Immunosuppression by avirulent,pseudohyphal forms ofCryptococcus neoformans

Our previous reports have shown that animals inoculated for 8 weeks with 1×10₅ pseudohyphal, avirulentC. neoformans exhibited prolonged survival upon challenge with virulent cryptococci. This paper described a transient phase of immunosuppression which occurs during the initial weeks of the immunization protocol. Animals injected with pseudohyphal organisms had depressed responses to T and B-cell mitogens. In addition, they had lowered responses to immunization with sheep erythrocytes. Both humoral and cell mediated responses were affected.     

The impact of maternal infection with Mycobacterium tuberculosis on the infant response to bacille Calmette–Guérin immunization

Bacille Calmette–Guérin (BCG) immunization provides variable protection against tuberculosis. Prenatal antigen exposure may have lifelong effects on responses to related antigens and pathogens. We therefore hypothesized that maternal latent Mycobacterium tuberculosis infection (LTBI) influences infant responses to BCG immunization at birth. We measured antibody (n = 53) and cellular (n = 31) responses to M. tuberculosis purified protein derivative (PPD) in infants of mothers with and without LTBI, in cord blood and at one and six weeks after BCG. The concentrations of PPD-specific antibodies declined between birth (median [interquartile range (IQR)]) 5600 ng ml⁻¹ [3300–11 050] in cord blood) and six weeks (0.00 ng ml⁻¹ [0–288]). Frequencies of PPD-specific IFN-γ-expressing CD4⁺T cells increased at one week and declined between one and six weeks (p = 0.031). Frequencies of IL-2- and TNF-α-expressing PPD-specific CD4⁺T cells increased between one and six weeks (p = 0.019, p = 0.009, respectively). At one week, the frequency of PPD-specific CD4⁺T cells expressing any of the three cytokines, combined, was lower among infants of mothers with LTBI, in crude analyses (p = 0.002) and after adjusting for confounders (mean difference, 95% CI −0.041% (−0.082, −0.001)). In conclusion, maternal LTBI was associated with lower infant anti-mycobacterial T-cell responses immediately following BCG immunization. These findings are being explored further in a larger study.     

Long-Term Central and Effector SHIV-Specific Memory T Cell Responses Elicited after a Single Immunization with a Novel Lentivector DNA Vaccine

Prevention of HIV acquisition and replication requires long lasting and effective immunity. Given the state of HIV vaccine development, innovative vectors and immunization strategies are urgently needed to generate safe and efficacious HIV vaccines. Here, we developed a novel lentivirus-based DNA vector that does not integrate in the host genome and undergoes a single-cycle of replication. Viral proteins are constitutively expressed under the control of Tat-independent LTR promoter from goat lentivirus. We immunized six macaques once only with CAL-SHIV-IN⁻ DNA using combined intramuscular and intradermal injections plus electroporation. Antigen-specific T cell responses were monitored for 47 weeks post-immunization (PI). PBMCs were assessed directly ex vivo or after 6 and 12 days of in vitro culture using antigenic and/or homeostatic proliferation. IFN-γ ELISPOT was used to measure immediate cytokine secretion from antigen specific effector cells and from memory precursors with high proliferative capacity (PHPC). The memory phenotype and functions (proliferation, cytokine expression, lytic content) of specific T cells were tested using multiparametric FACS-based assays. All immunized macaques developed lasting peripheral CD8⁺ and CD4⁺ T cell responses mainly against Gag and Nef antigens. During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes. These responses contracted but then reemerged later in absence of antigen boost. Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI. Altogether, our study demonstrated that a single immunization with a replication-limited DNA vaccine elicited persistent vaccine-specific CM and EM CD8⁺ and CD4⁺ T cells with immediate and readily inducible effector functions, in the absence of ongoing antigen expression.     

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10⁴ homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4⁺ and CD8⁺ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4⁺ and CD8⁺ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.     

Pseudohyphal Growth of Cryptococcus neoformans Is a Reversible Dimorphic Transition in Response to Ammonium That Requires Amt1 and Amt2 Ammonium Permeases

Cryptococcus neoformans is a human-pathogenic basidiomycete that commonly infects HIV/AIDS patients to cause meningoencephalitis (7, 19). C. neoformans grows as a budding yeast during vegetative growth or as hyphae during sexual reproduction. Pseudohyphal growth of C. neoformans has been observed rarely during murine and human infections but frequently during coculture with amoeba; however, the genetics underlying pseudohyphal growth are largely unknown. Our studies found that C. neoformans displays pseudohyphal growth under nitrogen-limiting conditions, especially when a small amount of ammonium is available as a sole nitrogen source. Pseudohyphal growth was observed with Cryptococcus neoformans serotypes A and D and Cryptococcus gattii. C. neoformans pseudohyphae bud to produce yeast cells and normal smooth hemispherical colonies when transferred to complete media, indicating that pseudohyphal growth is a conditional developmental stage. Subsequent analysis revealed that two ammonium permeases encoded by the AMT1 and AMT2 genes are required for pseudohyphal growth. Both amt1 and amt2 mutants are capable of forming pseudohyphae; however, amt1 amt2 double mutants do not form pseudohyphae. Interestingly, C. gattii pseudohypha formation is irreversible and involves a RAM pathway mutation that drives pseudohyphal development. We also found that pseudohyphal growth is related to the invasive growth into the medium. These results demonstrate that pseudohyphal growth is a common reversible growth pattern in C. neoformans but a mutational genetic event in C. gattii and provide new insights into understanding pseudohyphal growth of Cryptococcus.     

Comparative immunogenicity in rhesus monkeys of DNA plasmid,recombinant vaccinia virus,and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene

Cellular immune responses, particularly those associated with CD3⁺ CD8⁺ cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4⁺ and CD8⁺ T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.     

Immune response of Japanese flounder Paralichthys olivaceus to outer membrane protein of Edwardsiella tarda

The humoral and cellular immune responses of Japanese flounder, Paralichthys olivaceus, were investigated following intraperitioneal injection with outer membrane protein (OMP) of Edwardsiella tarda in Freund's incomplete adjuvant (FIA). The specific serum antibody titre against OMP of E. tarda were measured using ELISA for 14 weeks, and the total serum antibody concentrations were also determined according to the sandwich ELISA standard model constructed using purified IgM. Both of the specific and total antibodies had an increase and reached their peaks 4 weeks after immunization. Simultaneously, the percentages of sIg + lymphocytes in blood, spleen, pronephros and mesonephros were detected by flow cytometry. It was shown that the percentages of sIg + lymphocytes in all lymphoid organs reached their peak levels 4 weeks after immunization, and then decreased gradually. To investigate the protection against infection, three challenges were performed in the same way at day 14, 30 and 100 after immunization, fish challenged at day 30 showed a higher relative percentage survival (RPS) of 71 compared to the 14-day group (30) and 100-day group (53), which indicated a positive correlation between the survival and the levels of the antibody.     

Acute Effects of Pathogenic Simian-Human Immunodeficiency Virus Challenge on Vaccine-Induced Cellular and Humoral Immune Responses to Gag in Rhesus Macaques

Simian-human immunodeficiency virus (SHIV) infection in macaques provides a convenient model for testing vaccine efficacy and for understanding viral pathogenesis in AIDS. We immunized macaques with recombinant, Salmonella typhimurium (expressing Gag) or soluble Gag in adjuvant to generate T-cell-dependent lymphoproliferative or serum antibody responses. Immunized animals were challenged by intrarectal inoculation with SHIV89.6PD. Virus infection was accompanied by rapid losses of lymphoproliferative responses to Gag or phytohemagglutinin. By 8 weeks, mitogen responses recovered to near normal levels but antigen-specific immunity remained at low or undetectable levels. Serum antibody levels were elevated initially by virus exposure but soon dropped well below levels achieved by immunization. Our studies show a rapid depletion of preexisting Gag-specific CD4⁺ T cells that prevent or limit subsequent antiviral cellular and humoral immune responses during acute SHIV infection.     

Ascaris suum: Immunosuppression in mice during acute infection

Mice inoculated by stomach intubation with 10,000 embryonated Ascaris suum eggs, 4, 11, or 21 days before an intraperitoneal (ip) immunization with 2 × 10⁸ sheep erythrocytes (SRBC) had reduced numbers of direct (IgM) splenic hemolytic plaques measured at 4 days after immunization and only a marginal reduction in indirect plaques (IgG) measured at 9 days after immunization. Lower dosages of Ascaris eggs or simultaneous inoculation of Ascaris eggs and SRBC did not suppress antibody responses to SRBC. No reduction in a secondary antibody response to SRBC injected 4 days after Ascaris inoculation was observed. IgM and IgG hemagglutinin titers, as distinguished by 2-mercaptoethanol sensitivity, were suppressed in mice injected ip with 10⁸ SRBC 10 days following inoculation of 10,000 Ascaris eggs, but titers in both Ig classes were similar in infected and control mice injected with 2 × 10⁹ SRBC. At Day 20, antibody titers following ip injection of 1.0 or 100 μg of ovalbumin in alum were reduced in mice infected with 10,000 Ascaris eggs 4 days before antigen injection.Contact hypersensitivity to oxazalone was not altered in mice sensitized at 5 or 14 days after inoculation of 10,000 Ascaris eggs. The delayed hypersensitivity response, measured by footpad swelling, to an optimum intravenous sensitizing dosage of SRBC was inhibited in mice sensitized 10 days after Ascaris infection, but not inhibited in mice sensitized at 21 or 32 days after infection. In contrast, the delayed hypersensitivity response to subcutaneous sensitization with SRBC 10 days after Ascaris infection was not altered.     

A putative phospholipase C is involved in Pichia fermentans dimorphic transition

Background

Pichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi.

Methods

Pichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition.

Results

Five genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18.

Conclusion

Phospholipase C is part of a putative “methionine sensing machinery” that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition.

General significance

Phospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.     

DNA Vaccination Partially Protects against African Swine Fever Virus Lethal Challenge in the Absence of Antibodies

The lack of available vaccines against African swine fever virus (ASFV) means that the evaluation of new immunization strategies is required. Here we show that fusion of the extracellular domain of the ASFV Hemagglutinin (sHA) to p54 and p30, two immunodominant structural viral antigens, exponentially improved both the humoral and the cellular responses induced in pigs after DNA immunization. However, immunization with the resulting plasmid (pCMV-sHAPQ) did not confer protection against lethal challenge with the virulent E75 ASFV-strain. Due to the fact that CD8⁺ T-cell responses are emerging as key components for ASFV protection, we designed a new plasmid construct, pCMV-UbsHAPQ, encoding the three viral determinants above mentioned (sHA, p54 and p30) fused to ubiquitin, aiming to improve Class I antigen presentation and to enhance the CTL responses induced. As expected, immunization with pCMV-UbsHAPQ induced specific T-cell responses in the absence of antibodies and, more important, protected a proportion of immunized-pigs from lethal challenge with ASFV. In contrast with control pigs, survivor animals showed a peak of CD8⁺ T-cells at day 3 post-infection, coinciding with the absence of viremia at this time point. Finally, an in silico prediction of CTL peptides has allowed the identification of two SLA I-restricted 9-mer peptides within the hemagglutinin of the virus, capable of in vitro stimulating the specific secretion of IFNγ when using PBMCs from survivor pigs. Our results confirm the relevance of T-cell responses in protection against ASF and open new expectations for the future development of more efficient recombinant vaccines against this disease.     

Sublingual Immunization of Trivalent Human Papillomavirus DNA Vaccine in Baculovirus Nanovector for Protection against Vaginal Challenge

Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×10⁸ copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×10⁹ copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×10¹⁰ copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×10⁹ copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses.     

Multiple TORC1-associated proteins regulate nitrogen starvation-dependent cellular differentiation in Saccharomyces cerevisiae

Background

The budding yeast Saccharomyces cerevisiae undergoes differentiation into filamentous-like forms and invades the growth medium as a foraging response to nutrient and environmental stresses. These developmental responses are under the downstream control of effectors regulated by the cAMP/PKA and MAPK pathways. However, the upstream sensors and signals that induce filamentous growth through these signaling pathways are not fully understood. Herein, through a biochemical purification of the yeast TORC1 (Target of Rapamycin Complex 1), we identify several proteins implicated in yeast filamentous growth that directly associate with the TORC1 and investigate their roles in nitrogen starvation-dependent or independent differentiation in yeast.

Methodology

We isolated the endogenous TORC1 by purifying tagged, endogenous Kog1p, and identified associated proteins by mass spectrometry. We established invasive and pseudohyphal growth conditions in two S. cerevisiae genetic backgrounds (Σ1278b and CEN.PK). Using wild type and mutant strains from these genetic backgrounds, we investigated the roles of TORC1 and associated proteins in nitrogen starvation-dependent diploid pseudohyphal growth as well as nitrogen starvation-independent haploid invasive growth.

Conclusions

We show that several proteins identified as associated with the TORC1 are important for nitrogen starvation-dependent diploid pseudohyphal growth. In contrast, invasive growth due to other nutritional stresses was generally not affected in mutant strains of these TORC1-associated proteins. Our studies suggest a role for TORC1 in yeast differentiation upon nitrogen starvation. Our studies also suggest the CEN.PK strain background of S. cerevisiae may be particularly useful for investigations of nitrogen starvation-induced diploid pseudohyphal growth.     

Pooled Segregant Sequencing Reveals Genetic Determinants of Yeast Pseudohyphal Growth

The pseudohyphal growth response is a dramatic morphological transition and presumed foraging mechanism wherein yeast cells form invasive and surface-spread multicellular filaments. Pseudohyphal growth has been studied extensively as a model of conserved signaling pathways controlling stress responses, cell morphogenesis, and fungal virulence in pathogenic fungi. The genetic contribution to pseudohyphal growth is extensive, with at least 500 genes required for filamentation; as such, pseudohyphal growth is a complex trait, and linkage analysis is a classical means to dissect the genetic basis of a complex phenotype. Here, we implemented linkage analysis by crossing each of two filamentous strains of Saccharomyces cerevisiae (Σ1278b and SK1) with an S288C-derived non-filamentous strain. We then assayed meiotic progeny for filamentation and mapped allelic linkage in pooled segregants by whole-genome sequencing. This analysis identified linkage in a cohort of genes, including the negative regulator SFL1, which we find contains a premature stop codon in the invasive SK1 background. The S288C allele of the polarity gene PEA2, encoding Leu409 rather than Met, is linked with non-invasion. In Σ1278b, the pea2-M409L mutation results in decreased invasive filamentation and elongation, diminished activity of a Kss1p MAPK pathway reporter, decreased unipolar budding, and diminished binding of the polarisome protein Spa2p. Variation between SK1 and S288C in the mitochondrial inner membrane protein Mdm32p at residues 182 and 262 impacts invasive growth and mitochondrial network structure. Collectively, this work identifies new determinants of pseudohyphal growth, while highlighting the coevolution of protein complexes and organelle structures within a given genome in specifying complex phenotypes.     

A yeast tRNA mutant that causes pseudohyphal growth exhibits reduced rates of CAG codon translation

In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG‐decoding tRNA^Gln_CUG. A mutant allele, sup70‐65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNA^Gln_CUG anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70‐65 tRNA^Gln_CUG is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG‐rich ORFs in the tRNA^Gln_CUG‐depleted sup70‐65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70‐65 pseudohyphal phenotype was partly complemented by overexpressing CAA‐decoding tRNA^Gln_UUG, an inefficient wobble‐decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5′ end of an ORF can reduce eukaryote translational expression, and that the mutant tRNA_CUG^Gln constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency.     

Isolation and Characterization of EPD1, an Essential Gene for Pseudohyphal Growth of a Dimorphic Yeast, Candida maltosa

Additional copies of the centromeric DNA (CEN) region induce pseudohyphal growth in a dimorphic yeast, Candida maltosa (T. Nakazawa, T. Motoyama, H. Horiuchi, A. Ohta, and M. Takagi, J. Bacteriol. 179:5030–5036, 1997). To understand the mechanism of this transition, we screened the gene library of C. maltosa for sequences which could suppress this morphological change. As a result, we isolated the 5′ end of a new gene, EPD1 (for essential for pseudohyphal development), and then cloned the entire gene. The predicted amino acid sequence of Epd1p was highly homologous to those of Ggp1/Gas1/Cwh52p, a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae, and Phr1p and Phr2p of Candida albicans. The expression of EPD1 was moderately regulated by environmental pH. A homozygous EPD1 null mutant showed some morphological defects and reduction in growth rate and reduced levels of both alkali-soluble and alkali-insoluble β-glucans. Moreover, the mutant could not undergo the transition from yeast form to pseudohyphal form induced by additional copies of the CEN sequence at pH 4 or by n-hexadecane at pH 4 or pH 7, suggesting that EPD1 is not essential for yeast form growth but is essential for transition to the pseudohyphal form. Overexpression of the amino-terminal part of Epd1p under the control of the GAL promoter suppressed the pseudohyphal development induced by additional copies of the CEN sequence, whereas overexpression of the full-length EPD1 did not. This result and the initial isolation of the 5′ end of EPD1 as a suppressor of the pseudohyphal growth induced by the CEN sequence suggest that the amino-terminal part of Epd1p may have a dominant-negative effect on the functions of Epd1p in the pseudohyphal growth induced by the CEN sequence.     

In Vivo Induction of Functionally Suppressive Induced Regulatory T Cells from CD4+CD25- T Cells Using an Hsp70 Peptide

Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experimental autoimmune diseases. The heat shock protein (Hsp) 70, with its expression elevated in inflamed tissue, is a suitable candidate antigen because administration of both bacterial and mouse Hsp70 peptides has been shown to induce strong immune responses and to reduce inflammation via the activation or induction of Hsp specific Tregs. Although two subsets of Tregs exist, little is known about which subset of Tregs are activated by Hsp70 epitopes. Therefore, we set out to determine whether natural nTregs (derived from the thymus), or induced iTregs (formed in the periphery from CD4⁺CD25^- naïve T cells) were targeted after Hsp70-peptide immunization. We immunized mice with the previously identified Hsp70 T cell epitope B29 and investigated the formation of functional iTregs by using an in vitro suppression assay and adoptive transfer therapy in mice with experimental arthritis. To study the in vivo induction of Tregs after peptide immunization, we depleted CD25⁺ cells prior to immunization, allowing the in vivo formation of Tregs from CD4⁺CD25^- precursors. This approach allowed us to study in vivo B29-induced Tregs and to compare these cells with Tregs from non-depleted immunized mice. Our results show that using this approach, immunization induced CD4⁺CD25⁺ T cells in the periphery, and that these cells were suppressive in vitro. Additionally, adoptive transfer of B29-specific iTregs suppressed disease in a mouse model of arthritis. This study shows that immunization of mice with Hsp70 epitope B29 induces functionally suppressive iTregs from CD4⁺CD25^- T cells.     

Inhaled bordetella pertussis vaccine decreases airway responsiveness in guinea pigs

Bordetella pertussis (BP) has been used as adjuvant for experimental animal immunization, but its effects on airway responsiveness are uncertain. Three groups of guinea pigs were used: animals with a single exposure to inhaled BP vaccine (strain 134, total dose 1.24 × 10¹²germs), animals submitted to a sensitization procedure through inhalation of ovalbumin plus BP and healthy control animals. Four weeks after inhalation of BP or after the beginning of sensitization, dose- or concentration-response curves to histamine were constructed in vivo and in vitro (tracheal and parenchymal preparations). We found that BP alone produced lower responses to histamine than control guinea pigs in vivo (insufflation pressure, p = 0.0003) and in tracheal tissues (p = 0.04), but not in parenchymal preparations. Sensitization did not modify the responsiveness compared with their respective controls. These results suggest that some BP component(s), probably pertussis toxin, causes a long lasting airway hyporesponsiveness in guinea pigs.