Bacillus Calmette-Guérin (BCG) vaccine generates immunoregulatory cells in the cervical lymph nodes in guinea pigs injected intra dermally.

This work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining Bacillus Calmette-Guérin (BCG) vaccinated site on the dorsum of the ear in guinea pigs. It is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (PPD or leprosin) despite being responsive to whole mycobacteria. Besides, T cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to PPD in the presence of autologous antigen presenting cells. Interestingly, addition of as low as 20% nylon wool adherent cells to these, sharply decreased the proliferation by 83%. Looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the T cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. Since BCG induced granulomas resolve much faster than granulomas induced by other mycobacteria such as Mycobacterium leprae the present experimental findings add to the existing evidence that intradermal BCG vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in Covid-19 patients.

Differential innate immune responses induced by Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in various types of antigen presenting cells.

Mycoplasma (M.) hyopneumoniae is the etiological agent of enzootic pneumonia in pigs and is closely related to M. hyorhinis, which can be isolated from the healthy mucosal surfaces of the upper respiratory tract. In rare cases it can also cause arthritis and polyserositis. Since the innate immune system is an important first line of defense and promotes adaptive immune responses, we characterized the innate immune response of various antigen presenting cells (APCs) to M. hyopneumoniae and M. hyorhinis, which differ in their pathogenicity in vivo. Porcine peripheral blood mononuclear cells were infected with different multiplicities of infection (MOI) of live and inactivated porcine mycoplasmas. Both Mycoplasma species induced strong tumour necrosis factor (TNF) responses in monocytes, with a stronger activation by M. hyorhinis. This higher stimulatory activity was also confirmed for CD40 upregulation. Conventional and plasmacytoid dendritic cells (cDC and pDC, respectively) did not or poorly respond to mycoplasmas in terms of TNF expression but more efficiently in terms of CD40 upregulation. Again, these responses were generally stronger with M. hyorhinis than with M. hyopneumoniae. Both Mycoplasma species also activated B cells in terms of CD25 upregulation, proliferation, and IgM secretion. Interestingly, while the induction of CD25 and in particular proliferation was higher with M. hyorhinis, the IgM secretion did not differ between the two species with the exception of the highest dose of M. hyopneumoniae,which appeared to suppress IgM responses. Taken together, our results provide a comparative analysis of innate immune response with different porcine APCs and demonstrate Mycoplasma species-dependent differences, which could relate to their different pathogenicity in vivo.

Sophisticated specificity in the innate immune response.

Immunologists are sometimes guilty of describing the innate immune response as 'non-specific'. What we really mean is that the pattern recognition receptors of innate immune cells are not able to recombine and mutate to bind the spectacular range of molecular patterns that can be recognised by B and T cells. So, while it may be accurate to describe the innate immune response as less specific than adaptive immunity, even this belies the emerging complexity of the receptors and receptor complexes that control inflammatory responses. This complexity is necessary to recognise danger, and therefore successfully initiate proportionate inflammatory responses to cellular damage or against potential pathogens.

Induction of CD8 T cell cytotoxicity by fecal bacteria from healthy individuals and colorectal cancer patients.

Commensal microbiota modulates the anti-tumor immune response and alters the tumor infiltration of T cells in numerous human malignancies. Moreover, the existence of commensals and microbial metabolites has been directly observed inside numerous epithelial tumors. Their effects on the host immune system, independent of the pre-existing malignancy, are not completely understood. To resolve this issue, we compared immune modulatory roles of the fecal bacteria from healthy individuals and the fecal bacteria from colorectal cancer (CRC) patients. Peripheral blood mononuclear cells that were provided by healthy donors were used as study systems. Overall, fecal bacteria could potently activate the degranulation and cytotoxicity of CD8+ T cells. Interestingly, fecal bacteria from CRC patients in general induced higher degranulation and higher cytotoxicity than fecal bacteria from healthy individuals. These effects were dependent on the presence of antigen-presenting cells, such as monocytes and B cells, as fecal bacteria added directly to isolated CD8+ T cells failed to induce high cytotoxicity. Additionally, fecal bacteria from CRC patients induced stronger upregulation of CD80 and NOS2 expression in monocytes than fecal bacteria from healthy individuals. On the other hand, the viability of CD8+ T cells was significantly reduced with increasing levels of bacterial stimulation. Overall, we demonstrated that fecal bacteria from CRC patients could upregulate degranulation and cytotoxicity of CD8+ T cells in a manner that was dependent on antigen-presenting cells, and was more proinflammatory than fecal bacteria from healthy individuals.

Salmonella enterica Serovar Typhimurium Interacts with CD209 Receptors To Promote Host Dissemination and Infection.

Salmonella enterica serovar Typhimurium, a Gram-negative bacterium, can cause infectious diseases ranging from gastroenteritis to systemic dissemination and infection. However, the molecular mechanisms underlying this bacterial dissemination have yet to be elucidated. A study indicated that using the lipopolysaccharide (LPS) core as a ligand, S Typhimurium was able to bind human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (hCD209a), an HIV receptor that promotes viral dissemination by hijacking antigen-presenting cells (APCs). In this study, we showed that S Typhimurium interacted with CD209s, leading to the invasion of APCs and potentially the dissemination to regional lymph nodes, spleen, and liver in mice. Shielding of the exposed LPS core through the expression of O-antigen reduces dissemination and infection. Thus, we propose that similar to HIV, S Typhimurium may also utilize APCs via interactions with CD209s as a way to disseminate to the lymph nodes, spleen, and liver to initiate host infection.

Pathogen Molecular Pattern Receptor Agonists: Treating Cancer by Mimicking Infection.

Immunotherapies such as checkpoint blockade have achieved durable benefits for patients with advanced stage cancer and have changed treatment paradigms. However, these therapies rely on a patient's own a priori primed tumor-specific T cells, limiting their efficacy to a subset of patients. Because checkpoint blockade is most effective in patients with inflamed or "hot" tumors, a priority in the field is learning how to "turn cold tumors hot." Inflammation is generally initiated by innate immune cells, which receive signals through pattern recognition receptors (PRR)-a diverse family of receptors that sense conserved molecular patterns on pathogens, alarming the immune system of an invading microbe. Their immunostimulatory properties can reprogram the immune suppressive tumor microenvironment and activate antigen-presenting cells to present tumors antigens, driving de novo tumor-specific T-cell responses. These features, among others, make PRR-targeting therapies an attractive strategy in immuno-oncology. Here, we discuss mechanisms of PRR activation, highlighting ongoing clinical trials and recent preclinical advances focused on therapeutically targeting PRRs to treat cancer.

A Mechanistic Understanding of Pyroptosis: The Fiery Death Triggered by Invasive Infection.

Immune cells and skin and mucosal epithelial cells recognize invasive microbes and other signs of danger to sound alarms that recruit responder cells and initiate an immediate "innate" immune response. An especially powerful alarm is triggered by cytosolic sensors of invasive infection that assemble into multimolecular complexes, called inflammasomes, that activate the inflammatory caspases, leading to maturation and secretion of proinflammatory cytokines and pyroptosis, an inflammatory death of the infected cell. Work in the past year has defined the molecular basis of pyroptosis. Activated inflammatory caspases cleave Gasdermin D (GSDMD), a cytosolic protein in immune antigen-presenting cells and epithelia. Cleavage separates the autoinhibitory C-terminal fragment from the active N-terminal fragment, which moves to the cell membrane, binds to lipids on the inside of the cell membrane, and oligomerizes to form membrane pores that disrupt cell membrane integrity, causing death and leakage of small molecules, including the proinflammatory cytokines and GSDMD itself. GSDMD also binds to cardiolipin on bacterial membranes and kills the very bacteria that activate the inflammasome. GSDMD belongs to a family of poorly studied gasdermins, expressed in the skin and mucosa, which can also form membrane pores. Spontaneous mutations that disrupt the binding of the N- and C-terminal domains of other gasdermins are associated with alopecia and asthma. Here, we review recent studies that identified the roles of the inflammasome, inflammatory caspases, and GSDMD in pyroptosis and highlight some of the outstanding questions about their roles in innate immunity, control of infection, and sepsis.

Bacillus anthracis spore movement does not require a carrier cell and is not affected by lethal toxin in human lung models.

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores escape from the alveolus to regional lymph nodes, germinate and enter the circulatory system to cause disease. The roles of carrier cells and the effects of B. anthracis toxins in this process are unclear. We used a human lung organ culture model to measure spore uptake by antigen presenting cells (APC) and alveolar epithelial cells (AEC), spore partitioning between these cells, and the effects of B. anthracis lethal toxin and protective antigen. We repeated the study in a human A549 alveolar epithelial cell model. Most spores remained unassociated with cells, but the majority of cell-associated spores were in AEC, not in APC. Spore movement was not dependent on internalization, although the location of internalized spores changed in both cell types. Spores also internalized in a non-uniform pattern. Toxins affected neither transit of the spores nor the partitioning of spores into AEC and APC. Our results support a model of spore escape from the alveolus that involves spore clustering with transient passage through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur.

A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells.

Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory.

Human Invariant Natural Killer T cells possess immune-modulating functions during Aspergillus infection.

Aspergillus fumigatus is the most common cause for invasive fungal infections, a disease associated with high mortality in immune-compromised patients. CD1d-restricted invariant natural killer T (iNKT) cells compose a small subset of T cells known to impact the immune response toward various infectious pathogens. To investigate the role of human iNKT cells during A. fumigatus infection, we studied their activation as determined by CD69 expression and cytokine production in response to distinct fungal morphotypes in the presence of different CD1d(+) antigen presenting cells using flow cytometry and multiplex enzyme-linked immunosorbent assay (ELISA). Among CD1d(+) subpopulations, CD1d(+)CD1c(+) mDCs showed the highest potential to activate iNKT cells on a per cell basis. The presence of A. fumigatus decreased this effect of CD1d(+)CD1c(+) mDCs on iNKT cells and led to reduced secretion of TNF-α, G-CSF and RANTES. Production of other Th1 and Th2 cytokines was not affected by the fungus, suggesting an immune-modulating function for human iNKT cells during A. fumigatus infection.

Mechanism of Action of Probiotic Bacteria on Intestinal and Systemic Immunities and Antigen-Presenting Cells.

Immunomodulation has been shown to be one of the major functions of probiotic bacteria. This review is presented to provide detailed information on the immunomodulatory properties of probiotics in various animal models and clinical practices. Probiotics can regulate helper T (Th) responses and release of cytokines in a strain-specific manner. For example, Lactobacillus rhamnosus GG can induce beneficial Th1 immunomodulatory effect in infants with cow's milk allergy and relieve intestinal inflammation in atopic children by promoting IL-10 generation. Mechanism of action of probiotics on antigen-presenting cells at gastrointestinal tract is also postulated in this review. Probiotic bacterial cells and their soluble factors may activate dendritic cells, macrophages, and to certain extent monocytes via toll-like-receptor recognition and may further provoke specific Th responses. They are speculated to elicit immunomodulatory effects on intestinal and systemic immunities.

Salmonella impairs CD8 T cell response through PD-1: PD-L axis.

We have shown that Salmonella remains for a long period of time within B cells, plasma cells, and bone marrow B cell precursors, which might allow persistence and dissemination of infection. Nonetheless, how infected cells evade CD8 T cell response has not been characterized. Evidence indicates that some pathogens exploit the PD-1: PD-L (PD-L1 and PD-L2) interaction to inhibit CD8 T cells response to contribute the chronicity of the infection. To determine whether the PD-1: PD-L axis plays a role during Salmonella infection; we evaluated PD-1 expression in antigen-specific CD8 T cells and PD-1 ligands in Salmonella-infected cells. Our results show that infected B cells and macrophages express continuously co-stimulatory (CD40, CD80, and CD86) and inhibitory molecules (PD-L1 and PD-L2) in early and late stages of chronic Salmonella infection, while antigen-specific CD8 T cells express in a sustained manner PD-1 in the late stages of infection. Blocking this axis restores the ability of the CD8 T cells to proliferate and eliminate primary infected APCs. Therefore, a continuous PD-1: PDL interaction might be a mechanism employed by Salmonella to negatively regulate Salmonella-specific CD8 T cell cytotoxic response in order to remain within the host for a long period of time.

Liposomal delivery of lipoarabinomannan triggers Mycobacterium tuberculosis specific T-cells.

Lipoarabinomannan (LAM) is a major cell wall component of Mycobacterium tuberculosis (Mtb). LAM specific human T-lymphocytes release interferon-γ (IFNγ) and have antimicrobial activity against intracellular Mtb suggesting that they contribute to protection. Therefore the induction of LAM-specific memory T-cells is an attractive approach for the design of a new vaccine against tuberculosis. A prerequisite for the activation of LAM-specific T-cells is the efficient uptake and transport of the glycolipid antigen to the CD1 antigen presenting machinery. Based on the hydrophobicity of LAM we hypothesized that packaging of LAM into liposomes will support the activation of T-lymphocytes. We prepared liposomes containing phosphatidylcholine, cholesterol, stearylated octaarginine and LAM via thin layer hydration method (LIPLAM). Flow cytometry analysis using fluorescently labelled LIPLAM showed an efficient uptake by antigen presenting cells. LAM delivered via liposomes was biologically active as demonstrated by the down-regulation of peroxisome proliferator activated receptor gamma (PPARγ) protein expression. Importantly, LIPLAM induced higher IFNγ production by primary human T-lymphocytes than purified LAM (2-16 times) or empty liposomes. These results suggest that the delivery of mycobacterial glycolipids via liposomes is a promising approach to promote the induction of M. tuberculosis specific T-cell responses.

Host Langerin (CD207) is a receptor for Yersinia pestis phagocytosis and promotes dissemination.

Yersinia pestis is a Gram-negative bacterium that causes plague. After Y. pestis overcomes the skin barrier, it encounters antigen-presenting cells (APCs), such as Langerhans and dendritic cells. They transport the bacteria from the skin to the lymph nodes. However, the molecular mechanisms involved in bacterial transmission are unclear. Langerhans cells (LCs) express Langerin (CD207), a calcium-dependent (C-type) lectin. Furthermore, Y. pestis possesses exposed core oligosaccharides. In this study, we show that Y. pestis invades LCs and Langerin-expressing transfectants. However, when the bacterial core oligosaccharides are shielded or truncated, Y. pestis propensity to invade Langerhans and Langerin-expressing cells decreases. Moreover, the interaction of Y. pestis with Langerin-expressing transfectants is inhibited by purified Langerin, a DC-SIGN (DC-specific intercellular adhesion molecule 3 grabbing nonintegrin)-like molecule, an anti-CD207 antibody, purified core oligosaccharides and several oligosaccharides. Furthermore, covering core oligosaccharides reduces the mortality associated with murine infection by adversely affecting the transmission of Y. pestis to lymph nodes. These results demonstrate that direct interaction of core oligosaccharides with Langerin facilitates the invasion of LCs by Y. pestis. Therefore, Langerin-mediated binding of Y. pestis to APCs may promote its dissemination and infection.

Malassezia yeasts activate the NLRP3 inflammasome in antigen-presenting cells via Syk-kinase signalling.

Although being a normal part of the skin flora, yeasts of the genus Malassezia are associated with several common dermatologic conditions including pityriasis versicolour, seborrhoeic dermatitis (SD), folliculitis, atopic eczema/dermatitis (AE/AD) and dandruff. While Malassezia spp. are aetiological agents of pityriasis versicolour, a causal role of Malassezia spp. in AE/AD and SD remains to be established. Previous reports have shown that fungi such as Candida albicans and Aspergillus fumigatus are able to efficiently activate the NLRP3 inflammasome leading to robust secretion of the pro-inflammatory cytokine IL-1ß. To date, innate immune responses to Malassezia spp. are not well characterized. Here, we show that different Malassezia species could induce NLRP3 inflammasome activation and subsequent IL-1ß secretion in human antigen-presenting cells. In contrast, keratinocytes were not able to secrete IL-1ß when exposed to Malassezia spp. Moreover, we demonstrate that IL-1ß secretion in antigen-presenting cells was dependent on Syk-kinase signalling. Our results identify Malassezia spp. as potential strong inducers of pro-inflammatory responses when taken up by antigen-presenting cells and identify C-type lectin receptors and the NLRP3 inflammasome as crucial actors in this process.

Neonatal mucosal immune stimulation by microbial superantigen improves the tolerogenic capacity of CD103(+) dendritic cells.

Food allergy represents failure to develop tolerance to dietary proteins. Food allergy has increased in prevalence in parallel with decreased exposure to microbes during infancy. In mice, neonatal peroral exposure to the strongly T cell stimulating superantigen staphylococcal enterotoxin A (SEA), enhances the capacity to develop oral tolerance to a novel antigen encountered in adult life. A population of antigen-presenting cells in the gut, the CD103(+) dendritic cells (DCs), is thought to be involved in oral tolerance development, as they convert naïve T cells into FoxP3(+) regulatory T cells (Treg). This function depends on their capacity to convert vitamin A to retinoic acid, carried out by the retinal aldehyde dehydrogenase (RALDH) enzyme. Here, newborn mice were treated with superantigen and DC function and tolerogenic capacity was examined at six weeks of age. We observed that, in mice fed superantigen neonatally, the CD11c(+) DCs had increased expression of RALDH and in vitro more efficiently induced expression Foxp3 expression to stimulated T cells. Further, these mice showed an accumulation of FoxP3(+) T cells in the small intestinal lamina propria and had a more Ag-specific FoxP3(+) T cells after oral tolerance induction in vivo. Moreover, the improved oral tolerance, as shown by increased protection from food allergy, was eradicated if the Vitamin A metabolism was inhibited. These observations contribute to the understanding of how a strong immune stimulation during the neonatal period influences the maturation of the immune system and suggests that such stimulation may reduce the risk of later allergy development.

Role of the mannose receptor in phagocytosis of Enterococcus faecalis strain EC-12 by antigen-presenting cells.

The aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Fluorescein isothiocyanate (FITC)-labeled EC-12 was cocultured with peritoneal macrophage and the amount of EC-12 phagocytosed by peritoneal macrophages was measured using a microplate fluorometer. Peritoneal macrophages from toll-like receptor (TLR)2-, TLR7-, and MyD88-deficient knockout (KO) mice exhibited similar levels of EC-12 phagocytosis to those from wild-type mice. Similarly, dectin-1 neutralization of peritoneal macrophages had no effect on EC-12 phagocytosis. However, blockade of the mannose receptor (MR) significantly decreased the amount of EC-12 phagocytosed by peritoneal macrophages; the same effect was observed in bone marrow-derived macrophages and dendritic cells. Our findings suggest that MR plays a major role in EC-12 phagocytosis by the APCs.

Synergistic activation of innate and adaptive immune mechanisms in the treatment of gonadotropin-sensitive tumors.

Human chorionic gonadotropin (hCG) prolongs the secretion of progesterone from the corpus luteum, providing a critical stimulus for the sustenance of pregnancy. hCG (or individual subunits) is also secreted by a variety of trophoblastic and non-trophoblastic cancers and has been associated with poor prognosis. Early clinical studies have indicated merit in anti-hCG vaccination as potential immunotherapy, but anti-tumor efficacy is believed to be compromised by sub-optimal immunogenecity. In the present study, enhanced tumorigenesis was observed when SP2/O cells were subcutaneously injected in either male or female BALB/c x FVB/J(ßhCG/-) F1 transgenic mice, establishing the growth-promoting effects of the gonadotropin for implanted tumors in vivo. The utility of Mycobacterium indicus pranii (MIP) was evaluated, as an innate anti-tumor immunomodulator as well as adjuvant in mice. MIP elicited the secretion of the inflammatory cytokines IFNγ, IL-6, IL-12p40, KC and TNFα from murine antigen presenting cells. When MIP was incorporated into an anti-hCG vaccine formulation previously employed in humans (a ßhCG-TT conjugate adsorbed on alum), elevated T cell recall proliferative and cytokine responses to hCG, ßhCG and TT were observed. MIP increased vaccine immunogenicity in mice of diverse genetic background (including in traditionally low-responder murine strains), leading to enhanced titres of bioneutralizing anti-hCG antibodies which exhibited cytotoxicity towards tumor cells. Individual administration of MIP and ßhCG-TT to BALB/c mice subcutaneously implanted with SP2/O cells resulted in anti-tumor effects; significantly, immunization with ßhCG-TT supplemented with MIP invoked synergistic benefits in terms of tumor volume, incidence and survival. The development of novel vaccine formulations stimulating both adaptive and innate anti-tumor immunity to induce collaborative beneficial effects may fill a niche in the adjunct treatment of hCG-sensitive tumors that are resistant to conventional therapy.

A mutant in the Listeria monocytogenes Fur-regulated virulence locus (frvA) induces cellular immunity and confers protection against listeriosis in mice.

Listeria monocytogenes is a Gram-positive intracellular pathogen that is responsible for listeriosis, a potentially fatal, food-borne illness. Due to its cytoplasmic location during infection, this pathogen can mediate a long-lasting cellular immune response, which makes attenuated strains strong candidates for vaccine development. Recently, our group identified and characterized frvA (Fur-regulated virulence factor A), and deletion of this gene resulted in disruption of iron homeostasis and a strong attenuation in virulence. Despite significant attenuation in the mouse infection model, the frvA mutant was capable of intracellular growth in antigen-presenting cells. Indeed, mice immunized with L. monocytogenes ΔfrvA were able to effectively stimulate specific CD8(+) T cells to the listerial epitopes LLO(91-99) and P60(217-225) at levels comparable with L. monocytogenes strain EGDe. Most notably, mice immunized with ΔfrvA then subsequently challenged with the wild-type strain were completely protected from listerial infection. On the basis of these results, we advocate the use of ΔfrvA as a live attenuated listerial vaccine, and propose that this mutant may serve as a platform for the development of a future vaccine delivery vehicle.

Update on immune mechanisms associated with sublingual immunotherapy: practical implications for the clinician.

Sublingual immunotherapy (SLIT) is established as a safe and efficacious treatment for patients with type I respiratory allergies. The ability of SLIT to elicit antigen (allergen)-specific tolerance is linked to the peculiar biology of oral antigen-presenting cells. In the absence of danger signals, Langerhans cells, myeloid dendritic cells, and macrophages located in oral tissues, tonsils, and draining cervical lymph nodes are biased toward the induction of T(H)1 and IL-10-producing CD4(+) regulatory T cells, thus supporting tolerance as opposed to inflammation. Sublingual administration does not lead to any detectable systemic exposure of intact allergens nor to IgE neosensitization. Oral tissues contain limited numbers of mast cells located in submucosal areas, thereby explaining the well-established safety profile of SLIT, with mostly local but rare systemic reactions. The induction of CD4(+) regulatory T cells and blocking anti-inflammatory IgGs or IgAs are considered important for tolerance induction after SLIT. Specific molecular signatures associated with tolerogenic dendritic cells were recently reported during the onset of SLIT efficacy in the peripheral blood of patients exhibiting clinical benefit. Collectively, these observations confirm the induction of strong allergen-specific suppressive/tolerogenic immune responses during SLIT and pave the ground for the identification of biomarkers of efficacy. Practical implications of this emerging scientific knowledge are presented (1) to support the rational design of second-generation sublingual vaccines based on purified allergens, vector systems and/or adjuvants and (2) to help the clinician in decision making during his/her practice.