Characterization of exosome subpopulations from RBL-2H3 cells using fluorescent lipids.

Fluorescent lipid probes were used to track lipid trafficking between parent RBL cells and exosomes. We have checked the intracellular labeling of exosomes ("in vivo labeling") from parent cell incubated with either Bodipy-Cer, Bodipy-PC, or NBD-PC. Bodipy-PC labeled equally cells and exosomes, whereas Bodipy-Cer, a Golgi marker, was enriched in exosomes. Golgi membranes participated effectively in exosome biogenesis since cell incubation with brefeldin A leads to a modified phospholipid/protein ratio in exosomes. At the opposite, NBD-PC, a plasma membrane marker weakly labeled exosome membranes. Sorting of subpopulations indicated that the MHC-II containing exosomes were enriched in Bodipy-PC, whereas tetraspanin(CD 63 or CD81)-containing exosomes are essentially labeled with Bodipy-Cer and Bodipy-PC. These results indicated that RBL released two main subpopulations of exosomes that can be discriminated by their protein and lipid contents. When the bulk of exosomes was labeled after their purification ("in vitro labeling") with either of the above-mentioned lipid probes, the Bodipy-Cer was the only one to incorporate noticeably in all the subpopulations, indicating that the previous results obtained during "in vivo labeling" monitored real intracellular lipid trafficking between organelles and exosomes. Bodipy-Cer was further used as a tool to measure the respective amounts of each subpopulations. CD63, MHC II, and CD81-containing exosomes accounted for 47%, 32%, and 21%, respectively, of total exosomes.

Fcgamma receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization.

Dendritic cells (DCs) express several receptors for the Fc portion of immunoglobulin (Ig)G (FcgammaR), which mediate internalization of antigen-IgG complexes (immune complexes, ICs) and promote efficient major histocompatibility complex (MHC) class II-restricted antigen presentation. We now show that FcgammaRs have two additional specific attributes in murine DCs: the induction of DC maturation and the promotion of efficient MHC class I-restricted presentation of peptides from exogenous, IgG-complexed antigens. Both FcgammaR functions require the FcgammaR-associated gamma chain. FcgammaR-mediated MHC class I-restricted antigen presentation is extremely sensitive and specific to immature DCs. It requires proteasomal degradation and is dependent on functional peptide transporter associated with antigen processing, TAP1-TAP2. By promoting DC maturation and presentation on both MHC class I and II molecules, ICs should efficiently sensitize DCs for priming of both CD4(+) helper and CD8(+) cytotoxic T lymphocytes in vivo.

Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-alpha subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation.

Stimulation of CD4(+) helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-alpha/Ig-beta heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-alpha-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-alpha cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide- MHC class II complexes through antigen targeting by BCR subunits.

syk protein tyrosine kinase regulates Fc receptor gamma-chain-mediated transport to lysosomes.

B- and T-cell receptors, as well as most Fc receptors (FcR), are part of a large family of membrane proteins named immunoreceptors and are expressed on all cells of the immune system. Immunoreceptors' biological functions rely on two of their fundamental attributes: signal transduction and internalization. The signals required for these two functions are present in the chains associated with immunoreceptors, within conserved amino acid motifs called immunoreceptor tyrosine-based activation motifs (ITAMs). We have examined the role of the protein tyrosine kinase (PTK) syk, a critical effector of immunoreceptor-mediated cell signalling through ITAMs, in FcR-associated gamma-chain internalization and lysosomal targeting. A point mutation in the immunoreceptor-associated gamma-chain ITAM affecting syk activation, as well as overexpression of a syk dominant negative mutant, inhibited signal transduction without affecting receptor coated-pit localization or internalization. In contrast, blocking of gamma-chain-mediated syk activation impaired FcR transport from endosomes to lysosomes and selectively inhibited the presentation of certain T-cell epitopes. Therefore, activation of the PTK syk is dispensable for receptor internalization, but necessary for cell signalling and for gamma-chain-mediated FcR delivery to lysosomes.

Type II and III receptors for immunoglobulin G (IgG) control the presentation of different T cell epitopes from single IgG-complexed antigens.

T cell receptors on CD4(+) lymphocytes recognize antigen-derived peptides presented by major histocompatibility complex (MHC) class II molecules. A very limited set of peptides among those that may potentially bind MHC class II is actually presented to T lymphocytes. We here examine the role of two receptors mediating antigen internalization by antigen presenting cells, type IIb2 and type III receptors for IgG (FcgammaRIIb2 and FcgammaRIII, respectively), in the selection of peptides for presentation to T lymphocytes. B lymphoma cells expressing recombinant FcgammaRIIb2 or FcgammaRIII were used to assess the presentation of several epitopes from two different antigens. 4 out of the 11 epitopes tested were efficiently presented after antigen internalization through FcgammaRIIb2 and FcgammaRIII. In contrast, the 7 other epitopes were efficiently presented only when antigens were internalized through FcgammaRIII, but not through FcgammaRIIb2. The capacity to present these latter epitopes was transferred to a tail-less FcgammaRIIb2 by addition of the FcgammaRIII-associated gamma chain cytoplasmic tail. Mutation of a single leucine residue at position 35 of the gamma chain cytoplasmic tail resulted in the selective loss of presentation of these epitopes. Therefore, the nature of the receptor that mediates internalization determines the selection of epitopes presented to T lymphocytes within single protein antigens.

New evidence for two MHC class II-restricted antigen presentation pathways by overexpression of a small G protein.

Exogenous Ags may be presented by MHC class II molecules through two distinct pathways distinguished by their sensitivity to drugs that inhibit the protein synthesis. Using this approach, we previously showed that the subunits Ig-alpha and Ig-beta, associated to B cell Ag receptor, targeted Ags either to newly synthesized or to preexisting pools of MHC class II molecules, respectively. To further characterize these two Ag presentation pathways, we altered the intra-Golgi transport of newly synthesized MHC class II by stably overexpressing, in B cells, mutants of a small G protein involved in the intra-Golgi transport, Rab6. Overexpression of GTP-bound rab6 (Q72L) mutant proteins reduced the cell surface arrival of MHC class II molecules and consequently slowed down Ag presentation dependent upon newly synthesized class II molecules. In contrast, this mutant had no effect on Ag presentation dependent upon preexisting pools of class II molecules, and the overexpression of an inactive GDP-bound form of rab6 (T27N) did not affect any Ag presentation pathway. MHC class II-restricted Ag presentation pathways can therefore be distinguished by their sensitivity to the overexpression of proteins modifying the intracellular transport of newly synthesized class II molecules.

Role of B cell receptor Ig alpha and Ig beta subunits in MHC class II-restricted antigen presentation.

The ability of the B cell antigen receptors (BCRs) to enhance MHC class II-restricted antigen presentation was ascribed to mig-associated Ig alpha/Ig beta heterodimers. The relative role of Ig alpha and Ig beta subunits in antigen presentation was investigated by fusing their cytoplasmic tails to the extracellular and transmembrane domains of Fc receptors. Ig alpha and Ig beta chimera mediate antigen internalization and increase the efficiency of antigen presentation, but they drive antigens to different endosomal compartments. Furthermore, antigens internalized by either chimera are degraded and presented with different kinetics. The cytoplasmic tail of Ig alpha targets antigen towards a major population of newly synthesized MHC class II located in class II-rich compartments. In contrast, Ig beta targets antigen towards a minor population of recycling MHC class II molecules, located in transferrin receptor-containing endosomes. Altogether, our data indicate that the composition of BCR could be therefore an important way to modulate the immune response.

Vaccination against spontaneous abortion in mice by preimmunization with an anti-idiotypic antibody.

CBA/J females mated with DBA/2 display a high level of fetal wastage which can be corrected by anti-Balb/c vaccination. A batch of CBA/J anti-(CBA/J anti-Balb/c) antiserum was raised. This serum was characterized as anti-idiotypic by various techniques, including a solid-phase radioimmunoassay. Such a serum proved to confer protection against resorptions when injected into CBA/J mice mated with DBA/2. However, the kinetics of the effect pointed to the need for administration in early pregnancy for successful protection. The significance of these data, and the possible mechanism(s) by which the serum acts, are discussed.

Immunoactive products of placenta. V: Soluble factors from murine placenta can block effector stages of maternal antipaternal cell-mediated immunity.

Supernatants from short-term cultures of placental or trophoblast-enriched cell suspensions derived from 14-17-day isopregnant mice display suppressive activity on NK lysis in vitro. The soluble factor is produced by trypsin-sensitive cells and requires protein synthesis. Its activity is destroyed by treatment with insoluble trypsin. The suppression is not strain restricted, but appears species-restricted. The factor acts at the level of the NK effectors themselves. Furthermore, such supernatants also are able to inhibit CTL-mediated lysis at the effector stage, in an MHC nonrestricted, nonspecific fashion. The effect is not seen with supernatants from control fetal tissues. At least two mechanisms could be involved: inhibition of homing toward allogeneic targets, and a direct effect on effector cell lytic action. These factors could play an important role in protecting the placenta from the deleterious effects of maternal antipaternal immunity and could explain the survival of the fetus in a presensitized maternal host.

Immunoactive products of placenta: VIII. Translation products of messenger RNA extracted from murine placenta or A6B9IC5 teratocarcinoma are immunosuppressive in vitro.

RNA was extracted from placentae of synpregnant C3H mice (day 14 of gestation) and from A6B9IC5 teratocarcinoma. After isolation of poly-(A)+ RNA on an oligo d(T) cellulose column, putative messenger RNA was injected into Xenopus laevi oocytes. Supernatants and homogenates from the oocytes were assayed for immunoregulatory activity in MLR, CML, and NK assays and found to be immunosuppressive. These data indicate that immunomodulators could be obtained from the placenta by molecular biology technology in sufficient quantities for eventual clinical applications.