FcgammaRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection.

The high-affinity receptor for IgG, FcgammaRI, shares its capacity to bind IgG2a immune complexes (IgG2a-IC) with the low-affinity receptor FcgammaRIII and complement factors, hampering the definition of its biological role. Moreover, in vivo, FcgammaRI is occupied by monomeric IgG2a, reducing its accessibility to newly formed IgG2a-IC. By using a variety of FcgammaR(-/-) mice, we demonstrate that in the absence of FcgammaRI, the IgG2a-IC-induced cellular processes of phagocytosis, cytokine release, cellular cytotoxicity, and antigen presentation are impaired. FcgammaRI(-/-) mice showed impaired hypersensitivity responses, strongly reduced cartilage destruction in an arthritis model, and impaired protection from a bacterial infection. We conclude that FcgammaRI contributes substantially to a variety of IgG2a-IC-dependent immune functions and immunopathological responses.

In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity.

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8(+) cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8(+) class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8(+) T-cell epitopes, bound to 1-microm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503-7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4(+) T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.

Immune responses induced by recombinant BCG strains according to level of production of a foreign antigen: malE.

A variety of viral, bacterial and parasitic antigens have been expressed in BCG and the capacity of these recombinant bacteria to induce immune responses has been well documented. However, little is known about the parameters influencing the induction of immune responses by recombinant BCG (rBCG), such as level of production and localization of the recombinant antigen. In the present study, we have constructed several rBCG strains expressing the malE gene from Escherichia coli which is either secreted or targeted to the cytoplasm or plasma membrane. Expression of malE was quantified by ELISA and localization was analyzed by flow cytometry. Even when using the same promoter, levels of cytoplasmic or membrane MalE production were far less than those from secreting strains using either mycobacterial or E. coli secretion signals. Stronger and more rapid immune responses were induced by rBCG strains with the highest levels of secreted MalE compared to cytoplasmic or membrane constructs, including both good humoral and proliferative responses in BALB/c, C57BL6 and even C3H mice, previously shown to be poor MalE responders. These results suggest that the levels of foreign antigen production play an important role in the induction of immune responses by rBCG strains.

mAb against hen egg-white lysozyme regulate its presentation to CD4(+) T cells.

Specific antibodies increase antigen uptake and presentation by antigen-presenting cells via the B cell receptor in B cells or FcgammaR in dendritic cells. To determine whether the interaction between antibody and antigen could influence the set of peptides presented by MHC II molecules, we analyzed the presentation of different CD4(+) T cell epitopes of hen egg-white lysozyme (HEL) after the capture of immune complexes formed between HEL and seven different specific mAb. The 103-117 T cell epitope (I-E(d)) was specifically and selectively up-regulated by the D1.3 and F9.13.7 mAb that binds to proximal loops in the native structure of HEL. Furthermore, Ii-independent T cell epitopes exposed on the HEL surface (116-129 and 34-45, I-A(k) restricted) which require a mild processing involving the recycling of MHC II molecules were selectively up-regulated by mAb that overlap those T cell epitopes (D1.3 and D44.1). However, F10.6.6, somatically derived from the same germ line genes as D44.1 and exhibiting an higher affinity for HEL, was without effect on the presentation of the 34-45 epitope. An Ii-dependent T cell epitope buried into the tertiary structure of HEL (45-61, I-A(k) restricted) and requiring the neosynthesis of MHC II was up-regulated by high-affinity mAb recognizing epitopes located at the N- or C-terminus of the T cell epitope. These results strongly suggest that (i) the spatial relationship linking the T cell epitope and the B cell epitope recognized by the mAb, (ii) the intrinsic processing requirements of the T cell epitope, and (iii) the antibody affinity influences the presentation of a given T cell epitope.

Engineering parvovirus-like particles for the induction of B-cell, CD4(+) and CTL responses.

An antigen delivery system based on hybrid recombinant parvovirus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine (PPV) or canine parvovirus (CPV) expressed in insect cells with the baculovirus system has been developed. PPV:VLPs containing a CD8(+) epitope from the LCMV nucleoprotein evoked a potent CTL response and were able to protect mice against a lethal infection with the virus. Also, PPV:VLPs containing the C3:T epitope from poliovirus elicited a CD4(+)3 log(10) units) against poliovirus. The possibility of combining different types of epitopes in different positions of a single particle to stimulate different branches of the immune system paves the way to the production of more potent vaccines in a simple and cheap way.

Role of HLA-G versus HLA-E on NK function: HLA-G is able to inhibit NK cytolysis by itself.

Recent studies have shown that endogenous HLA-E molecules are stabilized on the cell surface upon the expression of HLA-G which contains within its leader sequence, a nonapeptide capable of binding with the HLA-E/beta2m complex. Since HLA-E was found to be the major ligand for the CD94/NKG2A inhibitory receptor, we determined the role of HLA-G versus HLA-E on NK lysis inhibition. We showed that K562 cells transfected with HLA-G1 cDNA are protected from NK lysis by direct interaction between HLA-G1 and killing inhibitory receptor(s). This NK lysis inhibition is not dependent on HLA-E expression, since no HLA-E protein was detected on K562 cells; HLA-G1 is therefore able to inhibit NK lysis by itself.

HLA-G inhibits the allogeneic proliferative response.

HLA-G is a non-classical MHC class I molecule expressed at the feto/maternal interface where it plays a role in materno-fetal tolerance by inhibiting NK cells. Expression of killing inhibitory receptors capable of interacting with HLA-G on T lymphocytes led us to hypothesize that HLA-G molecules could also modulate T cell responses, analyzed here in the context of the allogeneic proliferative response. Using LCL-HLA-G transfectants as stimulators of T cells present among peripheral mononuclear cells and K562-HLA-G1 transfectants as inhibitors in a classical mixed lymphocyte reaction, we showed that HLA-G is able to inhibit T cell allo-proliferation. These findings provide new insight into the role of HLA-G in preventing allograft rejection.

HLA-G-mediated inhibition of antigen-specific cytotoxic T lymphocytes.

In the present study, we demonstrate that the non-classical MHC class I molecule HLA-G impairs specific cytolytic T cell functions in addition to its well-established inhibition of NK lysis. The antigen-specific cytotoxic T lymphocyte (CTL) response analyzed was mediated by CD8(+) T cells specific for the influenza virus matrix epitope, M58-66, presented by HLA-A2. The transfection of HLA-G1 cDNA in target cells carrying the M58-66 epitope reduced their lysis by these virus-specific CTL. This HLA-G-mediated inhibition of antigen-specific CTL lysis was (i) peptide dose dependent, (ii) reversed by blocking HLA-G with a specific mAb and (iii) still observed despite the blockade of HLA-E/CD94/NKG2A interaction. By inhibiting both CTL and NK functions, HLA-G appears to have an extensive role in immune tolerance.

Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses.

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.

Activation of B cells by 1 microm particulate lysozyme or peptides: a Th-dependent pathway requiring CD40-CD40 ligand interaction.

Many antigens encountered by the immune system are included in complex structures such as bacteria or parasites. We previously developed an in vivo model to study the immunogenicity of particulate antigens, based on covalent linkage of proteins or peptides to 1 microm latex particles and showed that these antigens cannot be presented to MHC class II-restricted specific T cells by B cells. However, they induce strong CD4+ T cell responses when injected to mice without adjuvant. The present study demonstrates that four out of the five proteins tested did not stimulate antibody synthesis when linked to 1 microm microparticles, although a strong IgG production was induced by the same proteins administered in soluble form with adjuvant. In contrast, lysozyme and two synthetic peptides containing B and T cell viral epitopes induced strong and long-lasting specific antibody responses when linked to 1 micrometer synthetic beads. The isotypic pattern of antibodies induced by particulate lysozyme was similar to that induced by the soluble protein in alum. Studies using CD4+ T cell-depleted mice revealed that the induction of antibodies by particulate lysozyme strictly required Th cell activity. Moreover, the T-B cell cooperation involved in B cell activation by antigens linked to beads required CD40-CD40 ligand interaction. Thus, these particulate antigens provide a useful tool to study the mechanisms of induction of antibody response against complex bacterial or parasitic antigens. Moreover, they may represent attractive candidates to elaborate efficient new vaccines using short synthetic peptides.