Association of Single Nucleotide Polymorphisms in Cytotoxic T-Lymphocyte Antigen 4 and Susceptibility to Autoimmune Type 1 Diabetes in Tunisians

In addition to HLA and insulin genes, the costimulatory molecule CTLA-4 gene is a confirmed type 1 diabetes (T1D) susceptibility gene. Previous studies investigated the association of CTLA-4 genetic variants with the risk of T1D, but with inconclusive findings. Here, we tested the contributions of common CTLA-4 gene variants to T1D susceptibility in Tunisian patients and control subjects. The study subjects comprised 228 T1D patients (47.8% females) and 193 unrelated healthy controls (45.6% females). Genotyping for CTLA-4 CT60A/G (rs3087243), +49A/G (rs231775), and −318C/T (rs5742909) was performed by PCR-restriction fragment length polymorphism (RFLP) analysis. The minor-allele frequencies (MAF) for the three CTLA-4 variants were significantly higher in T1D patients, and significantly higher frequencies of homozygous +49G/G and homozygous CT60G/G genotypes were seen in patients, which was confirmed by univariate regression analysis (taking the homozygous wild type as a reference). Of the eight possible three-locus CTLA-4 haplotypes (+49A/G, −318C/T, and CT60A/G) identified, multivariate regression analysis confirmed the positive association of ACG (odds ratio [OR], 1.93; 95% confidence interval [CI], 1.26 to 2.94), GCG (OR, 2.40; 95% CI, 1.11 to 5.21), and GTA (OR, 4.67; 95% CI, 1.52 to 14.39) haplotypes with T1D, after confounding variables were adjusted for. Our results indicate that CTLA-4 gene variants are associated with increased T1D susceptibility in Tunisian patients, further supporting a central role for altered T-cell costimulation in T1D pathogenesis.Type 1 (insulin-dependent) diabetes (T1D) is the most prevalent form of diabetes in children and young adults and results from autoimmune CD4⁺ and CD8⁺ T-cell-directed destruction of insulin-producing pancreatic β islet cells in genetically susceptible individuals (3, 12), leading to irreversible hyperglycemia and related complications (13). There is a strong genetic component to T1D pathogenesis, evidenced by its clustering in families and by the contributions of a number of susceptibility gene variants to its pathogenesis (10, 12, 29). They include the human leukocyte antigen (HLA) locus, in particular the class II region (DR and DQ), which accounts for 40 to 50% of T1D familial clustering (1, 12, 18), and non-HLA susceptibility loci, several of which were mapped by genome-scanning (11, 29) and/or candidate gene (7, 18, 31) approaches. They include insulin promoter gene variants, which reportedly may modulate immunological tolerance by controlling the expansion of the autoreactive cell pool (26), and the T-cell costimulator cytotoxic T-lymphocyte antigen 4 (CTLA-4) transmembrane glycoprotein, which plays a key role in the fine tuning of T-cell immunity (9, 32, 33).CTLA-4 is a 40-kDa transmembrane glycoprotein expressed on resting and activated T cells and nonlymphoid cells (33), and along with the related CD28 costimulatory molecule, it regulates T-cell activation (and is itself primarily mediated by engagement of the T-cell receptor [TCR]) but does recognize major histocompatibility complex (MHC)-bound antigenic peptides (9, 33). CTLA-4 negatively regulates T-cell activation and effector function, in part by inhibiting Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ]) cytokine production and IL-2 receptor α-chain (p55; Tac) expression by engaging antigen-presenting cell (APC)-bound B7.1 (CD80) and B7.2 (CD86) ligands (9, 33). Functionally, CTLA-4 attenuates T-cell signaling by interference with intracellular signal transduction events, including TCR signaling, and reduced CTLA-4 expression and/or activity results in uncontrolled T-cell-associated autoimmunity and lymphoproliferative disease (9, 21). In this regard, it was shown that CTLA-4 polymorphisms significantly influence the risk of autoimmune diseases, including Graves'' disease, systemic lupus erythematosus, autoimmune hypothyroidism, celiac disease, and type 1 diabetes (15, 21, 32).First observed in Italian subjects (25), and confirmed subsequently by case control and family studies, CTLA-4 polymorphic variants were linked with T1D pathogenesis (14, 20, 31, 32). While this association was detected in different ethnic groups (14, 23, 30), it appears more likely to be Caucasian selective (10, 29, 33) and absent from non-Caucasians (5, 6, 8, 19, 22). A recent report from the Type I Diabetes Genetics Consortium bearing on 2,300 affected sib pair families demonstrated that among the 24 single nucleotide polymorphisms (SNPs) genotyped in the CTLA-4 region, only the +49A/G and CT60 SNPs were replicated in the nine combined collections (27). In the present study, we investigated the association of three common CTLA-4 SNPs (−318C/T; +49A/G, and CT60A/G) and the corresponding haplotypes with T1D in Tunisian Arab patients.     

Generation of Antibody Responses to Pneumococcal Capsular Polysaccharides Is Independent of CD1 Expression in Mice

Streptococcus pneumoniae is a bacterial microorganism that frequently causes serious infection, particularly in children and the elderly. Protection against infection with S. pneumoniae is based mainly on the generation of antibodies to the pneumococcal capsular polysaccharides (caps-PS), but the mechanisms responsible for the generation of anticapsular antibodies remain incompletely understood. The aim of the present study was to evaluate the role of CD1-restricted T cells in the antibody response to caps-PS. When immunized with Pneumo23, wild-type mice and CD1 knockout mice on BALB/c and C57BL/6 backgrounds generated immunoglobulin M (IgM) and IgG antibody responses to soluble caps-PS that were comparable. Similar results were obtained after immunization with heat-inactivated S. pneumoniae. The IgM and IgG antibody response of wild-type mice to Pneumo23 was not affected by an antagonizing monoclonal anti-CD1 antibody treatment. In summary, our data provide evidence that the antibody response to caps-PS is generated independently of CD1 expression.Streptococcus pneumoniae is a major human pathogen. Infections with S. pneumoniae result in substantial morbidity and mortality, particularly in young children, the elderly, and immunocompromised patients (26). In various animal species and in humans, protection against S. pneumoniae infection is mediated by antibodies against pneumococcal capsular polysaccharides (caps-PS) and surface proteins (2, 4, 21).caps-PS are classified as T lymphocyte-independent type 2 (TI-2) antigens (24). While T lymphocytes are not required for the generation of antibody responses against TI-2 antigens, they can influence the antibody response to these antigens (24). In the case of caps-PS, the role of T lymphocytes in the generation of antibody responses might be more important than was initially thought. There is now evidence that T lymphocytes may support the antibody response to TI-2 antigens via several pathways (14). The magnitude of the antibody response to caps-PS is regulated both positively and negatively by distinct subsets of thymus-derived T lymphocytes. It has been reported that CD4⁺ T cells have positive effects on the antibody response to caps-PS, whereas CD8⁺ T cells have a suppressive effect. The presence of these two distinct types of T cells with opposing regulatory functions with respect to the immune response to soluble caps-PS has been demonstrated in vivo in mice and in vitro with human lymphocytes (1, 8, 10). SCID/SCID mice reconstituted with B lymphocytes and CD4⁺ T lymphocytes mounted a higher specific immunoglobulin M (IgM) antibody response to soluble pneumococcal caps-PS than SCID/SCID mice reconstituted with only B lymphocytes (12, 15). Murine spleen cells depleted of CD8⁺ T lymphocytes mounted a higher immune response to soluble caps-PS than total murine spleen cells, whereas spleen cells depleted of CD4⁺ T cells elicited only a weak antibody response (15). Similarly, the human IgM and IgG antibody response to soluble pneumococcal caps-PS was strongly dependent on CD4⁺ T cells (13). Several reports have provided evidence that CD4⁺ T cells enhance the IgG antibody response to pneumococcal polysaccharides after immunization of mice with intact S. pneumoniae (19, 37). The antipolysaccharide antibody response after immunization with conjugated polysaccharide serotype 3 was higher in CD8-deficient mice than in control mice, a finding attributed to CD8 T lymphocyte-mediated suppression of the antipolysaccharide immune response (34).In a provocative study, Kobrynski et al. (20) reported that CD1-restricted T cells and major histocompatibility complex (MHC) class I-dependent CD8⁺ cells are essential for the anti-caps-PS immune response. These findings set forth a new paradigm for humoral responses to caps-PS in which CD1 expression as well as a subset of CD8⁺ cells is required to provide helper function for antibody production against TI-2 caps-PS, akin to the role of MHC class II-restricted CD4⁺ cells for the generation of antibody responses to protein antigens (20). The MHC class I-like protein CD1 is expressed on antigen-presenting cells and is required for the presentation of lipids and glycolipids to T lymphocytes (25, 28, 29).The findings of Kobrynski et al. (20), suggesting that CD8⁺ T cells are essential for the IgG antibody response to caps-PS, are at odds with many other experimental data (1, 8, 10, 12, 13, 15, 19, 34, 37) that support the concept that CD4⁺ T cells have a positive effect on the antipolysaccharide immune response. Because of this controversy and because Kobrynski et al. (20) did not investigate the role of CD1 expression in the generation of IgM anti-caps-PS antibody responses, we reevaluated the role of CD1 expression in the IgM and IgG antibody response to pneumococcal polysaccharides. Our results revealed that CD1 expression was not required for the generation of IgM and IgG antibody responses to caps-PS.     

Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401

A more effective vaccine against Mycobacterium tuberculosis is needed, and a number of M. tuberculosis vaccine candidates are currently in preclinical or clinical phase I and II studies. One of the strategies to select M. tuberculosis (protein) targets to elicit a CD8⁺ or CD4⁺ T-cell response is to gauge the binding of candidate peptides to major histocompatibility complex (MHC) class I or class II molecules, a prerequisite for successful peptide presentation and to expand antigen-specific T cells. We scanned 61 proteins from the M. tuberculosis proteome for potential MHC class II-presented epitopes that could serve as targets for CD4⁺ T-cell responses. We constructed a peptide microarray consisting of 7,466 unique peptides derived from 61 M. tuberculosis proteins. The peptides were 15-mers overlapping by 12 amino acids. Soluble recombinant DRB1*0101 (DR1), DRB1*1501 (DR2), and DRB1*0401 (DR4) monomers were used to gauge binding to individual peptide species. Out of 7,466 peptides, 1,282, 674, and 1,854 peptides formed stable complexes with HLA-DR1, -DR2, and -DR4, respectively. Five hundred forty-four peptides bound to all three MHC class II molecules, 609 bound to only two, and 756 bound to only a single MHC class II molecule. This allowed us to rank M. tuberculosis proteins by epitope density. M. tuberculosis proteins contained “hot spots,” i.e., regions with enriched MHC class II binding epitopes. Two hundred twenty-two peptides that formed MHC class II-peptide complexes had previously been described as exclusively recognized by IgG in sera from patients with active pulmonary tuberculosis, but not in sera from healthy individuals, suggesting that these peptides serve as B-cell and CD4⁺ T-cell epitopes. This work helps to identify not only M. tuberculosis peptides with immunogenic potential, but also the most immunogenic proteins. This information is useful for vaccine design and the development of future tools to explore immune responses to M. tuberculosis.CD4⁺ T cells play a central role in Mycobacterium tuberculosis-directed cellular immune responses (2, 6, 7, 12). It is most likely that an effective tuberculosis (TB) vaccine would target the expansion of CD8⁺ and CD4⁺ T cells, which recognize M. tuberculosis peptides presented by major histocompatibility complex (MHC) class I and class II molecules.The MHC locus is the most variable gene locus in the human genome, and the variability of MHC class II alleles in different populations is well documented (24). Certain MHC class II alleles have been shown to be associated with M. tuberculosis infection (1, 11, 15, 16, 23): DRB1*0803 and DQB1*0601 were found to be associated with TB disease progression, development of drug resistance, and disease severity in Koreans (16). In South Africa, DRB1*1302 and DQB1*0301 to -0304 were apparently associated with active TB compared to control individuals lacking these alleles (23). The prevalence of HLA-DRB1*0401 and HLA-DRB1*0801 was significantly decreased in Mexican patients with pulmonary TB compared to their prevalence in healthy controls (35).The association of some MHC class II alleles with “better disease outcome” could be due to the fact that these alleles are “better” at binding and presenting a certain repertoire of peptide epitopes to CD4⁺ T cells than other alleles. The identification of peptides binding to molecularly defined MHC class II alleles could therefore represent an important first step in identifying potential targets for TB vaccine design and the development of new diagnostic assays. More recently, De Groot and colleagues used a bioinformatics approach, followed by validation with functional assays to identify CD4⁺ T-cell epitopes that were used to construct an epitope-based M. tuberculosis vaccine (5).Only a few M. tuberculosis MHC class II binding peptides have been identified so far, and 7% of the M. tuberculosis open reading frames have been explored for both B-cell and T-cell epitopes (3). We described a peptide microarray assay that allowed us to visualize HIV peptide binding to molecularly defined MHC class II alleles (9). The assay has the major advantage that a high number of candidate peptides can be screened within a short time frame. In the current report, we describe M. tuberculosis peptide binding to the three most frequently encountered MHC class II alleles in different populations; DRB1*0101 (DR1), DRB1*1501 (DR2), and DRB1*0401 (DR4). DR1, DR2, and DR4 exhibit population frequencies of 15.4%, 32.9%, and 20.9% among Caucasians. In the Botswana population, HLA-DRB1*01, -DRB1*02, and -DRB1*04 show population frequencies of 21.7%, 21.3%, and 14.4%, respectively. The candidate test peptides are derived from 61 M. tuberculosis proteins that have been tested for IgG and IgA recognition in patients with active pulmonary TB. The data sets contribute to defining “immunogenicity” in M. tuberculosis candidate target proteins, visualize MHC class II epitope “hot spots,” and allow us to link B-cell targets and potential MHC class II-presented M. tuberculosis epitopes.     

Human Brucellosis Is Characterized by an Intense Th1 Profile Associated with a Defective Monocyte Function

In animal models, a defective Th1 response appears to be critical in the pathogenesis of brucellosis, but the Th1 response in human brucellosis patients remains partially undefined. Peripheral blood from 24 brucellosis patients was studied before and 45 days after antibiotherapy. Twenty-four sex- and age-matched healthy donors were analyzed in parallel. Significantly increased levels of interleukin 1β (IL-1β), IL-2, IL-4, IL-6, IL-12p40, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), but not of IL-10, in serum and/or significantly increased percentages of samples with detectable levels of these cytokines, measured by enzyme-linked immunosorbent assays (ELISA), were found for untreated brucellosis patients, but these levels were reduced and/or normalized after treatment. Flow cytometry studies showed that the intracytoplasmic expression of IFN-γ, IL-2, and TNF-α, but not that of IL-4, by phorbol myristate-activated CD4⁺ CD3⁺ and CD8⁺ CD3⁺ T lymphocytes was significantly increased in untreated brucellosis patients and was also partially normalized after antibiotherapy. The percentage of phagocytic cells, the mean phagocytic activity per cell, and the phagocytic indices for monocytes at baseline were defective and had only partially reverted at follow-up. T lymphocytes from untreated brucellosis patients are activated in vivo and show Th1 cytokine production polarization, with strikingly high serum IFN-γ levels. In spite of this Th1 environment, we found deficient effector phagocytic activity in peripheral blood monocytes.Brucellosis is a zoonotic disease of worldwide distribution. Despite its control in many countries, it remains endemic in the Mediterranean and Middle Eastern regions (20, 28, 41, 42). Brucella melitensis is the most frequent cause of human brucellosis in these geographical areas (19). In Spain, it has been reported that the majority (more than 97.5%) of isolates were identified as Brucella melitensis (13, 44, 45).Brucella organisms are facultatively intracellular Gram-negative coccobacilli that reside and replicate in a vacuolar compartment within myelomonocytic cells of the infected host (14, 15, 47). The response to Brucella involves the whole gamut of the immune system, from innate to adaptive immunity (21). In murine models, passive transfer of immune cells resulted in an effective anti-Brucella defensive response mediated by CD4⁺ and CD8⁺ T lymphocytes (5, 6, 32, 37, 51, 52). Furthermore, the pattern of T-lymphocyte cytokine secretion is considered to be critical for the effectiveness of the protective anti-Brucella immune response (3, 7). It has been postulated that Th1 cytokines confer resistance, while Th2 cytokines facilitate the development of brucellosis (2, 3, 24, 25, 40, 43, 52). In animal models, gamma interferon (IFN-γ) induces macrophage activation and control of Brucella infection (16, 18, 43). In Brucella-infected mice, administration of recombinant IFN-γ enhances host resistance, resulting in a deep decrease in the number of viable bacteria (51). Moreover, host IFN-γ depletion results in an increase in the number of viable bacteria (17, 37, 52). Several abnormalities in the immune system have been found in human brucellosis (27, 46, 49). It has been found that T and NK lymphocytes show defective functions in brucellosis patients (46, 49). Since mice are naturally resistant to Brucella infections, it is possible to suggest that the immune response elicited by Brucella in humans might have different characteristics. Thus, susceptibility to, or protection from, human brucellosis conferred by T-lymphocyte cytokines has not been established.In this work, we have further investigated the pattern of T-lymphocyte and monocyte responses to human Brucella infection. We have prospectively studied (i) the levels of Th1, Th2, and regulatory cytokines in serum, (ii) the distribution, activation stage, and pattern of Th1/Th2 cytokine production by T lymphocytes, and (iii) the phagocytic activity of monocytes in a group of brucellosis patients before and after antimicrobial treatment.     

Helminth Infection Can Reduce Insulitis and Type 1 Diabetes through CD25- and IL-10-Independent Mechanisms

Parasitic helminth infection has been shown to modulate pathological inflammatory responses in allergy and autoimmune disease. The aim of this study was to examine the effects of infection with a helminth parasite, Heligmosomoides polygyrus, on type 1 diabetes (T1D) in nonobese diabetic (NOD) mice and to elucidate the mechanisms involved in this protection. H. polygyrus inoculation at 5 weeks of age protected NOD mice from T1D until 40 weeks of age and also inhibited the more aggressive cyclophosphamide-induced T1D. Moreover, H. polygyrus inoculation as late as 12 weeks of age reduced the onset of T1D in NOD mice. Following H. polygyrus inoculation of NOD mice, pancreatic insulitis was markedly inhibited. Interleukin-4 (IL-4), IL-10, and IL-13 expression and the frequency of CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells were elevated in mesenteric and pancreatic lymph nodes. Depletion of CD4⁺ CD25⁺ T cells in vivo did not abrogate H. polygyrus-induced T1D protection, nor did anti-IL-10 receptor blocking antibody. These findings suggest that infection with H. polygyrus significantly inhibits T1D in NOD mice through CD25- and IL-10-independent mechanisms and also reduces the severity of T1D when administered late after the onset of insulitis.Helminth parasites infect about 1.5 billion people worldwide, especially in developing countries, and cause chronic infection that leads to malnutrition, anemia, impaired growth, and significant mortality. Intestinal nematode parasites can produce strong polarized Th2-type responses in mice. This immune response is characterized by eosinophilia, mucosal mast cell hyperplasia, elevated immunoglobulin E (IgE) secretion, and increased production of Th2 cytokines, such as interleukin-4 (IL-4) and IL-13. Recent studies have suggested that helminth infection can regulate infectious, allergic, or autoimmune inflammatory diseases. Helminth infection enhances susceptibility to certain infectious diseases, like tuberculosis (11, 35) and viral hepatitis (10, 17). Conversely, helminth infection is protective in murine models of asthma (19), multiple sclerosis (40), and inflammatory bowel disease (42).Type 1 diabetes (T1D) is a life-threatening disease that affects approximately 1 out of 400 children in westernized societies (18). Over the past 3 decades, the rate of T1D has increased by approximately 4% per year in both Europe and the United States (8, 12, 39). This increase in disease incidence may result in part from a dysregulated immune system due to lack of exposure to certain environmental pathogens, such as helminth parasites (5, 7, 32). Studies with nonobese diabetic (NOD) mice showed that inoculation with Trichinella spiralis, Heligmosomoides polygyrus, or Schistosoma mansoni markedly reduced the rate of T1D and suppressed lymphoid infiltration in the islets (9, 37). T1D was also prevented in NOD mice by injection of whole eggs or soluble antigens from the schistosome egg antigen or the schistosome worm antigen, but only if treatment was started at 4 weeks of age (49). Moreover, the addition of oral insulin B chain to schistosome egg antigen-treated mice augmented the induction of regulatory T cells (Tregs) that secreted IL-4, IL-10, and transforming growth factor beta (TGF-β) (27).We were interested in further examining potential mechanisms contributing to the control of T1D during infection with the intestinal nematode parasite H. polygyrus. This strictly enteric parasite triggers a potent Th2-type response without eliciting an associated Th1-type response (4). We found that H. polygyrus infection exerted significant protection against T1D in NOD mice when administered at 5 and 7 weeks of age and even when given as late as 12 weeks of age (30% protection). This was associated with reduced lymphoid infiltration in the islets and an increased frequency of CD25⁺ Tregs with augmented Th2-type responses, including induction of alternatively activated macrophages (AAMΦs) and IL-10 mRNA in pancreatic lymph nodes (PLN). When H. polygyrus-inoculated NOD mice were treated with cyclophosphamide (Cyp), an agent known to accelerate T1D, T1D prevention was sustained. Similarly, when H. polygyrus-inoculated NOD mice were treated with anti-CD25 monoclonal antibody (MAb) in vivo, we observed no change in insulitis between this group and those receiving a control monoclonal Ig. Furthermore, in Cyp-treated NOD mice, administration of an anti-IL-10 receptor (IL-10R) blocking MAb did not abrogate H. polygyrus-induced protection from T1D. These findings suggest that H. polygyrus inoculation suppressed T1D even after the development of insulitis and that suppression of T1D in H. polygyrus-treated NOD mice is accomplished through CD25- and IL-10-independent mechanisms.     

Cholera Caused by Vibrio cholerae O1 Induces T-Cell Responses in the Circulation

Considerable effort is being made to understand the acute and memory antibody responses in natural cholera infection, while rather less is known about the roles of cellular immune responses involving T and B lymphocytes. We studied responses in adult patients hospitalized with cholera caused by Vibrio cholerae O1. Peripheral blood mononuclear cells from patients (n = 15) were analyzed by flow cytometry after stimulation with V. cholerae O1 membrane protein (MP) or toxin-coregulated pilus antigen (TcpA). The gamma interferon (IFN-γ) and interleukin-13 (IL-13) responses in stimulated-lymphocyte supernatants were studied. The responses were compared with those of healthy controls (n = 10). Patients responded with increased frequencies of gut-homing CD4⁺ T cells (CD4⁺ β7⁺), gut-homing CD8⁺ T cells (CD8⁺ β7⁺), and gut-homing B cells (CD19⁺ β7⁺) at the early and/or late convalescent stages compared to the acute stage. After stimulation with MP or TcpA, proliferation of CD4⁺ and CD8⁺ T cells was increased at the acute stage and/or early convalescent stage compared to healthy controls. Increased IL-13 and IFN-γ responses were observed after antigenic stimulation at the acute and convalescent stages compared to healthy controls. Thus, increases in the levels of gut-homing T and B cells, as well as involvement of CD8 and CD4 Th1-mediated (IFN-γ) and CD4 Th2-mediated (IL-13) cytokine responses, take place in acute dehydrating disease caused by V. cholerae O1. Further studies are needed to determine if such responses are also stimulated after immunization with oral cholera vaccines and if these responses play a role in protection following exposure to cholera.Vibrio cholerae O1 is a common causative agent of acute watery diarrhea in children and adults in the developing world (1, 3, 10, 19). After colonizing the proximal small intestine, this bacterium produces cholera toxin, which induces a profuse secretory diarrhea. Cholera remains a key public health problem that results in epidemics in resource-poor settings.It is believed that the immune response to cholera is initiated by antigen presentation in the Peyer''s patches of the gastrointestinal mucosa, followed by migration of the stimulated antigen-specific B cells to regional lymph nodes and differentiation of these cells into specific antibody-secreting cells (28). Stimulation of the common mucosal immune system leads to production of both local and systemic antibodies (2, 15, 27) to virulence antigens of V. cholerae (25, 28).Natural cholera infection is believed to give rise to long-term protection against subsequent disease. Robust systemic and mucosal antibodies are produced to the V. cholerae lipopolysaccharide, to cholera toxin, and to colonization factors, including the major subunit of the toxin-coregulated pilus, TcpA (2, 24, 25, 28). We have recently shown that there is induction of memory B-cell responses following infection, which may play a role in longer-lasting protection (14). In addition, recent evidence suggests that an innate component of the immune system may also play a role in the host response to cholera (9, 22, 26). Studies with experimental animals have shown that the mucosal immune response to cholera toxin is T cell dependent and that CD4 T helper cells have an important role (7, 12, 13). However, not much is known about the role of the adaptive cellular immune responses in patients with cholera. The aim of the present study was to decipher the role of T- and B-cell-mediated immune responses in natural cholera infection in adults hospitalized with dehydrating illness, who were followed from the acute stage to convalescence.     

Avirulent Uracil Auxotrophs Based on Disruption of Orotidine-5′-Monophosphate Decarboxylase Elicit Protective Immunity to Toxoplasma gondii

The orotidine-5′-monophosphate decarboxylase (OMPDC) gene, encoding the final enzyme of the de novo pyrimidine biosynthesis pathway, was deleted using Toxoplasma gondii KU80 knockouts to develop an avirulent nonreverting pyrimidine auxotroph strain. Additionally, to functionally address the role of the pyrimidine salvage pathway, the uridine phosphorylase (UP) salvage activity was knocked out and a double knockout of UP and OMPDC was also constructed. The nonreverting ΔOMPDC, ΔUP, and ΔOMPDC ΔUP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The ΔUP knockout strain was replication competent and virulent. In contrast, the ΔOMPDC and ΔOMPDC ΔUP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the ΔOMPDC strain but not the ΔOMPDC ΔUP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the ΔOMPDC and ΔOMPDC ΔUP knockout strains exhibited extreme attenuation in murine virulence (∼8 logs). Genetic complementation of the ΔOMPDC strain using a functional OMPDC allele restored normal replication and type I parental strain virulence phenotypes. A single immunization of mice with either the live critically attenuated ΔOMPDC strain or the ΔOMPDC ΔUP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting ΔOMPDC and ΔOMPDC ΔUP strains provide new tools for the dissection of the host response to infection and are promising candidates for safe and effective Th1 vaccine platforms that can be easily genetically engineered.Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates in a wide variety of cell types. Infections are widespread in humans, and while infections in healthy individuals are typically asymptomatic, severe disease can occur in utero or in individuals with severe immune suppression (22, 29, 31). A chronic infection is established and is characterized by quiescent cysts containing bradyzoites in tissues such as brain, muscle, and eye (42). Chronic infection can reactivate in AIDS and cause toxoplasmic encephalitis (9, 31) or recurrent ocular toxoplasmosis, recently recognized as a prevalent retinal infection in the United States (23, 27). Current treatments are poorly tolerated and are ineffective against chronic stages of infection, and there are no vaccines. Targeting of the T. gondii de novo pyrimidine synthesis pathway is one potential approach to developing more-effective vaccination strategies based on live attenuated strains with defined genetic disruptions (14).The key uracil phosphoribosyltransferase (UPRT) activity in the pyrimidine salvage pathway can easily be disrupted, and loss of UPRT has no apparent effect on parasite growth in vitro or virulence in vivo (4, 8). In the absence of any pyrimidine salvage pathway, T. gondii still possesses a complete six-step pathway for the de novo biosynthesis of UMP, the precursor molecule of all essential pyrimidines (1, 14, 34, 38). Insertional disruption of the first step of the biosynthetic pathway, encoded by the carbamoyl phosphate synthetase II (CPSII) gene, produced a severe uracil auxotrophy exemplified by the cps1-1 strain of T. gondii, which was incapable of de novo pyrimidine synthesis (13, 16). After invasion of a host cell, the cps1-1 uracil auxotrophic mutant was starved for pyrimidines and ceased to proliferate, since uracil is not readily available for salvage in mammals (12, 13, 32). The cps1-1 mutant strain also exhibited an extreme attenuation of virulence in both immune-competent and severely immune-deficient homozygous gamma interferon (IFN-γ) knockout mice (13).Immunization of mice with the live attenuated type I cps1-1 strain elicits a potent CD8⁺ T-cell-dependent lifelong protective immunity against infection with type I strains (13, 18) and against infection with type II strains and chronic infection (19). In contrast, immunization with T. gondii extracts or killed noninvasive intact parasites does not elicit significant immunity to reinfection with T. gondii (2, 40). Only actively infected host cells have been shown to prime CD8⁺ T-cell-dependent immunity in T. gondii infection (10, 20, 21).Immunity is effectively elicited by immunization with the cps1-1 strain in C57BL/6 (18), BALB/c (13), and tyk2^−/− signaling-deficient mice (39) and surprisingly also in MyD88^−/− deficient mice (41). Remarkably, macrophages primed in vivo by cps1-1 immunization but not naive macrophages also exhibit extremely efficient ex vivo IFN-γ-mediated innate cellular immunity augmenting intracellular rupture and clearance of type II and type III strains of T. gondii (30, 45-47), whereas virulent type I strains resist this cps1-1-induced innate killing mechanism (46).The lifelong immunity elicited by vaccination with strain cps1-1 is dependent on CD8⁺ T cells and interleukin 12 (IL-12) (41, 44) and is also dependent on IFN-γ (13, 18), although systemic IFN-γ is not required for priming (18). Surprisingly, cps1-1 rapidly elicits functional IL-12p70 both locally and systemically following vaccination (18). In contrast to current models of viral or intracellular bacterial infections, CD8⁺ T-cell-intrinsic IL-12 signaling is required for development of IFN-γ-producing CD8⁺ cytotoxic-T-lymphocyte populations and for the generation of memory CD8⁺ T cells in response to cps1-1 (43, 44). Vaccination with cps1-1 induced four distinct effector CD8⁺ T-cell types based on KLRG1 and CD62L expression levels (44). The rapidly elicited and abundant populations of antigen-specific effector CD8⁺ T cells induced by vaccination with cps1-1 are cytolytic in vitro and in vivo (28). The nonreplicating cps1-1 vaccine model has significantly advanced the understanding of the host response, innate immunity, and protective adaptive immunity to T. gondii infection (3, 10, 13, 18, 19, 28, 30, 39, 41, 43-47).The cps1-1 strain is not easily amenable to further genetic manipulation, and this strain exhibits an extremely low frequency of reversion to the virulent parental (strain RH) phenotype (13). To further address the potential of the six-step pyrimidine synthesis pathway to develop improved avirulent, nonreverting, genetically defined, and genetically manipulatable strains, we deleted the sixth and final enzyme of the pathway by targeted disruption of the orotidine-5′-monophosphate (OMP) decarboxylase, the OMPDC gene, in the RH strain KU80 knockout background, which now enables highly efficient gene targeting (17, 24). Targeted disruption of OMPDC via double-crossover homologous recombination induced severe pyrimidine auxotrophy and resulted in the generation of nonreverting strains that were essentially completely attenuated in their virulence in mice. A single immunization with ΔOMPDC knockout strains effectively induced a potent protective immunity to subsequent lethal challenge infection with T. gondii.     

Clinical and Immunological Insights on Severe,Adverse Neurotropic and Viscerotropic Disease following 17D Yellow Fever Vaccination

Yellow fever (YF) vaccines (17D-204 and 17DD) are well tolerated and cause very low rates of severe adverse events (YEL-SAE), such as serious allergic reactions, neurotropic adverse diseases (YEL-AND), and viscerotropic diseases (YEL-AVD). Viral and host factors have been postulated to explain the basis of YEL-SAE. However, the mechanisms underlying the occurrence of YEL-SAE remain unknown. The present report provides a detailed immunological analysis of a 23-year-old female patient. The patient developed a suspected case of severe YEL-AVD with encephalitis, as well as with pancreatitis and myositis, following receipt of a 17D-204 YF vaccination. The patient exhibited a decreased level of expression of Fc-γR in monocytes (CD16, CD32, and CD64), along with increased levels of NK T cells (an increased CD3⁺ CD16^+/− CD56^+/−/CD3⁺ ratio), activated T cells (CD4⁺ and CD8⁺ cells), and B lymphocytes. Enhanced levels of plasmatic cytokines (interleukin-6 [IL-6], IL-17, IL-4, IL-5, and IL-10) as well as an exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within NK cells (gamma interferon positive [IFN-γ⁺], tumor necrosis factor alpha positive [TNF-α⁺], and IL-4 positive [IL-4⁺]), CD8⁺ T cells (IL-4⁺ and IL-5⁺), and B lymphocytes (TNF-α⁺, IL-4⁺, and IL-10⁺). The analysis of CD4⁺ T cells revealed a complex profile that consisted of an increased frequency of IL-12⁺ and IFN-γ⁺ cells and a decreased percentage of TNF-α⁺, IL-4⁺, and IL-5⁺ cells. Depressed cytokine synthesis was observed in monocytes (TNF-α⁺) following the provision of antigenic stimuli in vitro. These results support the hypothesis that a strong adaptive response and abnormalities in the innate immune system may be involved in the establishment of YEL-AND and YEL-AVD.Yellow fever (YF) is a mosquito-borne viral hemorrhagic fever and is one of the most lethal viral diseases that remains an important public health problem in the regions of Africa and South America where it remains endemic (17). The easiness of international travel also makes YF a serious health threat to millions of travelers to areas where YF is endemic (8).Highly effective, live attenuated 17D YF vaccines have been available for decades and are considered to be among the safest vaccines worldwide (24). YF vaccines are produced by inoculation of embryonated chicken eggs with stock virus, harvesting, and freeze-drying with thermostabilizing components (1). The YF vaccines are delivered as a single subcutaneous dose and induce neutralizing antibodies in 90 to 99% of recipients after inoculation (4).The 17D YF vaccines are well tolerated and cause a very low rate of adverse events postvaccination (21). Less than 25% of vaccinees develop mild systemic symptoms, which may include headache, myalgia, discomfort at the site of vaccination, or low-grade fever, 2 to 6 days after vaccination (17). Despite the strong safety profile of the 17D YF vaccines, reports of rare yellow fever vaccine-associated serious adverse events (YEL-SAEs) have been described in the literature, including severe allergic reactions, neurotropic adverse disease (YEL-AND), and viscerotropic disease (YEL-AVD) (5, 12, 13, 16, 22).The mechanisms underlying the development of these adverse events remain unknown. Distinct hypotheses have been postulated to explain the basis of YEL-SAE, which includes viral and host features (1, 2, 7, 9, 10, 11). The reversion of the 17D YF vaccine virus into a more virulent form and other genetic mutations do not seem to be probable causes of the adverse events, since the YF virus isolated from patients with YEL-SAEs have rarely shown a nucleotide sequence different from that of the original 17D YF vaccine strain (7, 9). It is considered that host factors, mainly host immune responses to the 17D YF vaccine, may represent the major causes of YEL-SAEs (1, 2, 9, 10, 11).Increasing age, a history of thymus disorder, and immunocompromise are considered risk factors for the development of YEL-SAEs (2, 10, 13). However, several cases with unknown risk factors have been reported, suggesting the involvement of other host immunological features. A recent study with European patients with YEL-SAEs following 17D YF vaccination illustrated the release of sets of proinflammatory cytokines and chemokines different from the sets of cytokines released from vaccinees who did not experience side effects (1). Nevertheless, the specific role of distinct immunological events in the pathogenesis of YEL-SAEs still needs to be clarified.This report provides a detailed immunological analysis of a 23-year-old female patient who developed combined suspected neurotropic and viscerotropic severe adverse events following 17D-204 YF vaccination.     

Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite,Trypanosoma cruzi,Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination

A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4⁺ and CD8⁺ T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4⁺ and CD8⁺ T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-γ) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8⁺ T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-γ or IFN-γ/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-γ in the presence of highly cytotoxic T cells. Vaccinated IFN-γ-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-γ in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy.Genetic vaccination using naked DNA or recombinant viruses is being pursued as alternative vaccines. This strategy can be particularly important in the case of intracellular pathogens and neoplasic cells when the effectiveness relies heavily on the vaccination''s capacity to elicit specific immune responses mediated by cytotoxic CD8⁺ T cells (reviewed in references 27, 48, and 49). One of the most prolific areas of genetic vaccination development is the strategy known as the heterologous prime-boost regimen. This consists in the use of two different vectors both carrying a gene that encodes the same antigenic protein for priming and boosting immunizations. This strategy was first proposed in the early 1990s using a combination of recombinant viruses (influenza and vaccinia viruses) to induce protective immunity against malaria (34, 50). Subsequently, this approach was extended and simplified by incorporating naked DNA priming, followed by a booster injection of a recombinant poxviral vector (i.e., modified vaccinia Ankara); this was also used to generate sterile protective immunity in rodent malaria (53, 54). Collectively, these studies demonstrated that the heterologous prime-boost regimen proved more effective than the repeated use of any of these vectors individually. In subsequent years, the heterologous plasmid DNA prime-poxvirus vector boost regimen was adopted worldwide as a powerful mean to elicit strong effector CD8⁺ Tc1-mediated immune responses against viral, parasitic, and neoplastic antigens in rodents and nonhuman primates (4; reviewed in references 29, 44, and 65). Based on the preclinical studies, a number of clinical human trials have also been initiated. However, to our knowledge, heterologous prime-boost regimens using plasmid DNA and recombinant poxviruses have not yet provided meaningful protective immunity to humans (21, 28, 42, 45).Although there are a number of possible vector combinations that significantly improve cell-mediated immunity, particularly of specific CD8⁺ T cells, heterologous prime-boost vaccination using naked plasmid DNA for priming, followed by a booster injection of recombinant replication-deficient adenovirus 5 has recently received significant attention. This strategy has proved successful in some relevant experimental models such as simian immunodeficiency virus and malaria, providing considerable protective immunity (2, 13, 14, 26, 33, 41, 62).The fact that protective CD8⁺ T cells could be induced in mice and nonhuman primates against both virus and parasites made it an interesting strategy against other microbial infections. We initially attempted to build on this strategy by generating protective CD4 Th1 and cytotoxic CD8⁺ T cells against the infection with a human intracellular protozoan parasite, Trypanosoma cruzi. Both CD4⁺ and CD8⁺ T cells were described as critical for acquired immunity against experimental infection with T. cruzi (38, 39, 56-59). Vaccination studies confirmed these observations by providing evidence that CD8⁺ T cells are particularly important to the development of protective immunity in mice immunized with recombinant plasmid DNA, proteins, or viruses (6, 19, 25, 31, 37, 43, 60). In addition to CD8⁺ T cells, CD4⁺ Th1 cells also play a role in immunity against T. cruzi after vaccination with plasmid DNA or recombinant protein (19, 30, 60).In our previous studies, we showed that multiple immunizations of highly susceptible A/Sn mice with a gene or a recombinant protein based on the amastigote surface protein 2 (ASP-2) of T. cruzi generated protective immunity against a lethal challenge with this parasite (6, 17, 19, 55, 60). In both cases, vaccinated animals depleted of CD8⁺ T cells prior to challenge became highly susceptible to infection (6, 60). Protective CD8⁺ T cells were directed to the immunodominant epitope TEWETGQI located within amino acids 320 to 327 of ASP-2 (6, 19).The present study was designed first to evaluate whether we could reduce the number of immunizing doses required to generate CD4⁺ and CD8⁺ T-cell-mediated protective immunity using a heterologous prime-boost regimen. A reduction in the number of doses may greatly improve the feasibility of human vaccination trials. The fact that mice immunized with recombinant AdASP-2 showed the highest levels of in vivo cytotoxicity mediated by CD8⁺ T cells prior to challenge, and some degree of protective immunity led us to study the role for perforin during vaccination. Subsequently, we determined whether the high susceptibility observed in perforin-deficient mice correlated with impaired effector T-cell immune responses and the expression of relevant activation markers by specific CD8⁺ T cells. Our final goal was to determine the importance of gamma interferon (IFN-γ), a critical mediator of adaptive immunity against T. cruzi infection, for protective immunity in the presence of highly cytotoxic T cells.     

Macrophage-Mediated Responses to Candida albicans in Mice Expressing the Human Immunodeficiency Virus Type 1 Transgene

The critical impairments of innate and adaptive immunity that cause susceptibility to mucosal candidiasis in human immunodeficiency virus (HIV) infection have not been fully determined. We therefore conducted an analysis of macrophage-mediated responses to Candida albicans in transgenic (Tg) mice expressing Nef, Env, and Rev of HIV type 1 (HIV-1) in CD4⁺ T cells, dendritic cells, and macrophages and developing an AIDS-like disease (CD4C/HIV^MutA Tg mice). Macrophages were successfully recruited to the oral and gastric mucosae of these Tg mice in response to chronic carriage of C. albicans and displayed polarization toward an alternatively activated phenotype. Functionally, peritoneal macrophages from uninfected Tg mice exhibited increased phagocytosis of C. albicans and enhanced production of interleukin 6 and monocyte chemoattractant protein 1, demonstrating that the HIV-1 transgene independently activates selected macrophage functions. Production of H₂O₂ by macrophages from Tg mice primed with gamma interferon and treated with phorbol 12-myristate 13-acetate or C. albicans was moderately reduced, but expression of the HIV-1 transgene did not alter production of nitric oxide or reduce killing of C. albicans. A knockout of the inducible nitric oxide synthase (NOS2) gene in these Tg mice did not augment oral or gastrointestinal burdens during chronic carriage of C. albicans or cause systemic dissemination, likely due to a redundancy provided by partially preserved production of H₂O₂ and oxygen-independent candidacidal mechanisms. Thus, the macrophage response to C. albicans is largely preserved in these Tg mice, and no functional macrophage defect appears to primarily determine the susceptibility to mucosal candidiasis.Oropharyngeal candidiasis (OPC) is the most frequent opportunistic fungal infection among human immunodeficiency virus (HIV)-infected patients (64). Although the incidence of OPC in HIV infection is sharply reduced by highly active antiretroviral therapy (45), it remains a common coinfection worldwide. The critical impairments of innate and adaptive immunity that are responsible for the onset and maintenance of mucosal candidiasis in HIV infection have not been fully determined (15, 25). A correlation has been established in HIV infection between symptomatic OPC and reduced CD4⁺ cell count (6, 46, 55), HIV viral load (6, 46), and the development of AIDS (55). Studies conducted with experimentally infected normal, nude, and cytokine-specific gene knockout mice indicated that host defense against OPC requires intact Th1- and Th17-mediated immune responses to Candida albicans, including production of interleukin 12 (IL-12), CD4⁺ T-cell augmentation of monocyte and polymorphonuclear leukocyte (PMN) functions, and mucosal production of nitric oxide (NO) (1, 7, 11, 17-21, 34, 61). Using a model of mucosal Candida infection in transgenic (Tg) mice expressing HIV-1 Nef in CD4⁺ T cells, dendritic cells, and macrophages which closely mimics the clinical and pathological features of candidal infection in human HIV infection (14), we have previously shown that altered CD4⁺ T-cell phenotype and function determine the susceptibility to chronic carriage of C. albicans in these Tg mice (37). However, PMNs from the Tg mice were unimpaired in their capacity to produce an oxidative burst and to phagocytose and kill C. albicans in vitro, and depletion of PMNs in these Tg mice did not alter the oral or gastrointestinal burdens of C. albicans or cause systemic dissemination (42). Accordingly, the defective anti-Candida effector mechanisms that render these Tg mice susceptible to mucosal candidiasis have not yet been identified.Oral colonization and infection of mice with C. albicans trigger macrophage recruitment to the mucosa of the oral cavity (9), stomach (10, 71), and cecum (12), suggesting that these cells play a role in resistance to mucosal candidiasis (68). Activated macrophages have the capacity to kill C. albicans by their production of the reactive oxygen intermediates (ROIs) O₂⁻ and H₂O₂, by the formation of peroxynitrite from O₂⁻ and the reactive nitrogen intermediate NO, and by oxygen-independent candidacidal mechanisms (68-71,73). The participation of macrophages in host resistance has been demonstrated by the enhanced susceptibility of severe combined immunodeficiency (SCID) mice to disseminated candidiasis of gastrointestinal origin after treatment with poly(I-C), an inhibitor of macrophage candidacidal activity (33).Treatment of human monocyte-derived macrophages with HIV-1 Nef protein or infection of these cells with HIV-1 alters cellular signal transduction pathways and specifically activates NF-κB, STAT1 and STAT3, mitogen-activated protein kinases, and genes for several inflammatory factors, including macrophage inflammatory protein 1α, macrophage inflammatory protein 1β, IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α) (5, 24, 40, 59). Therefore, the anticandidal properties of macrophages could be altered either directly by the expression of HIV-1 gene products within this cell population or indirectly by inadequate cytokine signaling from defective CD4⁺ T cells. In several investigations producing conflicting results, phagocytosis and killing of C. albicans by blood monocyte-derived macrophages from HIV-infected patients have been found to be either normal (56, 57) or reduced (13), possibly by HIV Nef (35, 62).With the recognition that classically activated (M1) macrophages (27, 44) primarily mediate the effector arm of a CD4⁺ T-cell-dependent protective Th1 adaptive immune response by their production of ROIs and reactive nitrogen intermediates which kill C. albicans (8, 43, 52, 54, 63), we asked whether a defective mucosal macrophage response to C. albicans contributes to the phenotype of chronic oral candidiasis in these Tg mice expressing HIV-1. The likelihood of such a defect was considered significant because CD4⁺ T cells are quantitatively and functionally defective in these Tg mice, and these alterations of CD4⁺ T cells determine at least in part the susceptibility of these animals to chronic carriage of C. albicans (37). We found that macrophages from these Tg mice display a polarization toward an alternatively activated phenotype and are successfully recruited to the mucosa in response to C. albicans. Although the production of H₂O₂ was modestly reduced, the production of NO and the killing of C. albicans by macrophages were both unaltered by expression of the HIV-1 transgene, and no further augmentation of oral burdens of C. albicans was found in NOS2^−/− gene-deficient Tg mice. Thus, the macrophage response to C. albicans is largely preserved in these Tg mice, and no functional macrophage defect appears to primarily determine susceptibility to mucosal candidiasis.     

Mobilization of CD34+ Progenitor Cells in Association with Decreased Proliferation in the Bone Marrow of Macaques after Administration of the Fms-Like Tyrosine Kinase 3 Ligand

Fms-like tyrosine kinase 3 ligand (FLT3-L) is critical for the differentiation and self-renewal of CD34⁺ progenitor cells in primates and has been used therapeutically to mobilize progenitor and dendritic cells in vivo. However, little is known regarding the expansion of progenitor cells outside of peripheral blood, particularly in bone marrow (BM), where progenitor cells primarily reside. Evaluation of FLT3-L-mediated cell mobilization during lentivirus infections, where the numbers of CD34⁺ progenitor cells are reduced, is limited. We enumerated frequencies and absolute numbers of CD34⁺ progenitor cells in blood and BM of naive and SIV- or SHIV-infected macaques during and after the administration of FLT3-L. Flow cytometric analyses revealed that, while CD34⁺ cells increased in the circulation, no expansion was observed in BM. Furthermore, in the BM intracellular Ki67, a marker of cell proliferation, was downregulated in CD34⁺ progenitor cells but was upregulated significantly in the bulk cell population. Although the exact mechanism(s) remains unclear, these data suggest that CD34⁺ cell mobilization in blood was the result of cellular emigration from BM and not the proliferation of CD34⁺ cells already in the periphery. It is possible that the decreased progenitor cell proliferation observed in BM is evidence of a negative regulatory mechanism preventing hyperproliferation and development of neoplastic cells.The cytokine receptor Fms-like tyrosine kinase 3 (FLT3) is expressed at high levels on both primitive and early lymphoid/myeloid CD34⁺ progenitor cells (3, 21). Interaction with its cognate ligand (FLT3-L), found in both soluble and membrane-bound isoforms, contributes to the regulation of self-renewal and differentiation potential of these cells (43, 44). However, dysregulation of FLT3/FLT3-L signaling can result in the development of various leukemias (1, 6, 22, 29), and increased serum levels of FLT3-L are often indicative of other hematologic and autoimmune abnormalities (17, 25, 39). Nonetheless, after both murine and human FLT3-L were cloned in the early 1990s (24), this hematopoietic cytokine was used effectively in vitro to expand and maintain CD34⁺ progenitor cells (26, 32, 33) and, in combination with other growth factors, was used to induce differentiation of myeloid lineage cells (4), dendritic cells (2, 15), natural killer (NK) cells (42), erythroid precursors (12), and even endothelial cells (41). In addition, FLT3-L was shown to specifically suppress apoptosis of CD34⁺ progenitor cells (27).Early in vivo studies in mice demonstrated that FLT3-L administration not only mobilized and expanded murine CD34⁺ progenitor cells but also promoted expansion of human CD34⁺ cells transferred into SCID mice (8, 9, 24). In nonhuman primates FLT3-L was used to expand dendritic cell subsets (7, 30, 35), to treat radiation-induced myelosuppression (13, 14, 19), and as an adjuvant for various vaccines (23, 40). Although CD34⁺ cells primarily reside in the bone marrow (BM), examination of mobilization of these cells in vivo in nonhuman primates has been limited and typically restricted to analyses of blood (5, 18, 28). CD34⁺ cell mobilization and hematopoiesis is of particular interest in macaque models of lentivirus infections because, during both HIV and SIV infections, BM damage and reduced hematopoiesis is evident early after infection and is associated with decreased numbers and clonogenic potential of CD34⁺ progenitors, despite low levels of infection and virus replication in these cells (10, 16, 20, 34, 36, 37). Therefore, in the present study we quantified and characterized mobilization of CD34⁺ progenitor cells in BM in relation to that observed in peripheral blood by examining BM aspirates taken at various times during and after FLT3-L administration to naive and SIV- or SHIV-infected macaques.     

Multiple T-Cell Responses to Human Immunodeficiency Virus Type 1 Are Enhanced by Dendritic Cells

Human immunodeficiency virus type 1 (HIV-1)-specific T-cell reactivity has been related to protection from disease progression. Optimal T-cell reactivity to HIV-1 presumably requires antigen processing and presentation by professional antigen-presenting cells, particularly dendritic cells (DC). Here we examined whether multiple HIV-1-specific T-cell functions are enhanced by stimulation with HIV-1 peptide-loaded DC derived from HIV-1-infected subjects on antiretroviral therapy. We first found that mature DC increased the number of gamma interferon (IFN-γ)-producing T cells detected by enzyme-linked immunospot assay to overlapping 15-mer peptides of HIV-1 Gag and Nef, compared to stimulation with peptide-loaded, immature DC or to peptides without DC. IFN-γ production was lower in response to large pools of the Gag and Nef peptides, regardless of presentation by DC. We further observed that HIV-1 peptide-loaded, mature DC stimulated greater CD8⁺ and CD4⁺ T-cell proliferation than did the peptides without DC and that T-cell proliferation was lower in response to larger pools of the peptides. The lower T-cell IFN-γ and proliferation responses to the larger peptide pools were related to lower T-cell viability. Finally, the number of polyfunctional CD8⁺ and CD4⁺ T cells stimulated by HIV-1 peptide-loaded, mature DC, defined as positive by intracellular staining for more than one immune mediator (IFN-γ, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1β, or CD107a), was greater than that stimulated by the peptides alone. These results indicate that DC can enhance multiple types of HIV-1-specific T-cell functions.Considerable evidence supports the idea that T-cell immunity to human immunodeficiency virus type 1 (HIV-1) is important in control of HIV-1 infection (10). Specific correlates of T-cell immunity that are associated with protection against or progression of HIV-1 infection have nonetheless been difficult to determine. Such immune correlates could be useful in defining the efficacy of prophylactic and therapeutic vaccines for HIV-1 infection. Many studies of T-cell immunity in HIV-1 infection have shown that the number of T cells exhibiting gamma interferon (IFN-γ) production in the enzyme-linked immunospot (ELISPOT) assay is decreased in association with progressive infection (4, 51). Proliferation of T cells in response to HIV-1 antigens as measured by uptake of the succinimidyl ester of carboxyfluorescein diacetate (CFSE) has also been related to less progressive HIV-1 infection (19, 33, 53). Recently, the quality of the CD8⁺ T-cell functional response to HIV-1 peptides as defined by intracellular cytokine staining (ICS) for more than one immune mediator, i.e., IFN-γ, interleukin 2 (IL-2), tumor necrosis factor alpha (TNF-α), macrophage inhibitory protein 1β (MIP-1β), and/or cytotoxic degranulation molecule CD107a (11, 44), has been associated with slow progression and better control of HIV-1 infection (5).Although these are all valid measures of anti-HIV-1 T-cell immunity, they usually do not account for a role of professional antigen-presenting cells (APC), particularly dendritic cells (DC), which are necessary for optimal processing and presentation of antigens to T cells (2). Indeed, it is likely that during HIV-1 infection, DC are required to take up, process, and present HIV-1 antigens via their major histocompatibility complex (MHC) class I and II molecules for priming and boosting of anti-HIV-1 CD8⁺ and CD4⁺ T-cell responses (40). We have previously shown that IFN-γ production by CD8⁺ T cells from HIV-1-infected persons is enhanced by stimulation with DC loaded with HIV-1 antigens and matured with CD40L or a cocktail of various proinflammatory cytokines and a Toll-like receptor 3 ligand (15, 20, 21). Myeloid DC loaded with peptides representing dominant epitopes of HIV-1 proteins stimulated significantly more epitope-specific, IFN-γ-producing CD8⁺ T cells than did peptides added directly to peripheral blood mononuclear cells (PBMC). There is little information, however, as to whether these professional APC can similarly enhance other T-cell functions that could be critical to control of HIV-1 infection, particularly their proliferative capacity and ability to produce multiple immune mediators. Moreover, many current approaches for measuring the magnitude and breadth of T-cell responses use pools of various numbers of synthetic peptides, usually 15 or 20 amino acids (aa) in length, which overlap by 10 to 11 aa (1, 3, 7, 9, 13, 14, 17, 24, 25, 27, 32, 37, 45, 48, 49), developed by Kern et al. (26) and Maecker et al. (31). Such studies have not accounted for a role of APC in processing that is required to reduce these peptides to their optimal, 8- to 10-mer length for presentation by MHC class I molecules to CD8⁺ T cells (43), or to 13- to 17-mers for presentation by MHC class II to CD4⁺ T cells (46). These are important considerations in determining correlates of T-cell immunity in HIV-1 infection and in response to HIV-1 vaccines.We have analyzed the magnitude of several types of T-cell responses during HIV-1 infection stimulated by autologous DC loaded with different-size pools of overlapping HIV-1 peptides. We assessed T-cell responses in HIV-1- infected persons for single-cell IFN-γ production by using a conventional ELISPOT assay; for CD8⁺ and CD4⁺ T-cell proliferation by using uptake of CFSE dye; and for production of IFN-γ, IL-2, TNF-α, MIP-1β, and CD107a by CD8⁺ and CD4⁺ T cells by using ICS. We found that, in addition to enhancing IFN-γ production detected by ELISPOT assay, DC loaded with HIV-1 peptide singlets or smaller pools of HIV-1 peptides enhanced HIV-1-specific T-cell proliferation and polyfunctional CD8⁺ and CD4⁺ T-cell responses.     

Yersinia enterocolitica Promotes Robust Mucosal Inflammatory T-Cell Immunity in Murine Neonates

Mucosal immunity to gastrointestinal pathogens in early life has been studied only slightly. Recently, we developed an infection model in murine neonates using the gastroenteric pathogen Yersinia enterocolitica. Here, we report that oral infection of neonatal mice with low doses of virulent Y. enterocolitica leads to vigorous intestinal and systemic adaptive immunity. Y. enterocolitica infection promoted the development of anti-LcrV memory serum IgG1 and IgG2a responses of comparable affinity and magnitude to adult responses. Strikingly, neonatal mesenteric lymph node CD4⁺ T cells produced Yersinia-specific gamma interferon (IFN-γ) and interleukin-17A (IL-17A), exceeding adult levels. The robust T- and B-cell responses elicited in neonates exposed to Y. enterocolitica were associated with long-term protection against mucosal challenge with this pathogen. Using genetically deficient mice, we found that IFN-γ and CD4⁺ cells, but not B cells, are critical for protection of neonates during primary Y. enterocolitica infection. In contrast, adults infected with low bacterial doses did not require either cell population for protection. CD4-deficient neonatal mice adoptively transferred with CD4⁺ cells from wild-type, IFN-γ-deficient, or IL-17AF-deficient mice were equally protected from infection. These data demonstrate that inflammatory CD4⁺ T cells are required for protection of neonatal mice and that this protection may not require CD4-derived IFN-γ, IL-17A, or IL-17F. Overall, these studies support the idea that Y. enterocolitica promotes the development of highly inflammatory mucosal responses in neonates and that intestinal T-cell function may be a key immune component in protection from gastrointestinal pathogens in early life.Host protection against microbial agents ultimately relies on the cooperative action of the innate and adaptive immune systems. In both human and murine neonates, adaptive immune responses are compromised compared to responses in developmentally mature hosts (5, 66). Factors that may contribute to the immunological immaturity reported during neonatal life include the following: the lack of antigen-specific immunological memory (5, 65), reduced levels of antigen presenting cells (APC) (46) and adaptive immune cells (21), delays in the development of lymph node germinal centers (57), and cell-intrinsic differences in immune responsiveness (4, 48, 67). Thus, neonatal immune responses following infection or vaccination often appear to be diminished compared to responses in adults. In particular, B-cell and CD4⁺ T helper (T_h) responses to a variety of antigens may be reduced in magnitude, quality, and duration (5, 65). Neonatal immunization with prototypic protein vaccine antigens often leads to mixed T_h1 and T_h2 primary responses (2), but the development of T_h1-associated memory (3) and production of T_h1-associated IgG2a antibodies are often reduced compared to these responses in adults (9). However, adult-like T_h1 immunity has been achieved in neonatal hosts after Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination (53, 72), DNA vaccines (55, 62), or attenuated vaccinia-derived vectors (44). These observations led to the recognition that immune responsiveness during early life could be greatly enhanced by optimizing the conditions of antigen exposure using highly inflammatory treatments.Activation of the neonatal immune system through microbe-associated molecular pattern receptors has demonstrated remarkable improvements in promoting effective immunity to vaccine antigens. For example, bacterially derived products such as mutated Escherichia coli enterotoxins LT-R192G (70) and LT-K63 (11, 14, 27, 34), CpG oligonucleotides (CpG) (8, 29, 31), and lyophilized bacterial extracts (12) have been described to markedly enhance neonatal vaccine responses. Another approach used to improve immune responses has been the delivery of specific antigens using live attenuated bacterial vectors such as Listeria monocytogenes (42, 50) and Salmonella species (16, 59). Both of these approaches have shown dramatic improvements in CD4⁺ and CD8⁺ IFN-γ production, mucosal IgA production, and systemic IgG1 and IgG2a antibodies to the delivered vaccine antigens. Recently, CD4⁺ T_h17-mediated immunity has been studied in response to vaccination with rotavirus antigen in adjuvant (70) and to Mycobacterium tuberculosis antigens in the presence of non-CpG oligonucleotides (36) or cationic liposomes (37). These vaccines promoted interleukin-17A (IL-17A) levels of the same magnitude in neonatal and adult CD4⁺ cells (36, 37, 70). Altogether, it has become apparent that under the proper stimulation conditions, all arms of the neonatal adaptive immune system can be induced to generate adult-like responses. Importantly, some of these immunization regimens promoted protective immunity against infection with fully pathogenic bacteria (16, 29, 31, 34, 42, 59).Despite the profound maturation of the neonatal immune system through vaccination with live attenuated L. monocytogenes and Salmonella vectors (16, 17, 42, 50, 59), neonatal immune responses to fully virulent pathogens are inefficient in controlling infection (15, 25, 31, 61). This exquisite susceptibility to infection during neonatal life includes both peripheral and mucosal routes of infection. In particular, neonatal animals succumb rapidly to pulmonary infection with Streptococcus pneumoniae (24) and gastrointestinal infection with enteropathogens including Vibrio cholerae (10), Aeromonas hydrophila (76), Shigella flexneri (23), enterotoxigenic E. coli (19), and Salmonella species (15, 61). Thus, mucosal immune responses to most pathogens studied to date are severely compromised in early life.In contrast to the vast majority of experimental systems, we recently demonstrated (20) that murine neonates are highly resistant to oral infection with the Gram-negative enteropathogen Yersinia enterocolitica. The resistance of neonatal mice infected with Y. enterocolitica was associated with robust innate inflammation, characterized by the recruitment of high levels of neutrophils and macrophages into the intestinal tissue (20). We hypothesized that the vigorous innate responses in neonates may promote similarly robust adaptive immunity. Here, we have compared the development of Yersinia-specific B- and CD4⁺ T-cell immunity in neonatal and adult mice. We demonstrate that highly protective intestinal and systemic adaptive immunity can be induced in neonatal mice. Remarkably, neonatal mice developed greater Yersinia-specific T_h1 and T_h17 responses in the mesenteric lymph nodes (MLN) than did adults. Experiments using genetically deficient mice with or without adoptive transfer of donor cells showed that CD4⁺ T cells, but not B cells, appeared to be necessary for resistance of infected neonates. Thus, we extend our earlier studies to further demonstrate the unprecedented inflammatory potential of the neonatal gastrointestinal immune system in response to a fully virulent enteric pathogen.     

Cellular Responses to Mycobacterial Antigens Are Present in Bronchoalveolar Lavage Fluid Used in the Diagnosis of Sarcoidosis

Considerable evidence supports the concept that CD4⁺ T cells are important in sarcoidosis pathogenesis, but the antigens responsible for the observed Th1 immunophenotype remain elusive. The epidemiologic association with bioaerosols and the presence of granulomatous inflammation support consideration of mycobacterial antigens. To explore the role of mycobacterial antigens in sarcoidosis immunopathogenesis, we assessed the immune recognition of mycobacterial antigens, the 6-kDa early secreted antigenic protein (ESAT-6) and catalase-peroxidase (KatG), by T cells derived from bronchoalveolar lavage (BAL) fluid obtained during diagnostic bronchoscopy. We report the presence of antigen-specific recognition of ESAT-6 and KatG in T cells from BAL fluid of 32/44 sarcoidosis subjects, compared to 1/27 controls (P < 0.0001). CD4⁺ T cells were primarily responsible for immune recognition (32/44 sarcoidosis subjects), although CD8⁺ T-cell responses were observed (25/41 sarcoidosis subjects). Recognition was significantly absent from BAL fluid cells of patients with other lung diseases, including infectious granulomatous diseases. Blocking of Toll-like receptor 2 reduced the strength of the observed immune response. The presence of immune responses to mycobacterial antigens in cells from BAL fluid used for sarcoidosis diagnosis suggests a strong association between mycobacteria and sarcoidosis pathogenesis. Inhibition of immune recognition with monoclonal antibody against Toll-like receptor 2 suggests that induction of innate immunity by mycobacteria contributes to the polarized Th1 immune response.Sarcoidosis is a multisystem granulomatous disease of unknown etiology, characterized by a Th1 immunophenotype. The epidemiology and immunology of sarcoidosis suggest that an infectious agent could be involved in its pathogenesis. Immunologic studies of sarcoidosis have detected oligoclonal T cells in the sarcoid granuloma, consistent with a major histocompatibility complex-restricted antigen-driven process (33, 42). Analysis of cells from bronchoalveolar lavage (BAL) fluid from patients with acute forms of sarcoidosis has demonstrated overrepresentation of specific T-cell receptor alleles in combination with DRB3*0101 and DRB1*0301 major histocompatibility complex restrictions, strongly implicating an antigen-specific response in sarcoidosis pathogenesis (21).Because sarcoidosis most commonly involves the lungs, eyes, and skin, the search for environmental causes has centered on exposures to airborne antigens (26). Several reports have noted associations with occupations such as agriculture, metalworking, firefighting, and the handling of building supplies (3, 30, 32, 38). The association of sarcoidosis with these occupations raises the possibility that disease may be caused by exposure to microbial bioaerosols, including inorganic particles, insecticides, and aerosols from moldy environments (3, 32, 35, 39). Sarcoidosis also demonstrates a seasonal and geographic variability (12, 29, 34, 50). The transmissibility, clustering of cases, and geographic and seasonal variability suggest that environmental factors are important in the acquisition of sarcoidosis. Moreover, these environmental risk factors correlate well with the typical distribution of environmental mycobacteria.The potential role of mycobacteria in sarcoidosis immunopathogenesis is supported by recent studies reporting detection of mycobacterial proteins and nucleic acids in sarcoidosis granulomas, as well as humoral and peripheral cellular immune responses to mycobacterial antigen in sarcoidosis subjects (4, 9, 13-17, 22, 44). However, in order to understand disease pathogenesis, it is crucial to analyze the immunologic response at the site of active sarcoidosis involvement. Although sarcoidosis is a systemic disease, it most commonly affects the lungs. Taking advantage of the observation that most patients undergo bronchoscopy with BAL as a routine investigation (49), we assessed the specific immune recognition of mycobacterial virulence factors by T cells from BAL fluid derived from sarcoidosis patients from geographically distinct locales.In order to investigate the role of mycobacterial antigen in lung involvement in sarcoidosis, we analyzed specific responses of BAL fluid-derived T cells to the mycobacterial 6-kDa early secreted antigenic protein (ESAT-6) and catalase-peroxidase (KatG). We chose ESAT-6 and KatG peptides due to prior reports of systemic cellular immune responses to these microbial virulence factors in sarcoidosis subjects (9, 13).     

CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28^−/−) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28^−/− mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro- and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.It has long been appreciated that cellular immunity is the most important resistance mechanism against fungal infections (14, 36, 64). CD4⁺ and CD8⁺ T-cell subpopulations have been described to have a fundamental role in the control of fungal growth, and disease severity is also controlled by regulatory T (Treg) cells, which prevent tissue pathology by controlling excessive inflammatory reactions (25, 45, 46, 65). Similar to other deep mycoses, the severity of paracoccidioidomycosis (PCM), the most severe pulmonary mycosis in Latin America, is controlled by cellular immunity and cytokine-activated phagocytes that are able to kill Paracoccidioides brasiliensis, the etiological agent of this infection (10, 20, 30, 60, 61). In humans and in murine models of PCM, resistance to the disease is associated with the secretion of gamma interferon (IFN-γ) and other type 1 cytokines, whereas impaired Th1 immunity and the prevalent secretion of Th2 cytokines correlate with a systemic and progressive disease (2, 6, 39, 59, 76). Studies with CD4⁺ and CD8⁺ T-cell-deficient mice revealed that both T-cell subsets are involved in the protective immunity against P. brasiliensis infection and indicated the prominent role of CD8⁺ T cells (3, 21, 25). Besides the prevalent Th2 immunity, recent investigations have described alternative mechanisms underlying T-cell dysfunction in humans and experimental PCM. Increased apoptosis and overexpression of Fas and FasL in T cells suggest that activation-induced cell death (AICD) is a mechanism that controls T-cell expansion during the active disease (13, 19). In addition, the increased expression of CTLA-4 and the expansion of Treg cells were associated with severe patterns of the disease (24, 45, 46, 56). Thus, in addition to cytokine imbalance, other regulatory mechanisms can actively participate in the unresponsiveness of T cells in P. brasiliensis-infected hosts.Optimal activation, proliferation, and cytokine production by antigen-specific T cells require two distinct signals from dendritic cells or other antigen-presenting cells (APCs). After T-cell receptor (TCR) occupancy by the antigen epitope/major histocompatibility (MHC) complex (first signal), a second signal is mediated by costimulatory molecules (43, 63), such as CD28 on T cells and their counter-receptors CD80 (B7-1) and CD86 (B7-2) expressed by APCs (1, 34). Soluble molecules, such as cytokines and chemokines, also participate in the activation process, which drives and controls T-cell numbers and fates (1). CD28 enhances the TCR-triggered activation of naïve T cells, promotes interleukin-2 (IL-2) secretion and prevents T-cell anergy (1, 37). Alternatively, CD28-independent T-cell activation can occur if a strong and sustained antigen-specific signal is available (40, 81). Like CD28, two other molecules, cytotoxic T-lymphocyte antigen-4 (CTLA-4) and mouse inducible costimulatory molecule (ICOS), are selectively expressed by T cells, but the expression of these molecules depends on previous cell activation (50, 71). More recently, evidence has emerged that CD28 family members are also crucial regulators of natural and induced regulatory (CD4⁺CD25⁺Foxp3⁺) T cells (9). These cells are induced in the thymus and in the periphery, respectively, and control self-tolerance and the activation of several components of innate and adaptive immunity (68). Treg cells can suppress immune responses through the production of immunosuppressive cytokines (mainly IL-10 and transforming growth factor β [TGF-β]), through the induction of the apoptosis of effector T cells and through the modification of the functional properties of antigen-presenting cells (70, 78).Immunoprotection against microorganisms has been shown to be either CD28 dependent or independent. CD28-deficient (CD28^−/−) mice are highly susceptible to infection with Salmonella enterica serovar Typhimurium due to the poor ability of these mice to secrete IFN-γ (51). During some viral and parasitic infections, CD28 was shown to be required to mediate CD8⁺ T-cell immunoprotection (8, 53). In contrast, CD28^−/− mice infected with Mycobacterium bovis or Listeria monocytogenes control the bacterial burden and develop cell-mediated immunity (35, 52). In primary and opportunistic fungal infections, CD28 costimulation controls protective immunity, the expansion and function of regulatory T cells, and the intensity of inflammatory reactions (5, 54, 55, 66, 84).Because CD28 is critical for T-cell activation in fungal infections, we investigated its role in a murine model of P. brasiliensis infection. We show that CD28 costimulation exerts contrasting roles in pulmonary PCM. Early in infection, CD28 expression results in efficient adaptive immunity that is able to control fungal growth. Late in infection, however, this costimulatory molecule induces significant expansion of regulatory T cells, diminished immunity, and uncontrolled fungal growth that eventually leads to the death of the mice. In contrast, the absence of CD28 costimulation results in impaired T-cell immunity, which appeared to be compensated by the absence of Treg cell expansion. This weak but persistent immunity was able to partially control fungal growth, organize granulomatous lesions, and guarantee the enhanced survival of the mice, suggesting the relative protection conferred by CD28-independent mechanisms.     

Discordant Brucella melitensis Antigens Yield Cognate CD8+ T Cells In Vivo

Brucella spp. are intracellular bacteria that cause the most frequent zoonosis in the world. Although recent work has advanced the field of Brucella vaccine development, there remains no safe human vaccine. In order to produce a safe and effective human vaccine, the immune response to Brucella spp. requires greater understanding. Induction of Brucella-specific CD8⁺ T cells is considered an important aspect of the host response; however, the CD8⁺ T-cell response is not clearly defined. Discovering the epitope containing antigens recognized by Brucella-specific CD8⁺ T cells and correlating them with microarray data will aid in determining proteins critical for vaccine development that cover a kinetic continuum during infection. Developing tools to take advantage of the BALB/c mouse model of Brucella melitensis infection will help to clarify the correlates of immunity and improve the efficacy of this model. Two H-2^d CD8⁺ T-cell epitopes have been characterized, and a group of immunogenic proteins have provoked gamma interferon production by CD8⁺ T cells. RYCINSASL and NGSSSMATV induced cognate CD8⁺ T cells after peptide immunization that showed specific killing in vivo. Importantly, we found by microarray analysis that the genes encoding these epitopes are differentially expressed following macrophage infection, further emphasizing that these discordant genes may play an important role in the pathogenesis of B. melitensis infection.Brucellosis is the world''s most common zoonosis, with more than half a million new human infections each year (44). Brucellosis has been endemic to the Mediterranean and Middle East since ancient times, since carbonized cheese and skeletal remains in Pompeii show evidence of Brucella spp. (8). Evidence of brucellosis also exists in the skeleton of a 2.4- to 2.8-million-year-old hominid (16). In areas of endemicity, domestic animal brucellosis severely affects regional economies, and vaccination campaigns cannot always reach nomadic herders. Human infections occur in these regions mainly from the ingestion of infected animal products, including unpasteurized milk and fresh cheeses (14). Antibiotic treatment exists but is costly and prolonged, lasting at least 6 weeks in moderate cases, and it may extend for years depending on complications that arise. Even after treatment, PCR data have revealed that low levels of bacteria are detectable years after the resolution of symptoms, and relapses occur in 5 to 30% of cases (20, 30, 55, 62). In areas where brucellosis is endemic, prevention of infection via vaccine would be far more cost-effective than the regimen of antibiotics suggested by the World Health Organization (WHO). Unfortunately, this disease flies below the radar of many of the major world health agencies, and the problem is compounded by frequent misdiagnosis and under-reporting (15, 20).Although brucellosis is eradicated from food sources here, in the post-Gulf War United States, awareness was raised to fund vaccine research concerning potential biological weapons. Brucella melitensis, B. abortus, and B. suis are considered category B select agents because of the ease of aerosolization, diverse symptoms, and chronic persistence. The spectrum of disease that results from Brucella infection suggests that Brucella spp. could be a biological weapon in the current absence of any human vaccine (43). Human symptoms begin with a general malaise and fever, followed by organ-specific “hot spots” of infection, for instance, endocarditis and orchitis. In the United States, infections are due to accidental infection with a live animal vaccine by veterinarians and laboratory workers. In fact, brucellosis is one of the most common laboratory-acquired infections, and the lack of a human vaccine discourages work with the agent (20, 37, 40).Three vaccines are currently recommended by the WHO for livestock, and all of them are live-attenuated Brucella strains: B. abortus S-19 and RB-51 for bovine brucellosis and B. melitensis Rev-1 for goat and sheep brucellosis. These vaccine constructs are not completely effective and pose safety risks, including abortifacient effects and residual virulence, making them unsuitable for human application (33). Heat-killed Brucella does not induce detectable interleukin-12 (IL-12) in vivo, and killed bacteria actively suppress IL-12 production in response to challenge with live bacteria by unknown mechanisms (24). Studies conducted in our laboratory, and confirmed by others, have shown that subunit vaccines can confer a degree of short-term protection but have not elicited long-term effective immunity (3, 39). Only live bacteria appear to induce cell-mediated immunity, whereas dead bacteria induce a nonprotective humoral response (31, 36).CD4⁺ T cells induce the production of IgG2 antibodies from B cells during the course of murine and ovine B. melitensis infections (9, 56). There is evidence that this humoral response is an indispensable aspect of the host defenses in that opsonization may be required for successful uptake by macrophages, although a humoral response is not protective (7, 18, 31). In addition, although opsonization may result in increased bacterial uptake by macrophages, bacterial survival is unchanged (18). Previous studies have shown that host protection can be mediated by gamma interferon (IFN-γ) produced by CD4⁺ T cells, although data have also shown that treatment of macrophages with optimal concentrations of IFN-γ still allows some intracellular Brucella to survive (19, 26, 57, 63). Brucella can escape complement-mediated killing and thrive inside the acidified phagosomes of macrophages, using the common bactericidal host mechanisms to its own advantage (11, 13, 28a). In addition, major histocompatibility complex (MHC) class II antigen presentation can be disrupted by Brucella lipopolysaccharide that has incorporated into the host cell membrane (28). In our lab and others, evidence supports that protection in animal models is engendered by CD8⁺ T cells (10, 12, 22, 27, 38, 42, 64). Therefore, we chose to investigate the Brucella antigens that are recognized by CD8⁺ T cells in the context of MHC class I molecules.In the United States, most select agent work is confined to biosafety level 3 and above, the logistics of which largely dictate the use of small-animal models in Brucella research. Mice are not a natural host of B. melitensis, making the optimization of this model a high priority. By exploring the CD8⁺ T-cell component of the BALB/c mouse response to B. melitensis infection, we are further refining the mouse as a valuable tool in Brucella research and vaccine development.Determining the epitopes recognized by Brucella-specific CD8⁺ T cells and the Brucella genes encoding the proteins containing these epitopes will help establish proteins critical for vaccine development (47, 48, 51, 52, 60). Epitopes were predicted from the Brucella genome using an algorithm based on allele-specific binding motifs and cleavage sites (49, 50). Select peptides were then tested for their capacity to bind their respective MHC alleles in vitro (54). Peptides subsequently deemed epitopes displayed a combination of immunogenicity, natural processing, and functional avidity, while eliciting CD8⁺ T cells that kill in vivo. Peptide immunogenicity was evaluated using peptide pools in adjuvant, whereas natural processing and functional avidity tests used nonreplicating but metabolically active whole B. melitensis to immunize mice. Our approach has identified the first B. melitensis-specific MHC class I CD8⁺ T-cell epitopes that are recognized in H-2^d mice and generate CD8⁺ T cells that kill in vivo. These present findings offer insight regarding the debate concerning Brucella correlates of immunity and provide guidance in designing a safe and viable human vaccine.