T cell reactivity against mycolyl transferase antigen 85 of M. tuberculosis in HIV-TB coinfected subjects and in AIDS patients suffering from tuberculosis and nontuberculous mycobacterial infections.

The mycolyl transferase antigen 85 complex is a major secreted protein family from mycobacterial culture filtrate, demonstrating powerful T cell stimulatory properties in most HIV-negative, tuberculin-positive volunteers with latent M.tuberculosis infection and only weak responses in HIV-negative tuberculosis patients. Here, we have analyzed T cell reactivity against PPD and Ag85 in HIV-infected individuals, without or with clinical symptoms of tuberculosis, and in AIDS patients with disease caused by nontuberculous mycobacteria. Whereas responses to PPD were not significantly different in HIV-negative and HIV-positive tuberculin-positive volunteers, responses to Ag85 were significantly decreased in the HIV-positive (CDC-A and CDC-B) group. Tuberculosis patients demonstrated low T cell reactivity against Ag85, irrespective of HIV infection, and finally AIDS patients suffering from NTM infections were completely nonreactive to Ag85. A one-year follow-up of twelve HIV-positive tuberculin-positive individuals indicated a decreased reactivity against Ag85 in patients developing clinical tuberculosis, highlighting the protective potential of this antigen.

Ly49E-dependent inhibition of natural killer cells by urokinase plasminogen activator.

The Ly49 natural killer (NK)-cell receptor family comprises both activating and inhibitory members, which recognize major histocompatibility complex (MHC) class I or MHC class I-related molecules and are involved in target recognition. As previously shown, the Ly49E receptor fails to bind to a variety of soluble or cell-bound MHC class I molecules, indicating that its ligand is not an MHC class I molecule. Using BWZ.36 reporter cells, we demonstrate triggering of Ly49E by the completely distinct, non-MHC-related protein urokinase plasminogen activator (uPA). uPA is known to be secreted by a variety of cells, including epithelial and hematopoietic cells, and levels are up-regulated during tissue remodeling, infections, and tumorigenesis. Here we show that addition of uPA to Ly49E-positive adult and fetal NK cells inhibits interferon-gamma secretion and reduces their cytotoxic potential, respectively. These uPA-mediated effects are Ly49E-dependent, as they are reversed by addition of anti-Ly49E monoclonal antibody and by down-regulation of Ly49E expression using RNA interference. Our results suggest that uPA, besides its established role in fibrinolysis, tissue remodeling, and tumor metastasis, could be involved in NK cell-mediated immune surveillance and tumor escape.

Intralesional regulatory T-cell suppressive function during human acute and chronic cutaneous leishmaniasis due to Leishmania guyanensis.

The levels of regulatory T cells (Treg cells), analyzed by Foxp3 mRNA expression, were determined in lesions from patients with acute cutaneous leishmaniasis (ACL) and chronic cutaneous leishmaniasis (CCL). We demonstrated that Treg cells preferentially accumulate in lesions from ACL patients during the early phase of infection (lesion duration of less than 1 month). In addition, levels of Foxp3 mRNA transcripts were significantly higher in specimens from patients with CCL than in those from patients with ACL, suggesting a critical role of intralesional Treg cells in CCL. Intralesional Treg cells from both ACL and CCL patients were shown to have suppressive functions in vitro, since they inhibited the gamma interferon (IFN-gamma) produced by CD4(+) CD25(-) T cells purified from peripheral blood mononuclear cells from the same patient in response to Leishmania guyanensis stimulation. Intralesional 2,3-indoleamine dioxygenase (IDO) mRNA expression was associated with that of Foxp3, suggesting a role for IDO in the suppressive activity of intralesional Treg cells. In addition, a role, albeit minor, of interleukin-10 (IL-10) was also demonstrated, since neutralization of IL-10 produced by intralesional T cells increased IFN-gamma production by effector cells in an in vitro suppressive assay. These results confirm the role of intralesional Treg cells in the immunopathogenesis of human Leishmania infection, particularly in CCL patients.

Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the contribution of tumor necrosis factor is minimal.

Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.

Cytomegalovirus (CMV)-specific cellular immune responses.

A large percentage of healthy individuals (50-90%) is chronically infected with Cytomegalovirus (CMV). Over the past few years, several techniques were developed in order to monitor CMV-specific T-cell responses. In addition to the identification of antigen-specific T cells with peptide-loaded MHC complexes, most of the current strategies to identify CMV-specific T cells are centered on the assessment of the functions of memory T cells including their ability to mediate effector function, to proliferate or to secrete cytokines following antigen-specific stimulation. The investigation of these functions has allowed the characterization of the CMV-specific T-cell responses that are present during different phases of the infection. Furthermore, it has also been shown that the combination of virus-specific CD4 and CD8 T-cell responses are critical components of the immune response in the control of virus replication.

T helper 1 (Th1) and Th2 characteristics start to develop during T cell priming and are associated with an immediate ability to induce immunoglobulin class switching.

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cgamma2a and Cgamma1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon gamma in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone. The addition of killed Bordetella pertussis to the hapten-protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten-protein can proceed in a node where there is substantial Th1 activity.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

Critical roles of PPAR beta/delta in keratinocyte response to inflammation.

The immediate response to skin injury is the release of inflammatory signals. It is shown here, by use of cultures of primary keratinocytes from wild-type and PPAR beta/delta(-/-) mice, that such signals including TNF-alpha and IFN-gamma, induce keratinocyte differentiation. This cytokine-dependent cell differentiation pathway requires up-regulation of the PPAR beta/delta gene via the stress-associated kinase cascade, which targets an AP-1 site in the PPAR beta/delta promoter. In addition, the pro-inflammatory cytokines also initiate the production of endogenous PPAR beta/delta ligands, which are essential for PPAR beta/delta activation and action. Activated PPAR beta/delta regulates the expression of genes associated with apoptosis resulting in an increased resistance of cultured keratinocytes to cell death. This effect is also observed in vivo during wound healing after an injury, as shown in dorsal skin of PPAR beta/delta(+/+) and PPAR beta/delta(+/-) mice.

Fatty acid amide hydrolase deficiency enhances intraplaque neutrophil recruitment in atherosclerotic mice.

OBJECTIVE: Endocannabinoid levels are elevated in human and mouse atherosclerosis, but their causal role is not well understood. Therefore, we studied the involvement of fatty acid amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in atherosclerotic plaque vulnerability. METHODS AND RESULTS: We assessed atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)FAAH(-/-) mice. Before and after 5, 10, and 15 weeks on high-cholesterol diet, we analyzed weight, serum cholesterol, and endocannabinoid levels, and atherosclerotic lesions in thoracoabdominal aortas and aortic sinuses. Serum levels of FAAH substrates anandamide, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were 1.4- to 2-fold higher in case of FAAH deficiency. ApoE(-/-)FAAH(-/-) mice had smaller plaques with significantly lower content of smooth muscle cells, increased matrix metalloproteinase-9 expression, and neutrophil content. Circulating and bone marrow neutrophil counts were comparable between both genotypes, whereas CXC ligand1 levels were locally elevated in aortas of FAAH-deficient mice. We observed enhanced recruitment of neutrophils, but not monocytes, to large arteries of ApoE(-/-) mice treated with FAAH inhibitor URB597. Spleens of ApoE(-/-)FAAH(-/-) mice had reduced CD4+FoxP3+regulatory T-cell content, and in vitro stimulation of splenocytes revealed significantly elevated interferon-γ and tumor necrosis factor-α production in case of FAAH deficiency. CONCLUSIONS: Increased anandamide and related FAAH substrate levels are associated with the development of smaller atherosclerotic plaques with high neutrophil content, accompanied by an increased proinflammatory immune response.

Heterologous prime-boost regimen adenovector 35-circumsporozoite protein vaccine/recombinant Bacillus Calmette-Guérin expressing the Plasmodium falciparum circumsporozoite induces enhanced long-term memory immunity in BALB/c mice.

BACKGROUND: Sustained antibody levels are a hallmark of immunity against many pathogens, and induction of long-term durable antibody titers is an essential feature of effective vaccines. Heterologous prime-boost approaches with vectors are optimal strategies to improve a broad and prolonged immunogenicity of malaria vaccines. RESULTS: In this study, we demonstrate that the heterologous prime-boost regimen Ad35-CS/BCG-CS induces stronger immune responses by enhancing type 1 cellular producing-cells with high levels of CSp-specific IFN-γ and cytophilic IgG2a antibodies as compared to a homologous BCG-CS and a heterologous BCG-CS/CSp prime-boost regimen. Moreover, the heterologous prime-boost regimen elicits the highest level of LLPC-mediated immune responses. CONCLUSION: The increased IFN-γ-producing cell responses induced by the combination of Ad35-CS/BCG-CS and sustained type 1 antibody profile together with high levels of LLPCs may be essential for the development of long-term protective immunity against liver-stage parasites.

Preclinical vaccine study of Plasmodium vivax circumsporozoite protein derived-synthetic polypeptides formulated in montanide ISA 720 and montanide ISA 51 adjuvants.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate previously assessed in animals and humans. Here, combinations of three synthetic polypeptides corresponding to amino (N), central repeat (R), and carboxyl (C) regions of the CS protein formulated in Montanide ISA 720 or Montanide ISA 51 adjuvants were assessed for immunogenicity in rodents and primates. BALB/c mice and Aotus monkeys were divided into test and control groups and were immunized three times with doses of 50 and 100 μg of vaccine or placebo. Antigen-specific antimalarial antibodies were determined by enzyme-linked immunosorbent assay, immunofluorescent antibody test, and IFN-γ responses by enzyme-linked immunosorbent spot (ELIspot). Both vaccine formulations were highly immunogenic in both species. Mice developed better antibody responses against C and R polypeptides, whereas the N polypeptide was more immunogenic in monkeys. Anti-peptide antibodies remained detectable for several months and recognized native proteins on sporozoites. Differences between Montanide ISA 720 and Montanide ISA 51 formulations were not significant.

gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection.

BACKGROUND & AIMS: Infection with Helicobacter induces a T helper type 1 response in mice and humans. Mice can be cured or protected from infection with Helicobacter by mucosal immunization with recombinant H. pylori urease B subunit (rUreB). This study characterizes the immune response of infected mice immunized with rUreB. METHODS: BALB/c mice were infected with H. felis. Two weeks later, they were orally immunized four times with rUreB and cholera toxin (CT) at weekly intervals. Controls were only infected or sham-immunized with CT. Animals were killed at various times after immunization. Splenic CD4(+) cells were obtained and cultured in vitro with rUreB to evaluate antigen-specific proliferation and induction of interferon gamma and interleukin 4 secretion. RESULTS: All rUreB-immunized mice (n = 8) were cured from infection 3 weeks after the fourth immunization. Immunization induced a proliferative response of splenic CD4(+) cells, a progressive decrease in interferon gamma secretion, and a concomitant increase in interleukin 4 secretion after each immunization. A simultaneous increase in rUreB specific serum immunoglobulin G1 levels was observed in infected/immunized mice. CONCLUSIONS: In BALB/c mice, therapeutic mucosal immunization with rUreB induces progressively a Th2 CD4(+) T cell response resulting in the elimination of the pathogen.

The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12.

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.