Sublingual vaccination and delivery systems.

Most infectious agents use mucosal tissues as entry portals, thus, mucosae are frequently defined as a first line of defense against pathogens. Mucosal protection generally operates through antibody-mediated and cytotoxic T-cell responses which can be triggered by mucosal vaccines. Sublingual vaccination provides many advantages such as systemic and mucosal responses (both locally and at remote mucosal sites), besides being a needle-free administration route with high patient compliance and limited adverse effects. Buccal mucosa complexity nonetheless represents a challenge for vaccine administration, hence, many efforts were recently deployed to improve vaccine components, mucoadhesion and/or penetration. Several innovative approaches indeed confirmed that a robust and protective immunity can be achieved by sublingual vaccines. This review will then specify the most recent delivery systems and improvements developed to increase sublingual vaccines efficiency. We will focus our description on the immune mechanisms involved and the requirements for optimal sublingual immunization and mucosal protection.

Antigen-bearing dendritic cells from the sublingual mucosa recirculate to distant systemic lymphoid organs to prime mucosal CD8 T cells.

Effector T cells are described to be primed in the lymph nodes draining the site of immunization and to recirculate to effector sites. Sublingual immunization generates effector T cells able to disseminate to the genital tract. Herein, we report an alternative mechanism that involves the recirculation of antigen-bearing dendritic cells (DCs) in remote lymphoid organs to prime T cells. Sublingual immunization with a muco-adhesive model antigen unable to diffuse through lymphatic or blood vessels induced genital CD8 T cells. The sublingual draining lymph nodes were not mandatory to generate these lymphocytes, and antigen-bearing DCs from distant lymph nodes and spleen were able to prime specific CD8 T cells in a time- and dose-dependent manner. This study demonstrates, for the first time, that antigen-bearing DCs originating from the site of immunization recirculate to distant lymphoid organs and provides insights into the mechanism of distant CD8 T-cell generation by sublingual immunization.

B cell and T cell immunity in the female genital tract: potential of distinct mucosal routes of vaccination and role of tissue-associated dendritic cells and natural killer cells.

The female genital mucosa constitutes the major port of entry of sexually transmitted infections. Most genital microbial pathogens represent an enormous challenge for developing vaccines that can induce genital immunity that will prevent their transmission. It is now established that long-lasting protective immunity at mucosal surfaces has to involve local B-cell and T-cell effectors as well as local memory cells. Mucosal immunization constitutes an attractive way to generate systemic and genital B-cell and T-cell immune responses that can control early infection by sexually transmitted pathogens. Nevertheless, no mucosal vaccines against sexually transmitted infections are approved for human use. The mucosa-associated immune system is highly compartmentalized and the selection of any particular route or combinations of routes of immunization is critical when defining vaccine strategies against genital infections. Furthermore, mucosal surfaces are complex immunocompetent tissues that comprise antigen-presenting cells and also innate immune effectors and non-immune cells that can act as 'natural adjuvants' or negative immune modulators. The functions of these cells have to be taken into account when designing tissue-specific antigen-delivery systems and adjuvants. Here, we will discuss data that compare different mucosal routes of immunization to generate B-cell and T-cell responses in the genital tract, with a special emphasis on the newly described sublingual route of immunization. We will also summarize data on the understanding of the effector and induction mechanisms of genital immunity that may influence the development of vaccine strategies against genital infections.

Requirement for Daxx in mature T-cell proliferation and activation.

The protein Daxx promotes Fas-mediated cell death through activation of apoptosis signal-regulating kinase 1, leading to the activation of the MAPKs JNK and p38. Owing to the in utero lethality of daxx-deficient mice, the in vivo role of Daxx has been so far difficult to analyze. We have generated transgenic mice expressing a dominant-negative form of Daxx (Daxx-DN) in the T-cell lineage. We show that Daxx is recruited to the Fas receptor upon FasL engagement and that Daxx-DN expression protects activated T cells from Fas-induced cell death, by preventing the death-inducing signal complex to be properly formed. Normal lymphocyte development and homeostasis are nevertheless observed. Interestingly, we report that both in vitro and in vivo stimulation of Daxx-DN T-lymphocytes leads to increased proliferative T-cell responses. This increased proliferation is associated with a marked increase in tyrosine phosphorylation of LAT and ZAP70 as Daxx-DN favor their recruitment to the T-cell receptor (TCR) complex. These findings identify Daxx as a critical regulator of T-lymphocyte homeostasis by decreasing TCR-induced cell proliferation and by promoting Fas-mediated cell death.

Tissue-specific differential antitumour effect of molecular forms of fractalkine in a mouse model of metastatic colon cancer.

BACKGROUND AND AIMS: Fractalkine, a chemokine that presents as both a secreted and a membrane-anchored form, has been described as having tumour-suppressive activities in standard subcutaneous models. Here, we investigate the antitumour effect of fractalkine, in its three molecular forms, in two orthotopic models of metastatic colon cancer (liver and lung) and in the standard subcutaneous model. METHODS: We have developed models of skin tumours, liver and pulmonary metastasis and compared the extent of tumour development between C26 colon cancer cells expressing either the native, the soluble, the membrane-bound fractalkine or none. RESULTS: The native fractalkine exhibits the strongest antitumour effect, reducing the tumour size by 93% in the skin and by 99% in the orthotopic models (p<0.0001). Its overall effect results from a critical balance between the activity of the secreted and the membrane-bound forms, balance that is itself dependent on the target tissue. In the skin, both molecular variants reduce tumour development by 66% (p<0.01). In contrast, the liver and lung metastases are only significantly reduced by the soluble form (by 96%, p<0.002) whereas the membrane-bound variant exerts a barely significant effect in the liver (p = 0.049) and promotes tumour growth in the lungs. Moreover, we show a significant difference in the contribution of the infiltrating leukocytes to the tumour-suppressive activity of fractalkine between the standard and the orthotopic models. CONCLUSIONS: Fractalkine expression by C26 tumour cells drastically reduces their metastatic potential in the two physiological target organs. Both molecular forms contribute to its antitumour potential but exhibit differential effects on tumour development depending on the target tissue.

Origin and differentiation of dendritic cells.

Despite extensive, recent research on the development of dendritic cells (DCs), their origin is a controversial issue in immunology, with important implications regarding their use in cancer immunotherapy. Although, under defined experimental conditions, DCs can be generated from myeloid or lymphoid precursors, the differentiation pathways that generate DCs in vivo remain unknown largely. Indeed, experimental results suggest that the in vivo differentiation of a particular DC subpopulation could be unrelated to its possible experimental generation. Nevertheless, the analysis of DC differentiation by in vivo and in vitro experimental systems could provide important insights into the control of the physiological development of DCs and constitutes the basis of a model of common DC differentiation that we propose.

In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection.

BACKGROUND: The role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN) cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum. RESULTS: Promastigote phagocytosis and killing occurs very early after infection, as demonstrated by electron microscopy analyses which show in BALB/c mouse spleen, but not in liver, numerous PN harbouring ultrastructurally degraded parasites. It is shown, using mAb RB6-8C5 directed against mature mouse granulocytes, that in chronically infected mice, long-term PN depletion did not enhance parasite counts neither in liver nor in spleen, indicating that these cells are not involved in the late phase of L. infantum infection. In acute stage of infection, in mouse liver, where L. infantum load is initially larger than that in spleen but resolves spontaneously, there was no significant effect of neutrophils depletion. By contrast, early in infection the neutrophil cells crucially contributed to parasite killing in spleen, since PN depletion, performed before and up to 7 days after the parasite inoculation, resulted in a ten-fold increase of parasite burden. CONCLUSIONS: Taken together these data show that neutrophil cells contribute to the early control of the parasite growth in spleen but not in liver and that these cells have no significant effect late in infection in either of these target organs.

Sustained parasite burden in the spleen of Leishmania infantum-infected BALB/c mice is accompanied by expression of MCP-1 transcripts and lack of protection against challenge.

We analyzed differential responses of spleen and liver, major organ targets for viscerotropic Leishmania species, to experimental infection and examined if resistance to challenge was organ-specific. In liver, parasites were spontaneously cleared and iNOS trancripts expression paralleled that of amastigote load. In the spleen, amastigote multiplication was only partly controlled, and iNOS transcripts expression was transient. Total numbers of spleen cells, B cells, and T cells were decreased, while F4/80(+) and Mac1(+) cells were conserved. Expression of splenic MCP-1 transcripts remained constant, indicating its possible contribution to immigration of Leishmania host cells and to sustained parasite load. Spleen cells produced both, Th1- and Th2-type cytokines and Th2-type response was dominant, compatible with the sustained MCP-1 expression. Challenge experiments showed that in contrast to the liver, where initial infection conferred a progressively established immunity, in the spleen there was no induced protection against reinfection. Organ-specific resistance against challenge could be important for designing antileishmanial vaccines.

Isolation of mouse thymic dendritic cells.

The method described in this chapter for the isolation of mouse thymic dendritic cells (DC) is an optimization of our previously published methods (1,2) and involves the following major steps: 1. Enzymatic digestion of thymic fragments with collagenase and DNase. 2. Separation of a very-low-density cell fraction (VLDF). 3. Magnetic depletion of T-lineage cells, B cells, macrophages, and granulocytes. 4. Positive selection of DCs by magnetic cell sorting (MACS).

Concept of lymphoid versus myeloid dendritic cell lineages revisited: both CD8alpha(-) and CD8alpha(+) dendritic cells are generated from CD4(low) lymphoid-committed precursors.

Two dendritic cell (DC) subsets have been identified in the murine system on the basis of their differential CD8alpha expression. CD8alpha(+) DCs and CD8alpha(-) DCs are considered as lymphoid- and myeloid-derived, respectively, because CD8alpha(+) but not CD8alpha(-) splenic DCs were generated from lymphoid CD4(low) precursors, devoid of myeloid reconstitution potential. Although CD8alpha(-) DCs were first described as negative for CD4, our results demonstrate that approximately 70% of them are CD4(+). Besides CD4(-) CD8alpha(-) and CD4(+) CD8alpha(-) DCs displayed a similar phenotype and T-cell stimulatory potential in mixed lymphocyte reaction (MLR), although among CD8alpha(-) DCs, the CD4(+) subset appears to have a higher endocytic capacity. Finally, experiments of DC reconstitution after irradiation in which, in contrast to previous studies, donor-type DCs were analyzed without depleting CD4(+) cells, revealed that both CD8alpha(+) DCs and CD8alpha(-) DCs were generated after transfer of CD4(low) precursors. These data suggest that both CD8alpha(+) and CD8alpha(-) DCs derive from a common precursor and, hence, do not support the concept of the CD8alpha(+) lymphoid-derived and CD8alpha(-) myeloid-derived DC lineages. However, because this hypothesis has to be confirmed at the clonal level, it remains possible that CD8alpha(-) DCs arise from a myeloid precursor within the CD4(low) precursor population or, alternatively, that both CD8alpha(+) and CD8alpha(-) DCs derive from an independent nonlymphoid, nonmyeloid DC precursor. In conclusion, although we favor the hypothesis that both CD8alpha(+) and CD8alpha(-) DCs derive from a lymphoid-committed precursor, a precise study of the differentiation process of CD8alpha(+) and CD8alpha(-) DCs is required to define conclusively their origin.

Langerhans cells develop from a lymphoid-committed precursor.

Langerhans cells (LCs) are specialized dendritic cells (DCs) strategically located in stratified epithelia, such as those of the skin, oral cavity, pharynx, esophagus, upper airways, urethra, and female reproductive tract, which are exposed to a wide variety of microbial pathogens. LCs play an essential role in the induction of T-lymphocyte responses against viruses, bacteria, and parasites that gain access to those epithelial surfaces, due to their high antigen capture and processing potential and their capacity to present antigen peptides to T cells on migration to the lymph nodes.(1) Although LCs have been classically considered of myeloid origin, recent reports, which demonstrate the existence of lymphoid DCs derived from multipotent lymphoid precursors devoid of myeloid differentiation potential,(2-5) raise the question of the lymphoid or myeloid origin of LCs. The present study shows that mouse lymphoid-committed CD4(low) precursors, with the capacity to generate T cells, B cells, CD8(+) lymphoid DCs, and natural killer cells,(26) also generate epidermal LCs on intravenous transfer, supporting the view that LCs belong to the lymphoid lineage. (Blood. 2000;96:1633-1637)

Langerhans cells acquire a CD8+ dendritic cell phenotype on maturation by CD40 ligation.

Dendritic cell (DC) reconstitution experiments and phenotypic analysis of DC subpopulations have allowed the definition in the mouse of two main DC categories: CD8+ lymphoid DCs and CD8- myeloid DCs. With regard to Langerhans cells (LCs), which represent immature DCs differentiating into mature DCs on migration to the lymph nodes after an antigenic stimulation, although classically considered as myeloid DCs, there is no experimental evidence of their origin. It has been recently shown that mouse LCs, negative for CD8 and LFA-1, undergo CD8/LFA-1 up-regulation on migration, suggesting that LCs belong to the CD8+ lymphoid DC lineage. To further reinforce this hypothesis, we have analyzed the modulation of CD8 expression by LCs on culture with molecules known to induce LC maturation. Our results show that LC acquired a CD8+ lymphoid phenotype on CD40 ligation.

Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice.

The p42 and p44 mitogen-activated protein kinases (MAPKs), also called Erk2 and Erk1, respectively, have been implicated in proliferation as well as in differentiation programs. The specific role of the p44 MAPK isoform in the whole animal was evaluated by generation of p44 MAPK-deficient mice by homologous recombination in embryonic stem cells. The p44 MAPK-/- mice were viable, fertile, and of normal size. Thus, p44 MAPK is apparently dispensable and p42 MAPK (Erk2) may compensate for its loss. However, in p44 MAPK-/- mice, thymocyte maturation beyond the CD4+CD8+ stage was reduced by half, with a similar diminution in the thymocyte subpopulation expressing high levels of T cell receptor (CD3high). In p44 MAPK-/- thymocytes, proliferation in response to activation with a monoclonal antibody to the T cell receptor in the presence of phorbol myristate acetate was severely reduced even though activation of p42 MAPK was more sustained in these cells. The p44 MAPK apparently has a specific role in thymocyte development.

Mucosal immunity and tolerance: relevance to vaccine development.

The mucosal immune system of mammals consists of an integrated network of lymphoid cells which work in concert with innate host factors to promote host defense. Major mucosal effector immune mechanisms include secretory antibodies, largely of immunoglobulin A (IgA) isotype, cytotoxic T cells, as well as cytokines, chemokines and their receptors. Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination or natural immunization by a mucosal route can effectively induce immune suppression. The diverse compartments located in the aerodigestive and genitourinary tracts and exocrine glands communicate via preferential homing of lymphocytes and antigen-presenting cells. Mucosal administration of antigens may result in the concomitant expression of secretory immunoglobulin A (S-IgA) antibody responses in various mucosal tissues and secretions, and under certain conditions, in the suppression of immune responses. Thus, developing formulations based on efficient delivery of selected antigens/tolerogens, cytokines and adjuvants may impact on the design of future vaccines and of specific immunotherapeutic approaches against diseases associated with untoward immune responses, such as autoimmune disorders, allergic reactions, and tissue-damaging inflammatory reactions triggered by persistent microorganisms.

Functional and phenotypic analysis of thymic B cells: role in the induction of T cell negative selection.

The phenotype of mouse thymic B cells and their capacity to induce T cell negative selection in vitro were analyzed. Thymic B cells expressed B cell markers such as IgM, Fc gamma receptor, CD44, heat-stable antigen, LFA-1 and CD40. In addition, they were positive for the activation molecule CD69 and displayed high levels of B7-2. Although thymic B cells expressed CD5 on their surface, no CD5-specific mRNA was detected. Moreover, thymic B cells induced a stronger deletion of TCR-transgenic (TG) thymocytes than splenic B cells, which had low CD69 and B7-2 levels. Interestingly, CD40-activated splenic B cells up-regulated CD69 and B7-2 and acquired a capacity to induce T cell deletion comparable to that of thymic B cells. Moreover, thymic B cells from CD40-deficient mice displayed lower CD69 and B7-2 levels than control thymic B cells, and lower capacity to induce the deletion of TCR TG thymocytes. These results support the hypothesis that CD40-mediated activation of thymic B cells determines a high efficiency of antigen presentation, suggesting that within the thymus B cells may play an important role in the elimination of autoreactive thymocytes.

B cell response after MMTV infection: extrafollicular plasmablasts represent the main infected population and can transmit viral infection.

The immune response to mouse mammary tumor virus (MMTV) relies on the presentation of an MMTV-encoded superantigen by infected B cells to superantigen-specific T cells. The initial extrafollicular B cell differentiation involved the generation of B cells expressing low levels of B220. These B220low B cells corresponded to plasmablasts that expressed high levels of CD43 and syndecan-1 and were CD62 ligand- and IgD-. Viral DNA was detected nearly exclusively in these B220low B cells by PCR, and retroviral type-A particles were observed in their cytoplasm by electron microscopy. An MMTV transmission to the offspring was also achieved after transfer of B220low CD62 ligand- CD43+ plasmablasts into noninfected females. These data suggest that B220low plasmablasts, representing the bulk of infected B cells, are capable of sustaining viral replication and may be involved in the transmission of MMTV.

Definition of dendritic cell subpopulations present in the spleen, Peyer's patches, lymph nodes, and skin of the mouse.

Dendritic cells (DC) are highly efficient antigen-presenting cells (APC) that have an essential function in the development of immune responses against microbial pathogens and tumors. Although during the past few years our understanding of DC biology has remarkably increased, a precise characterization of the different DC subpopulations remains to be achieved with regard to their phenotype and lineage relationships. In this report, we have extensively studied the DC subpopulations present in the thymus, spleen, Peyer's patches, lymph nodes (LN) and skin of the mouse. Thymus DC and 60% spleen DC have a lymphoid DC phenotype, ie, CD8(+) DEC-205(high) Mac-1(low), whereas 40% spleen DC have a myeloid DC phenotype, ie, CD8(-) DEC-205(low) Mac-1(high). Both CD8(+) and CD8(-) DC are leukocyte function-associated antigen-1 (LFA-1)high and highly adherent. Within Peyer's patches the majority of DC correspond to the CD8(+) DEC-205(high) Mac-1(low) lymphoid category. In the LN, together with CD8(+) and CD8(-) DC, an additional nonadherent CD8(int) LFA-1(int) subpopulation with lymphoid DC characteristics is described. Finally, in the skin both epidermal Langerhans cells (LC) and dermal DC are CD8(-)DEC-205(high) Mac-1 (high), and do not express LFA-1. Interestingly, LC migration experiments indicate that LC underwent the upregulation of CD8 and LFA-1 upon migration to the LN, supporting the hypothesis that LC belong to the CD8(+) lymphoid lineage.

Viral superantigen-induced negative selection of TCR transgenic CD4+ CD8+ thymocytes depends on activation, but not proliferation.

T-cell negative selection, a process by which intrathymic immunological tolerance is induced, involves the apoptosis-mediated clonal deletion of potentially autoreactive T cells. Although different experimental approaches suggest that this process is triggered as the result of activation-mediated cell death, the signal transduction pathways underlying this process is not fully understood. In the present report we have used an in vitro system to analyze the cell activation and proliferation requirements for the deletion of viral superantigen (SAg)-reactive Vbeta8.1 T-cell receptor (TCR) transgenic (TG) thymocytes. Our results indicate that in vitro negative selection of viral SAg-reactive CD4+ CD8+ thymocytes is dependent on thymocyte activation but does not require the proliferation of the negatively signaled thymocytes.

In vitro negative selection of viral superantigen-reactive thymocytes by thymic dendritic cells.

Intrathymic expression of endogenous mouse mammary tumor virus (MMTV)-encoded superantigens (SAg) induces the clonal deletion of T cells bearing SAg-reactive T-cell receptor (TCR) Vbeta elements. However, the identity of the thymic antigen-presenting cells (APC) involved in the induction of SAg tolerance remains to be defined. We have analyzed the potential of dendritic cells (DC) to mediate the clonal deletion of Mtv-7-reactive TCR alphabeta P14 transgenic thymocytes in an in vitro assay. Our results show that both thymic and splenic DC induced the deletion of TCR transgenic double positive (DP) thymocytes. DC appear to be more efficient than splenic B cells as negatively selecting APC in this experimental system. Interestingly, thymic and splenic DC display a differential ability to induce CD4+ SP thymocyte proliferation. These observations suggest that thymic DC may have an important role in the induction of SAg tolerance in vivo.

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.