwelling filarial nematodes. Although this diminished responsiveness is directed primarily against the intravascular microfilarial (MF) parasite stage and mediated in part by reduced APC function, the mechanisms involved are not fully understood. In this report, we demonstrate that human dendritic cells (DC) exposed to live MF up-regulate both the cell surface and gene expression of CD54 (ICAM-1). Moreover, live MF result in a 3-fold increase in DC death compared with MF-unexposed DC, primarily due to apoptosis. Notably, microarray and real-time RT-PCR data indicate that live MF concurrently up-regulate mRNA expression of proinflammatory molecules such as IL-8, RANTES, IL-1alpha, TNF-alpha, and IL-beta in DC, the presence of which is also detected at the protein level, while inhibiting the production of IL-12 (p40 and p70) and IL-10. Soluble excretory-secretory products from live MF diminished IL-12 and IL-10 production and induced DC death, although to a lesser degree. Moreover, exposure of DC to live MF resulted in a decrease in the ability of DC to promote CD4(+) T cell production of IFN-gamma and IL-5. Our findings clearly suggest that the interaction between live MF and DC is complex but contributes to the hyporesponsiveness and parasite persistence associated with the MF(+) state in the infected human. These data further suggest that MF induce an orchestrated response in APC that leads to a diminished capacity to function appropriately, which in turn has significant consequences for CD4(+) T cells.

A prospective study was undertaken to define early predictive immunological marker(s) of exposure to Leishmania in naïve subjects who have never been exposed to any Leishmania and who were also free of any cutaneous leishmaniasis lesions. These naïve subjects could have been exposed to Leishmania in a rain forest where Leishmania guyanensis and their natural vectors and mammalian host are cocirculating. The production of interferon (IFN)-gamma in response to the Leishmania homologue of the mammalian receptor for activated c kinase (LACK), a candidate for vaccine against leishmaniasis was analysed. At the end of their stay in the rain forest, LACK-specific CD8+ T cells were detected in subjects whose peripheral blood mononuclear cell (PBMC) produced IFN-gamma in response to soluble Leishmania antigens (SLA) and in those whose PBMC remained unresponsive to SLA. However, LACK-specific CD4+ T cells were detected only in PBMCs from individuals who became IFN-gamma responders to SLA. In subjects whose PBMC became positive to SLA, LACK-reactive CD4+ T cells producing high level of IFN-gamma were detectable before the SLA-reactive IFN-gamma producing CD4+ T cells, suggesting that the former readout assay could be used as an early predictive immunological marker of exposure to Leishmania in subjects who remained disease free.

Host responses to Pneumocystis carinii infection mediate impairment of pulmonary function and contribute to the pathogenesis of pneumonia. IL-10 is known to inhibit inflammation and reduce the severity of pathology caused by a number of infectious organisms. In the present studies, IL-10-deficient (IL-10 knockout (KO)) mice were infected with P. carinii to determine whether the severity of pathogenesis and the efficiency of clearance of the organisms could be altered in the absence of IL-10. The clearance kinetics of P. carinii from IL-10 KO mice was significantly enhanced compared with that of wild-type (WT) mice. This corresponded to a more intense CD4(+) and CD8(+) T cell response as well as an earlier neutrophil response in the lungs of IL-10 KO mice. Furthermore, IL-12, IL-18, and IFN-gamma were found in the bronchoalveolar lavage fluids at earlier time points in IL-10 KO mice suggesting that alveolar macrophages were activated earlier than in WT mice. However, when CD4(+) cells were depleted from P. carinii-infected IL-10 KO mice, the ability to enhance clearance was lost. Furthermore, CD4-depleted IL-10 KO mice had significantly more lung injury than CD4-depleted WT mice even though the intensity of the inflammatory responses was similar. This was characterized by increased vascular leakage, decreased oxygenation, and decreased arterial pH. These data indicate that IL-10 down-regulates the immune response to P. carinii in WT mice; however, in the absence of CD4(+) T cells, IL-10 plays a critical role in controlling lung damage independent of modulating the inflammatory response.

Amebic colitis is an important worldwide parasitic disease for which there is not a well-established animal model. In this work we show that intracecal inoculation of Entamoeba histolytica trophozoites led to established infection in 60% of C3H mice, while C57BL/6 or BALB/c mice were resistant, including mice genetically deficient for IL-12, IFN-gamma, or inducible NO synthase. Infection was a chronic and nonhealing cecitis that pathologically mirrored human disease. Characterization of the inflammation by gene chip analysis revealed abundant mast cell activity. Parasite-specific Ab and cellular proliferative responses were robust and marked by IL-4 and IL-13 production. Depletion of CD4(+) cells significantly diminished both parasite burden and inflammation and correlated with decreased IL-4 and IL-13 production and loss of mast cell infiltration. This model reveals important immune factors that influence susceptibility to infection and demonstrates for the first time the pathologic contribution of the host immune response in amebiasis.

Inoculation of Trypanosoma cruzi, Tulahuén strain, into C57BL/6 and BALB/c mice led to an acute infection characterized by marked parasitaemia, myocardial inflammation and thymocyte depletion. While C57BL/6 mice showed a progressive and lethal disease, BALB/c mice partly recovered. To characterize these murine models more effectively, we studied the parasite burden, serum levels of major infection outcome-related cytokines, the in vitro features of T. cruzi infection in peritoneal macrophages and the immunophenotype of thymic cells. The greater disease severity of T. cruzi-infected C57BL/6 mice was not linked to an increased parasite load, as parasitaemia, myocardial parasite nests and amastigote counts in peritoneal macrophages were not different from those in BALB/c mice. Cortical thymocyte loss was accompanied by the presence of apoptotic bodies and fragmented nuclear DNA, whereas fluorocytometric analysis at 17 days postinfection (p.i.) revealed a more pronounced loss of CD4+ CD8+ cells in C57BL/6 mice. This group displayed higher levels of TNF-alpha on days 14 and 21 p.i., in the presence of lower IL-1beta and IL-10 concentrations by days 14 and 21, and days 7 and 14 p.i., respectively. Day-21 evaluation showed higher concentrations of nitrate and TNF-alpha soluble receptors in C57BL/6 mice with no differences in IFN-gamma levels, with respect to the BALB/c group. Increased morbidity of C57BL/6 T. cruzi-infected mice does not seem to result from an aggravated infection but from an unbalanced relationship between pro- and anti-inflammatory mediators.

Trans-sialidase is a membrane-bound and shed sialidase from Trypanosoma cruzi, the protozoan parasite responsible for Chagas disease. We investigated the role of soluble trans-sialidase on host CD4+ T cell activation. Trans-sialidase activated naive CD4+ T cells in vivo. Both enzymatically active and inactive recombinant trans-sialidases costimulated CD4+ T cell activation in vitro. Costimulation resulted in increased mitogen-activated protein kinase activation, proliferation, and cytokine synthesis. Furthermore, active and inactive trans-sialidases blocked activation-induced cell death in CD4+ T cells from T. cruzi-infected mice. By flow cytometry, inactive trans-sialidase bound the highly sialylated surface Ag CD43 on host CD4+ T cells. Both costimulatory and antiapoptotic effects of trans-sialidases required CD43 signaling. These results suggest that trans-sialidase family proteins are involved in exacerbated host T lymphocyte responses observed in T. cruzi infection.

The parasite Toxoplasma gondii can infect most mammals and birds, sometimes causing severe pathology. Primary infection during pregnancy can result in abortion or fetal defects. Host immunity, particularly cellular immunity towards antigenic peptides, can control infection, but an efficient vaccine is not yet available. We have evaluated T-cell responses to a crude soluble toxoplasma antigen (ST-Ag) and to five recombinant peptide antigens of cells in whole-blood cultures from 22 pregnant women with preexisting infections and from 7 pregnant negative controls. Cells from all infected patients but from none of the controls responded specifically to ST-Ag by expressing surface CD25 on culture. Responses to the recombinant antigens showed considerable variation between individuals. rGRA1 elicited a response in 16 of the 22 samples (73%), rSAG1 in 13, rGRA7 in 9, rGRA6-CT in 4, and rGRA6-NT in only 1. Most responding cells were CD4(+). Cells from infected subjects cultured with ST-Ag all released high levels of gamma interferon (IFN-gamma) into the culture supernatant (4,343 +/- 2,536 pg/ml). Cells from 12 patients released IFN-gamma after culture with rGRA1 (130 +/- 98 pg/ml), those from 10 patients released it after culture with rSAG1 (183 +/- 128 pg/ml), and those from 4 patients released it after culture with rGRA7 (324 +/- 374 pg/ml). Intensity of IFN-gamma production in response to the latter two recombinant antigens correlated with responses to ST-Ag (r = 0.61 and 0.53, respectively; P < 0.01). Interleukin-4 was always absent from supernatants of cells stimulated with toxoplasma antigens. The heterogeneity of human responses to individual recombinant toxoplasma antigens should be considered in the design of potential vaccines.

It is thought that both helper and effector functions of CD4(+) T cells contribute to protective immunity to blood stage malaria infection. However, malaria infection does not induce long-term immunity and its mechanisms are not defined. In this study, we show that protective parasite-specific CD4(+) T cells were depleted after infection with both lethal and nonlethal species of rodent PLASMODIUM: It is further shown that the depletion is confined to parasite-specific T cells because (a) ovalbumin (OVA)-specific CD4(+) T cells are not depleted after either malaria infection or direct OVA antigen challenge, and (b) the depletion of parasite-specific T cells during infection does not kill bystander OVA-specific T cells. A significant consequence of the depletion of malaria parasite-specific CD4(+) T cells is impaired immunity, demonstrated in mice that were less able to control parasitemia after depletion of transferred parasite-specific T cells. Using tumor necrosis factor (TNF)-RI knockout- and Fas-deficient mice, we demonstrate that the depletion of parasite-specific CD4(+) T cells is not via TNF or Fas pathways. However, in vivo administration of anti-interferon (IFN)-gamma antibody blocks depletion, suggesting that IFN-gamma is involved in the process. Taken together, these data suggest that long-term immunity to malaria infection may be affected by an IFN-gamma-mediated depletion of parasite-specific CD4(+) T cells during infection. This study provides further insight into the nature of immunity to malaria and may have a significant impact on approaches taken to develop a malaria vaccine.

Acute and lethal ileitis can be elicited in certain strains of inbred mice after oral infection with the intracellular protozoan parasite Toxoplasma gondii. The development of this inflammatory process is dependent upon the induction of a robust Th1 response, including overproduction of IFN-gamma, TNF-alpha, and NO, as has been reported in other experimental models of human inflammatory bowel disease. In this study we have investigated the role of CD4(+) T cells from the lamina propria (LP) in the early inflammatory events after T. gondii infection using isolated and primary cultured intestinal cells from infected mice and immortalized mouse mIC(cl2) intestinal epithelial cells. Primed LP CD4(+) T cells isolated from parasite-infected mice produce substantial quantities of both IFN-gamma and TNF-alpha. IFN-gamma- and TNF-alpha-producing LP CD4(+) T cells synergize with infected mIC(cl2) and enhance the production of several inflammatory chemokines including macrophage-inflammatory protein-2, monocyte chemoattractant protein-1, monocyte chemoattractant protein-3, macrophage-inflammatory protein-1alphabeta, and IFN-gamma-inducible protein-10. Furthermore, primed LP CD4(+) T cells cocultured with infected mIC(cl2) inhibited replication of the parasite in the intestinal epithelial cells. Thus, LP CD4(+) T cells can interact with parasite-infected intestinal epithelial cells and alter the expression of several proinflammatory products that have been associated with the development of intestinal inflammation. The interaction between these two components of the gut mucosal compartment (CD4(+) T cells and enterocytes) may play a role in the immunopathogenesis of this pathogen-driven experimental inflammatory bowel disease model.

T-cell immune responses in patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) were studied during the active disease, at the end of therapy, and 1 to 17 years posttherapy (long-term follow-up). Lymphocyte proliferative responses, phenotypic characterization of CD4(+) and CD8(+) Leishmania-reactive T cells, and cytokine production were assayed. Patients with active ML and CL showed higher proportions of CD4(+) than CD8(+) T cells. In CL, the healing process was associated with a decrease of CD4(+) and an increase of CD8(+), leading to similar CD4(+) and CD8(+) proportions. This pattern was only seen in ML after long-term therapy. Long-term follow-up of patients with CL showed a positive CD4(+)/CD8(+) ratio as observed during the active disease, although the percentages of these T cell subsets were significantly lower. Patients with CL did not show significant differences between gamma interferon (IFN-gamma) and interleukin-5 (IL-5) production during the period of study. Patients with active ML presented higher IFN-gamma and IL-5 levels compared to patients with active CL. IL-4 was only detected during active disease. Patients long term after cure from ML showed increasing production of IFN-gamma, significant decrease of IL-5, and no IL-4 production. Two apparently beneficial immunological parameters were detected in tegumentary leishmaniasis: (i) decreasing proportions of CD4(+) Leishmania-reactive T cells in the absence of IL-4 production associated with cure of CL and ML and (ii) decreasing levels of IL-5 long after cure, better detected in patients with ML. The observed T-cell responses maintained for a long period in healed patients could be relevant for immunoprotection against reinfection and used as a parameter for determining the prognosis of patients and selecting future vaccine preparations.

Persistent immunity against Leishmania: infections in humans is mediated predominantly by CD4(+) T cells of the Th1 phenotype. Herein we report the expression cloning of eight Leishmania: Ags using parasite-specific T cell lines derived from an immune donor. The Ags identified by this technique include the flagellar proteins alpha- and beta-tubulin, histone H2b, ribosomal protein S4, malate dehydrogenase, and elongation factor 2, as well as two novel parasite proteins. None of these proteins have been previously reported as T cell-stimulating Ags from Leishmania: beta-tubulin-specific T cell clones generated against Leishmania: major amastigotes responded to Leishmania:-infected macrophages and dendritic cells. IFN-gamma enzyme-linked immunospot analysis demonstrated the presence of T cells specific for several of these Ags in PBMC from self-healing cutaneous leishmaniasis patients infected with either Leishmania: tropica or L. major. The responses elicited by Leishmania: histone H2b were particularly striking in terms of frequency of histone-specific T cells in PBMC (1 T cell of 6000 PBMC) as well as the percentage of responding donors (86%, 6 of 7). Ags identified by T cells from immune donors might constitute potential vaccine candidates for leishmaniasis.

c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase that plays important regulatory roles in helper T cell differentiation. In the current study, we used Jnk1-deficient mice to examine the function of JNK during an in vivo pathogenic infection, leishmaniasis, which is strongly influenced by Th1/Th2 effector mechanisms. The data show that Jnk1-deficient mice, despite their usually genetically resistant background, were unable to resolve Leishmania infections. Jnk1-/- mice displayed reduced delayed-type hypersensitivity in response to the pathogen, which was associated with a T cell defect. We found that, although these mice can direct an apparent Th1-response, there is also simultaneous generation of Leishmania-specific Th2 responses, which possibly down-modulate protective Th1-mediated immune function. These findings demonstrate that the negative regulation of Th2 cytokine production by the JNK1 signaling pathway is essential for generating Th1-polarized immunity against intracellular pathogens, such as Leishmania major.