Carola Rask

Impaired regulatory T cell function in germ-free mice

Regulatory T cells (Treg) are crucial for the maintenance of tolerance to auto‐antigens and harmless exogenous antigens. Here, we studied the role of the commensal microbiota for the development and function of Treg. CD4+CD25+ T cells were obtained from peripheral lymph nodes (PLN) and mesenteric lymph nodes (MLN) of germ‐free (GF) and conventional (conv) NMRI mice and tested for phenotype and functional suppressive capacity. CD4+CD25+ T cells from GF mice showed a lower relative gene expression of fork head box p3 gene (Foxp3) and were not as potent suppressors in vitro as CD4+CD25+ T cells from conv animals. Intracellular staining for Foxp3 and CTLA‐4 revealed proportional and regional differences in putative Treg subsets between conv and GF mice. Fewer of the CD4+CD25+ T cells in GF MLN expressed Foxp3 and CTLA‐4, while the expression of these markers was similar amongst the CD4+CD25+ T cells in PLN of conv and GF mice. The largest difference between conv and GF Treg was observed in the liver draining celiac lymph node, where GF mice had fewer putative Treg as compared to conv mice. We propose that the presence of a microbial flora favors the development of a fully functional Treg population.

Serum-derived exosomes from antigen-fed mice prevent allergic sensitization in a model of allergic asthma

Oral tolerance is an active process that starts with sampling of luminal antigens by the intestinal epithelial cells (IEC), followed by processing and assembly with major histocompatibility complex class II and subsequently a release of tolerogenic exosomes (tolerosomes) from the IEC. We have previously shown that tolerosomes can be isolated from serum shortly after an antigen feed, and will potently transfer antigen‐specific tolerance to naive recipients. Here we study the capacity of the tolerosomes to protect against allergic sensitization in a mouse model of allergic asthma. Serum or isolated serum exosomes from tolerized BALB/c donor mice were transferred to syngeneic recipients followed by sensitization and intranasal exposure to ovalbumin (OVA). Blood, bronchoalveolar lavage (BAL) and lymph nodes were sampled 24 hr after the final exposure. The number of eosinophils was counted in BAL fluid and the levels of immunoglobulin E (IgE) and OVA‐specific IgE were measured in serum. Mediastinal and coeliac lymph nodes were analysed by flow cytometry. The animals receiving serum from OVA‐fed mice displayed significantly lower numbers of airway eosinophils and lower serum levels of total IgE as well as of OVA‐specific IgE compared with controls. Moreover, the tolerant animals showed a significantly higher frequency of activated T cells with a regulatory phenotype in both mediastinal and coeliac lymph nodes. The results show that serum or isolated serum exosomes obtained from OVA‐fed mice and administered intraperitoneally to naive recipient mice abrogated allergic sensitization in the recipients.

Selective tolerization of Th1‐like cells after nasal administration of a cholera toxoid‐LACK conjugate

Recent reports have suggested that after infection of BALB/c mice with Leishmania major, CD4+ T cells responding to a single antigen, LACK (Leishmaniahomologue of receptors for activated C kinase), drive the differentiation of other responding T cells to the Th2 phenotype and so allow lesion development to occur. Transgenic mice expressing LACK in the thymus are tolerant to LACK and thus resolve infection with L. major. The oral administration of soluble protein to mice has been shown to result in the peripheral tolerance of antigen‐specific CD4+ T cells. We therefore sought to tolerize LACK reactive T cells in non‐transgenic BALB/c mice in order to determine the effectiveness of this tolerization approach as an alternative to standard vaccination protocols againist L. majorinfection. Surprisingly, oral or nasal administration of up to 8 mg of recombinant LACK did not affect the outcome of infection. We therefore conjugated LACK to cholera toxin β subunit (CTB‐LACK) which has previously been shown to improve the effectiveness of oral tolerance to conjugated antigens. Nasal administration of as little as 12 μg of CTB‐LACK effectively diminished the capacity of mice to mount a subsequent proliferative response to LACK and further delayed the onset of lesion development in infected mice. However, pretreatment with CTB‐LACK did not prevent the eventual onset and progression of disease in these mice. An examination of cytokine responsiveness to LACK after tolerization with CTB‐LACK revealed that while the Th1 response to LACK was suppressed, Th2 cytokine production was unaffected. Similar experiments using an ovalbumin‐CTB conjugate suggested that this selective tolerance of Th1 cells was not specific to the LACK protein but may be an effect common to CTB‐conjugated proteins.

Mucosal and systemic antibody responses after peroral or intranasal immunization: Effects of conjugation to enterotoxin B subunits and/or of co-administration with free toxin as adjuvant

The mucosa‐binding molecules cholera toxin (CT) from Vibrio cholerae and heat‐labile enterotoxin (LT) from Escherichia coli have previously been used as mucosal adjuvants and carriers for many types of antigen. However, since these molecules are toxic and cannot be used in human vaccines, it is important to study whether their non‐toxic mucosa‐binding B subunits, CTB and LTB, can be used as alternative safe mucosal adjuvants and/or carrier molecules. We have as a model protein antigen used human gammaglobulin (HGG) for admixture with or chemical conjugation to recombinantly produced CTB and LTB, respectively, and measured antigen‐specific local secretory IgA antibodies in saponin extracts from intestine and lung tissue by ELISA following intra‐nasal (i.n.) or per‐oral (p.o.) immunization. The results show that local antibody formation against HGG was increased after immunization with conjugated as compared to free HGG. However, while the conjugates alone gave rise to significant immune responses in the lung and also, to a lesser degree, in the intestine after i.n. immunization, co‐administration of a small amount of free CT/LT as adjuvant was needed to induce a significant immune response in the intestine after p.o. immunization. We also found that following i.n. immunization, the addition of CTB to HGG, without coupling, increased the mucosal immune response to some extent, indicating that CTB by itself can work as an adjuvant by the i.n. route of immunization. A striking finding was that, as a carrier, CTB was superior to LTB when the conjugates were used by the oral but not by the i.n. route of immunization. In conclusion, conjugation of an antigen to mucosa‐binding molecules such as CTB and/or LTB can dramatically increase their mucosal immunogenicity. This approach may thus be useful in the preparation of mucosal vaccines.

Neonatal exposure to staphylococcal superantigen improves induction of oral tolerance in a mouse model of airway allergy

The hygiene hypothesis suggests that lack of microbial stimulation in early infancy may lead to allergy, but it has been difficult to identify particular protective microbial exposures. We have observed that infants colonised in the first week(s) of life with Staphylococcus aureus have lower risk of developing food allergy. As many S. aureus strains produce superantigens with T‐cell stimulating properties, we here investigate whether neonatal mucosal exposure to superantigen could influence the capacity to develop oral tolerance and reduce sensitisation and allergy. BALB/c mice were exposed to staphylococcal enterotoxin A (SEA) as neonates and fed with OVA as adults, prior to sensitisation and i.n. OVA challenge. Our results show that SEA pre‐treated mice are more efficiently tolerised by OVA feeding, as shown by lower lung‐cell infiltration and antigen‐specific IgE response in the SEA pre‐treated mice, compared with sham‐treated mice. This was not due to deletion or anergy of lymphocytes by SEA treatment, because the SEA pre‐treated mice that were fed with PBS showed similar inflammatory response as the sham‐treated PBS‐fed mice. Our results suggest that strong T‐cell activation in infancy conditions the mucosal immune system and promotes development of oral tolerance.

Differential effect on cell-mediated immunity in human volunteers after intake of different lactobacilli.

Probiotics are live microorganisms which have beneficial effects on the host when ingested in adequate amounts. Probiotic bacteria may stimulate immune effector functions in a strain‐specific manner. In this blind placebo‐controlled trial, we investigated the effects on the immune system following daily intake of six different strains of lactobacilli or the Gram‐negative bacterium Pseudomonas lundensis for 2 or 5 weeks. Blood lymphocyte subsets were quantified by fluorescence activated cell sorter and the expression of activation and memory markers was determined. The bacterial strains were also examined for their capacity to adhere to human intestinal cells and to be phagocytosed by human peripheral blood mononuclear cells. Intake of Lactobacillus plantarum strain 299v increased the expression of the activation marker CD25 (P = 0·01) on CD8+ T cells and the memory cell marker CD45RO on CD4+ T cells (P = 0·03), whereas intake of L. paracasei tended to expand the natural killer T (NK T) cell population (P = 0·06). The phagocytic activity of granulocytes was increased following intake of L. plantarum 299v, L. plantarum HEAL, L. paracasei or L. fermentum. In contrast, ingestion of L. rhamnosus decreased the expression of CD25 and CD45RO significantly within the CD4+ cell population. The observed immune effects after in‐vivo administration of the probiotic bacteria could not be predicted by either their adherence capacity or the in‐vitro‐induced cytokine production. The stimulation of CD8+ T cells and NK T cells suggests that intake of probiotic bacteria may enhance the immune defence against, e.g. viral infections or tumours.

Stronger T Cell Immunogenicity of Ovalbumin Expressed Intracellularly in Gram-Negative than in Gram-Positive Bacteria

This study aimed to clarify whether Gram-positive (G+) and Gram-negative (G−) bacteria affect antigen-presenting cells differently and thereby influence the immunogenicity of proteins they express. Lactobacilli, lactococci and Escherichia coli strains were transformed with plasmids conferring intracellular ovalbumin (OVA) production. Murine splenic antigen presenting cells (APCs) were pulsed with washed and UV-inactivated OVA-producing bacteria, control bacteria, or soluble OVA. The ability of the APCs to activate OVA-specific DO11.10 CD4+ T cells was assessed by measurments of T cell proliferation and cytokine (IFN-γ, IL-13, IL-17, IL-10) production. OVA expressed within E. coli was strongly immunogenic, since 500 times higher concentrations of soluble OVA were needed to achieve a similar level of OVA-specific T cell proliferation. Furthermore, T cells responding to soluble OVA produced mainly IL-13, while T cells responding to E. coli-expressed OVA produced high levels of both IFN-γ and IL-13. Compared to E. coli, G+ lactobacilli and lactococci were poor inducers of OVA-specific T cell proliferation and cytokine production, despite efficient intracellular expression and production of OVA and despite being efficiently phagocytosed. These results demonstrate a pronounced difference in immunogenicity of intracellular antigens in G+ and G− bacteria and may be relevant for the use of bacterial carriers in vaccine development.