A genital tract peptide epitope vaccine targeting TLR-2 efficiently induces local and systemic CD8+ T cells and protects against herpes simplex virus type 2 challenge.

The next generation of needle-free mucosal vaccines is being rationally designed according to rules that govern the way in which the epitopes are recognized by and stimulate the genital mucosal immune system. We hypothesized that synthetic peptide epitopes extended with an agonist of Toll-like receptor 2 (TLR-2), that are abundantly expressed by dendritic and epithelial cells of the vaginal mucosa, would lead to induction of protective immunity against genital herpes. To test this hypothesis, we intravaginally (IVAG) immunized wild-type B6, TLR-2 (TLR2(-/-)) or myeloid differentiation factor 88 deficient (MyD88(-/-)) mice with a herpes simplex virus type 2 (HSV-2) CD8+ T-cell peptide epitope extended by a palmitic acid moiety (a TLR-2 agonist). IVAG delivery of the lipopeptide generated HSV-2-specific memory CD8+ cytotoxic T cells both locally in the genital tract draining lymph nodes and systemically in the spleen. Moreover, lipopeptide-immunized TLR2(-/-) and MyD88(-/-) mice developed significantly less HSV-specific CD8+ T-cell response, earlier death, faster disease progression, and higher vaginal HSV-2 titers compared to lipopeptide-immunized wild-type B6 mice. IVAG immunization with self-adjuvanting lipid-tailed peptides appears to be a novel mucosal vaccine approach, which has attractive practical and immunological features.

Advances in type I diabetes associated tolerance mechanisms.

Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells by autoreactive T cells. The polygenic trait for T1D risk implicates many genes that have an impact on fundamental immunological processes such as central and peripheral tolerance. Several pieces of evidence have suggested that many of the genetic loci that are directly linked to type 1 diabetes susceptibility modulate the generation and/or the activation of autoreactive T-lymphocytes. We and others have proposed a critical role for medullary thymic epithelial cells (mTEC) forming the Hassall's corpuscles in T-cell tolerance. Indeed, mTEC have been found to express promiscuous self-antigens, used directly or through thymic dendritic cells to drive either negative selection of insulin-reacting precursors or their differentiation into naturally occurring regulatory Foxp3+ CD4+ CD25+ T cells. In the periphery, naturally occurring Foxp3+ CD4+ CD25+regulatory T (Treg) cells represent the master cells in dominant peripheral T-cell tolerance. The development and function of Treg cells are ultimately linked to IL-2 and Foxp3 expression. This review addresses recent literature and emerging concepts of central and peripheral T-cell tolerance with regards to T1D.

B-Cell suppression in adult mice injected with anti-delta followed by anti-mu mAb.

Cross-linking of surface IgM or IgD B-cell receptors (BCR) with appropriate anti-Ig antibodies induces IgM(high) or IgD(high) B-cell depletion, respectively. The aim of this paper is to analyze how injections of anti-delta followed by anti-mu monoclonal antibodies (mAb) can deplete and suppress B cells and then induce T-independent type 2 antigen tolerance in adult mice even after treatment is stopped. The experimental protocol consisted of three daily injections of anti-delta mAb followed by repeated injections of anti-mu mAb. It shows that a sequential injection of anti-delta and anti-mu mAb induces B-cell depletion and T-independent type 2 response downregulation. Morever, the T-dependent response is maintained, except for the IgG3 isotype. After clearance of the anti-delta mAb from the circulation, B cells reappear as an IgD(+) IgM(-) B-cell population in the bone marrow (BM) and spleen. The origin of IgD(+) IgM(-) cells was studied in scid mouse transfer models. We show that IgD(+) IgM(-) B cells are not mature cells reexpressing sIgD but BM-derived cells that require a T-cell presence to be developed. The lack of sIgM expression by posttranscriptional regulation and the need of T-cell help for escaping anti-mu negative selection suggest strongly that this population had properties similar to those of anergized B cells. These results support the potential use of sequential injections of anti-delta and anti-mu in the prevention of xenograft rejection.

The experimental (in vitro) and clinical (in vivo) immunosuppressive effects of a rat IgG2b anti-human CD2 mAb, LO-CD2a/BTI-322.

BACKGROUND: CD2 is a cell surface glycoprotein expressed on most human T cells and natural killer (NK) cells, working as a cell adhesion and costimulatory molecule. The aim of this paper is to analyze the mechanism of action of a rat IgG2b anti-human CD2 monoclonal antibody (mAb) (LO-CD2a/BTI-322 mAb), which is a potent immunosuppressive agent and inducer of cell death. In vivo, this mAb is able to prevent or treat kidney allograft rejection. METHODS: The mechanisms by which the LO-CD2a/BTI-322 mAb is able to induce inhibition of cell activation and cell death were analyzed by mixed lymphocyte reactions and by flow cytometry. After in vivo treatment, levels of circulating mAb were measured by ELISA as well as anti-rat immunization and cytokine release. RESULTS: We show that the inhibition of cell activation induced by LO-CD2a/BTI-322 mAb after allogeneic or OKT3 stimulation is due to an Fcgamma receptor-dependent CD2 down-modulation and to T-cell depletion through an antibody-dependent cell-mediated cytotoxicity mechanism mediated by NK cells or activated monocytes. Peripheral T- and NK-cell depletion was observed after in vivo treatment with LO-CD2a/BTI322. Cytokine release (TNFalpha) was correlated with some side effects, but only after the first injection, and the effects were never severe or life threatening. CONCLUSION: The correlation between the in vitro and in vivo data suggests that T-cell depletion, especially of activated cells, and inhibition of cell activation after CD2 down-modulation are the main mechanisms of action of the LO-CD2a/BTI-322 mAb.

Differential effects of injections of anti-mu and anti-delta monoclonal antibodies on B-cell populations in adult mice: regulation of xenoreactive natural antibody-producing cells.

BACKGROUND: The depletion of differential B cell and xenoreactive natural antibodies (XNA) by anti-delta and anti-mu injections was analyzed in adult mice. Sequential treatment with anti-delta and then anti-mu induces a complete depletion of B cells and XNA and represents a potential approach to induce xenograft tolerance. METHODS: Adult mice were injected with anti-mu, anti-delta, anti-delta then anti-mu, or control isotype monoclonal antibodies from day 0 to day 14. The different B-cell populations were analyzed by FACS and immunohistology. Ig production was tested by ELISA. XNA were analyzed by FACS. RESULTS: Anti-mu injections induced a depletion of IgMhigh, immature B cells, marginal zone B cells, and B1 cells and an increase of IgG-XNA production. Anti-delta injections induced mature conventional IgDhigh B-cell depletion and increased IgM-XNA production. Interestingly, sequential injections of anti-delta then anti-mu induced a depletion of immature B cells, mature B cells (MZ, B2, and B1), and XNA. CONCLUSIONS: These results demonstrate that mature B-cell depletion in adult mice can be obtained by mAb injections and depends on the surface immunoglobulin cross-linking threshold. Indeed, anti-mu mAb depleted IgMhigh B cells (MZ and B1) and anti-delta, IgDhigh B cells (B2). The differential B-cell suppression shows that conventional B cells are responsible in the IgG-XNA production and MZ and B1 cells in the IgM-XNA production. Sequential repeated injections of anti-delta then anti-mu mAb depleted all B-cell populations and suppressed the whole XNA production.

Apoptosis of human naive NK cells mediated by a rat IgG2b anti CD2 mAb through a fractricidal ADCC reaction.

LO-CD2a/BTI-322, a rat anti human CD2 mAb, shows in vitro and in vivo immunosuppressive properties and induces T-cell depletion resulting partially from an antibody dependent cellular cytotoxicity (ADCC) mediated by NK cells. The aim of this paper is to study the in vitro effect of LO-CD2a/BTI-322 on NK cells, the majority of them also expressing the CD2 molecule. The addition of the mAb to purified naive NK cells induces apoptosis of CD2+ cells. The apoptosis is rapid, Fas ligand independent and completely inhibited by the calcium chelator EGTA, suggesting a fractricidal ADCC reaction and implying that NK cells are not resistant to lysis when used as target cells. At the end of the reaction, the CD2 - remaining cells are still capable of natural cytotoxicity against K562 cells, but at a lower rate than untreated cells.

Differential inhibition of B-cell development and xenoreactive natural antibody production by administration of anti-mu or anti-delta monoclonal antibodies in adult rats.

BACKGROUND: Given the role of xenoreactive natural antibodies (XNA) in the pathogenesis of xenograft rejection, we tested whether the administration of anti-mu or anti-delta monoclonal antibodies (mAbs) in adult rats would suppress the generation of XNA. METHODS: Adult LOU/C (Igkappa-1a) rats were treated with anti-mu or anti-delta mAbs after nonlethal total body irradiation and bone marrow transplantation from congenic LOU/C (Igkappa-1b) rats. The differentiation of donor bone marrow (BM)-driven Igkappa-1b+ B cells and XNA production were analyzed. RESULTS: Both anti-mu and anti-delta mAbs arrested B-cell differentiation in the BM. In anti-mu-treated rats, there was a total depletion of donor-driven, peripheral Igkappa-1b+ B cells, secreting cells, and circulating XNA of the Igkappa-1b allotype. In anti-delta-treated rats, a significant number of Igkappa-1b+ B cells, which did not express membrane IgD, "escaped" deletion and partially repopulated peripheral lymphoid organs. This B-cell population was active in the production of XNA, as revealed by the high serum levels of XNA in these animals. CONCLUSIONS: Anti-mu administration resulted in arrest of B-cell differentiation and in down-regulation of IgM and IgG XNA production in adult rats. These data suggest that the use of anti-mu mAbs may be a useful approach to suppress the production of XNA and prevent xenograft rejection. Furthermore, we suggest that the B-cell population responsible for the production of XNA in adult rats belongs to a B-cell lineage expressing low levels of membrane IgD and "escaping" deletion in the BM upon anti-delta treatment.

Glucocorticoids down-regulate dendritic cell function in vitro and in vivo.

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex-restricted presentation of the antigen and an additional co-stimulatory signal provided by the antigen-presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen-specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively down-regulates the expression of co-stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.