Responsiveness of T Cells to Mutant Major Histocompatibility Complex Class I Antigen

Proliferative response of splenic T cells of C57BL/6 mice to mutant major histocompatibility complex (MHC) class I antigen (H-2Kbm1) was examined with regard to the role of accessory cells. T cell proliferation in mixed lymphocyte culture (MLC) was not induced when accessory cells were removed from stimulator spleen cells by passage through Sephadex G-10 or nylon-wool column. Anti-Iab antibodies did not inhibit the proliferative response to class I antigen, whereas the same antibodies completely blocked the response to class II antigen (Iabm12). Accessory cells may not be mere presenters of MHC class I antigen because stimulator cells fixed with 0.05% paraformaldehyde lost the stimulating function. The proliferative response was partially recovered by the addition of recombinant interleukin 1 (IL-1) and/or IL-2 to MLC devoid of stimulator type accessory cells. It is concluded that stimulatory type accessory cells were obligatorily involved in the T cell proliferation, and the production of IL-1 by accessory cells is thought to play a critical role in this process.     

Identification of αβ and γδ T Cell Receptor-Positive Cells

Two lineages of T lymphocytes bearing the CD3 antigen can be defined on the basis of the nature of the heterodimeric receptor chain (alpha beta or gamma delta T cell receptor (TCR) expressed. Precise identification of alpha beta and gamma delta TCR+ cells is essential when studying the tissue distribution and function of these different T cells. In immunofluorescence studies gamma delta TCR+ cells have been identified as CD3+WT-31- or CD3+CD4-CD8- cells. However, this may not be the optimal procedure because gamma delta TCR+ cells are weakly WT-31+, and some are CD8+. The aim of this study was to evaluate a panel of monoclonal antibodies (MoAb) directed against different chains of the TCR-T3 complex for a more precise identification of alpha beta+ and gamma delta TCR+ cells in flow cytometric studies. We found that the MoAb anti-Ti-gamma A and delta-TCS-1, recognizing the TCR-gamma and the TCR-delta chain respectively, only reacted with a subpopulation of gamma delta TCR+ cells, whereas another TCR-delta chain recognizing MoAb anti-TCR-delta 1 reacted with all gamma delta TCR+ cells. All MoAb reported to belong to the CD3 group reacted with both alpha beta TCR+ and gamma delta TCR+ cells as expected. Our results indicate that all gamma delta TCR+ cells can be identified with the MoAb anti-TCR-delta 1. Because no MoAb recognizing the TCR-alpha or TCR-beta chains at the cell surface of intact cells are yet available, we suggest that alpha beta TCR+ cells could be identified as CD3+ anti-TCR-delta 1-cells.     

Selection for Polymorphism in the Antigen Recognition Site of Major Histocompatibility Complex Class II Molecules

The genetic basis for the extensive polymorphism of major histocompatibility complex (MHC) class II molecules was investigated by statistical analysis. Nucleotide sequences of human DQA1, DQB1, DRB1, and DRB3 genes and murine A alpha, A beta, and E beta genes were used. The results show that polymorphism is selected for in the antigen recognition site of class II molecules since replacement substitutions in this region were found to occur at a significantly higher frequency than expected in the absence of selection. In contrast, replacement substitutions are selected against in the remaining part of the first domain exon and in the second domain exon. Furthermore, comparing the sequence variability pattern among different class II alpha and beta sequences, using a variability index for each residue, showed that, with few exceptions, highly polymorphic residues occur in the antigen recognition site. There was a strong and highly significant correlation in the variability pattern in the homologous DRB/E beta sequences but not for DQB/A beta or DQA/A alpha sequences. This difference may be related to the fact that both alpha and beta chains of DQ/A molecules are polymorphic, while only beta chains of DR/E molecules vary.     

T Cells are Unresponsive to Transgenic MHC Class II Molecules Expressed on Pancreatic β Cells

It has been proposed that the autoimmune attack on the pancreatic β cells leading to insulin-dependent diabetes mellitus can be caused by the expression of MHC class II molecules on the β cells. Transgenic mice expressing normal levels of allogeneic MHC class II A^k on the β-cell surface (IP-A^k) do not develop either insulitis or diabetes, yet these mice are not tolerant to A^k when expressed on normal antigen-presenting cells. The authors have stimulated T cells from IP-A^k mice in vitro with A^k-expressing β cells. Mice were also primed in vivo in order to facilitate the antiallogeneic response. The authors found that neither IP-A^k positive nor IP-A^k negative mice were able to respond to A^k-expressing β cells, and that in vivo priming does not overcome this inability. They suggest that β cells do not act as antigen-presenting cells, probably due to inability of delivering costimulatory signals. This strengthens the notion that MHC class II expression per se is not sufficient to induce an autoimmune attack on the β cells.     

Protection against Ascending Infection of the Genital Tract by Chlamydia trachomatis Is Associated with Recruitment of Major Histocompatibility Complex Class II Antigen-Presenting Cells into Uterine Tissue

A mouse model of ascending infection following intravaginal inoculation with a strain of Chlamydia trachomatis isolated from humans has been used to identify immune mechanisms associated with protection against genital infection. BALB/c and C3H mice differed in their susceptibilities to infection and inflammatory disease. In both mouse strains, ascension of the organism and recruitment of bone marrow-derived mononuclear leukocytes were evident in uterine tissue 1 week postinfection. By 3 weeks the organism had been cleared and inflammation had been resolved in the BALB/c mice, but both persisted in the C3H animals. In athymic nude BALB/c mice both the organism and inflammation persisted, indicating the influence of the hosts’ immune response on the outcome of infection. Both BALB/c and C3H mice had a Th1 response in draining lymph nodes, with predominant production of gamma interferon and tumor necrosis factor alpha, low levels of interleukin-10, and no detectable levels of interleukin-4. However, the composition of the early uterine infiltrate differed in these two mouse strains. Cell surface labeling and analysis of light scatter properties by flow cytometry identified a population of large, CD45⁺ major histocompatibility complex class II mononuclear cells, which were a prominent feature of the infiltrates in BALB/c mice but were present in significantly lower numbers in C3H mice. These cells expressed the costimulatory molecules CD86 and CD40 and stimulated allogeneic T cells, suggesting that these mononuclear cells are a population of antigen-presenting cells and that they may play a role in clearing antigen and protecting against inflammatory disease in BALB/c mice. An additional level of immunological control may thus exist in genital chlamydial infection.Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which selectively colonizes epithelial cells in the human host. Infection of the genital tract with C. trachomatis serovars D through K is a major cause of sexually transmitted disease worldwide. Infection is insidious and, though often asymptomatic, can have serious consequences particularly for women. In some cases of cervical infection the organism ascends into the upper genital tract; this is a major cause of pelvic inflammatory disease with sequelae that include infertility and ectopic pregnancy (21).Left untreated, genital chlamydial infections are chronic, and repeated infections are common, indicating that the natural immune response is poorly protective. However, the incidence of genital chlamydial infection falls with increasing age; this might be due to cumulative serovar-specific immunity mediated by a local antibody (3). Antibodies can neutralize infectivity in vitro and in vivo (40) but have not been identified as the dominant protective mechanism in animal studies; antibodies play little part in protection against primary infections (12, 26, 31), although they can protect against severe pathology (7) and can play a subsidiary role in defense against reinfection (31).A predominant role for Th1 CD4⁺ T cells and the production of gamma interferon (IFN-γ) in controlling primary genital infection and preventing spread to other tissues has been implicated in cell transfer studies, antibody-mediated depletion experiments, and infections in gene knockout mice (8, 12, 15, 19, 30). However, in the absence of a functioning IFN-γ system a poorly defined compensatory mechanism can operate (12, 37) and a mechanism dependent on interleukin-12 (IL-12) but independent of IFN-γ may thus be important in the early stages of infection (25). IFN-γ is less important in protection against reinfection than against a primary challenge (8, 37). Thus, a successful immune response against chlamydial infection is flexible and complex, with different mechanisms involved as the infection progresses.Studies of infection in mouse strains with different susceptibilities to disease have proved useful in identifying protective immune mechanisms. We have identified mouse strain differences in disease susceptibility following intrauterine (i.u.) injection with a serovar-F strain of C. trachomatis from humans; C3H mice developed severe disease with prolonged salpingitis resulting in infertility, whereas BALB/c suffered less-severe inflammatory changes and remained fertile (32). Because direct injection of the organism into the upper genital tract did not allow us to distinguish between increased susceptibility to infection and sensitivity to pathological reactions, we developed a model of ascending infection following intravaginal (i.vag.) inoculation of this strain of C. trachomatis. Ascension of the organism into the uterus and oviducts of C3H mice was detected in association with inflammatory changes in genital tract tissue (29).In the present study we compare ascending infections in C3H and BALB/c mice and report increased susceptibility in C3H mice despite the development of a predominantly Th1 response and the production of IFN-γ in the lymph nodes of both mouse strains. Instead, clearance of the organism and protection against inflammatory disease appeared to be associated with recruitment of major histocompatibility complex (MHC) class II antigen-presenting cells (APC) into uterine tissue early in infection. These cells may possibly play a role in the defense against genital chlamydial infection.     

Differential Expression of Major Histocompatibility Complex Class II Variants in Cells Infiltrating the Meth A Sarcoma

CD4+ T cells are involved in immune responses against the Meth A sarcoma and infiltrate tumours arising from Meth A cells inoculated intradermally in (BALB/c x C57BL/6)F1 (H-2d/b) mice. In order to investigate whether the prerequisites for tumour antigen presentation to CD4+ T cells are fulfilled within the malignant lesion, expression of class II major histocompatibility complex (MHC) molecules and the costimulatory ligand B7 were examined. The I-Ab and I-Ed allomorphs were abundantly expressed by virtually all B cells and 50% of macrophages infiltrating the tumours. In striking contrast, the I-Ad variant was hardly detectable. This pattern of class II expression appeared to be unique for the tumour microenvironment. Thus the proportion of I-Ad+ spleen B cells and peritoneal macrophages did not significantly differ from the proportion expressing I-Ab and I-Ed, and these extratumoral I-Ad+ cells stained as brightly as did cells from healthy mice. Expression of B7 was weak by tumour-infiltrating cells. The profound capacity of the Meth A sarcoma to confer low local I-Ad and B7 expression might preclude T-cell-dependent anti-tumour immune responses and thus promote survival of malignant cells. Whereas MHC class II genes are generally found to be co-ordinately transcribed, this study demonstrates that the expression of MHC class II allelic variants can be differentially regulated in vivo.     

Expression of Class II Major Histocompatibility Complex Antigens on Adult T Cells in Xenopus is Metamorphosis- Dependent

Class II major histocompatibility complex (MHC) antigens are expressed predominantly on B lymphocytes and macrophages of tadpoles of the South African clawed frog, Xenopus laevis, as is the pattern in lymphocyte populations of most mammals. However, unlike most mammals, young postmetamorphic frogs show expression of class II MHC antigens on a high proportion of thymocytes and most peripheral T and B lymphocytes. Using the J-strain of Xenopus and the anticlass II monoclonal antibody, 14A2, we have studied, by indirect immunofluorescence, whether inhibition of metamorphosis would alter the pattern of expression of class II antigens during ontogeny. In control animals, class II antigens were virtually absent from thymic lymphocytes and peripheral T cells of normal untreated larvae, but could be found in increasing numbers in both populations after metamorphosis (10-12 weeks of age). In contrast, larvae, whose metamorphosis was inhibited by treatment with sodium perchlorate, had relatively few class II⁺ thymic lymphocytes throughout the 6-month period of study, and the proportion of class II⁺ splenic lymphocytes was approximately equal to that of IgM⁺ B lymphocytes. Thus, perchlorate-treated animals retained the larval pattern of class II epression, suggesting that emergence of class II⁺ T cells is dependent on metamorphosis.     

Inhibition of Class II Major Histocompatibility Complex Antigen Processing by Escherichia coli Heat-Labile Enterotoxin Requires an Enzymatically Active A Subunit

Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing. Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes. Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity.Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) are related ADP-ribosylating toxins with five identical B subunits that bind to cell surface ganglioside receptors and an enzymatically active A subunit that enters the cell and catalyzes the ADP-ribosylation of guanine nucleotide binding proteins of the adenylate cyclase complex, causing constitutive activation of adenylate cyclase and increased intracellular cyclic AMP (cAMP).LT and CT are potent mucosal adjuvants (7, 8, 12, 20, 22, 23, 29, 31–33). Some degree of A subunit enzymatic activity is required for oral adjuvant function (20, 23, 32, 33). While ADP-ribosyltransferase activity enhances adjuvanticity, it also confers toxicity. For an optimal adjuvant, reduced toxicity would be desirable, and mutant LT (6, 9–11, 15, 17, 21, 26, 34) and CT (5, 35) molecules have been constructed with altered A subunits, reduced ADP ribosylation activity, and reduced toxicity, yet with maintained adjuvant function (9–11, 13, 25, 26, 35). Mutation studies with LT revealed that residues at positions 7, 110, and 112 of LT A subunit (LTA) are important for ADP-ribosyltransferase activity (6, 21, 28), with Glu-112 providing a catalytic role. A conservative mutation (Asp to Glu) at position 112 produced a mutant toxin, rLT-E112D, with substantially reduced (<2% of wild type) but detectable ADP-ribosyltransferase activity (6).CT and LT affect many components of immune responses, including antigen presentation (3, 4, 18), with inhibitory as well as enhancing effects. We previously showed that CT enhances macrophage presentation of cell surface peptide-class II major histocompatibility complex (MHC-II) complexes to T cells but inhibits intracellular antigen processing (24). However, the effects of LT have not been similarly investigated. Furthermore, mutant LT molecules provide tools to determine the role of A subunit enzymatic activity in immunomodulation and toxicity.The present study was designed to investigate the effects of LT and mutant LT molecules on antigen processing and presentation by macrophages. In particular, we examined the effects of LT on the processing and presentation of a model antigen expressed in bacteria (a system to which LT has natural relevance) by using Escherichia coli strain HB101 expressing the Crl-HEL fusion protein (HB101.Crl-HEL) (27), which contains the HEL(48-61) epitope. LT, the mutant toxin rLT-E112D, and recombinant LTB (rLTB) (Table ​(Table1)1) were prepared as described previously (6, 15). rLTB was produced by using a vector encoding LTB and the A2 fragment of LT (LTA2), but subsequent chromatographic purification produced isolated rLTB, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Trypsin-cleaved LT was produced as described previously (15) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly purified CT was purchased from List Biologicals (Campbell, Calif.). Recombinant CTB (rCTB) was a gift from Jan Holmgren (University of Gøteborg, Gøteborg, Sweden) and was prepared as described previously (30).

TABLE 1

Toxin composition and enzymatic activity

Lyt-Phenotype Conversion of Cytotoxic T Lymphocytes Specific for the A and E Class II Major Histocompatibility Complex Molecules

The Lyt-1+ (high) Lyt-2+/- (low) primary cytotoxic T lymphocytes (CTL) specific for A(A alpha A beta) molecules and the Lyt-1+Lyt-2+ primary E(E alpha E beta)-specific CTL are both shown to become Lyt-1 Lyt-2+ effector cells after secondary in vitro stimulation. Thus CTL specific for class II major histocompatibility complex molecules exhibit the same Lyt-phenotype shift as class-I-specific CTL do. The data suggest that either both class-I-specific and class-II-specific CTL follow the same differentiation pathway or regulatory cellular interactions allow only Lyt-1-Lyt-2+ cells to differentiate to secondary CTL.     

Alternative Endogenous Protein Processing via an Autophagy-Dependent Pathway Compensates for Yersinia-Mediated Inhibition of Endosomal Major Histocompatibility Complex Class II Antigen Presentation

Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.Protein antigens are recognized by T cells as short peptide fragments bound either to major histocompatibility class (MHC) I or to MHC class II molecules on the surface of antigen-presenting cells (APCs). The location of antigens in distinct intracellular compartments of APCs influences their proteolytic processing as well as access to MHC molecules (16, 39). Classically, peptides generated in the cytosol (e.g., derived from viral proteins) by proteasomal degradation are bound to MHC class I molecules after transport across the endoplasmic reticulum membrane by the transporters associated with antigen processing (TAP) (90). Subsequently, MHC class I molecules present these endogenous antigenic peptides to CD8 T cells. In contrast, exogenous antigens (e.g., antigens derived from engulfed bacteria and soluble antigens) are directed into the endosomal/lysosomal pathway for degradation (63). In late endosomal compartments, degraded protein fragments interact with MHC class II molecules and are further trimmed into peptides for presentation to CD4 T cells.However, the synchrony of this system has been challenged by biochemical and functional studies of professional and nonprofessional APCs. Epitopes derived from a diverse pool of cytosolic antigens such as metabolic enzymes, cytoskeletal proteins, and viral and tumor antigens have been identified to be presented in the context of both murine and human MHC class II molecules (20, 51, 52, 53, 71, 88). Several different alternative pathways for delivering antigens into the MHC class II pathway have been described. Lich et al. reported that cytoplasmic processing by the proteasome and calpain is required for efficient processing of the autoantigen glutamate decarboxylase to CD4 T cells (51). Also, a proteasome/TAP-dependent pathway was shown to be important for the presentation of MHC class II peptides from the influenza virus (83). In contrast, other studies using human B lymphoblastoid cells revealed that TAP is not involved in the transport of cytosolic peptides to MHC class II molecules (52, 53). Alternatively, autophagy in the form of either macroautophagy (27, 30, 56, 60) or chaperone-mediated autophagy (19, 23, 24, 60, 91) results in the transport of cytosolic peptides and proteins directly into endosomes and lysosomes. Two essential components of the chaperone-mediated autophagy pathway are Lamp-2a and the heat shock cognate protein hsc70. The latter molecule is an accessory chaperone that intersects target proteins in the cytoplasm and facilitates their delivery to Lamp-2a, a lysosomal membrane protein.Pathogenic yersiniae are Gram-negative bacteria that cause a wide range of diseases in humans, ranging from bubonic plague, caused by Yersinia pestis, to self-limiting gastroenteritis and lymphadenitis, caused by the enteric pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica. Results from in vitro experiments and the mouse infection model revealed that the last two bacterial species have acquired a complex arsenal of effector proteins to overcome host defense mechanisms. These major pathogenicity factors are located on a 70-kb virulence plasmid, which encodes a protein export apparatus called the type III secretion system (T3SS) (21, 35). T3SS is a complex bacterial organelle that provides Gram-negative pathogens with a unique virulence mechanism enabling them to translocate bacterial effector proteins directly into the host cell cytosol. At least six effector proteins, Yersinia outer proteins (Yops), namely, YopE, YopH, YopM, YopO/YpkA, YopP/YopJ, and YopT, are injected into the cytosol of eukaryotic cells in a T3SS-dependent manner (22). The main function of these Yops is to inhibit the immune response of the host. Four Yops (YopE, YopH, YopO/YpkA, and YopT) are involved in inhibiting phagocytosis of yersiniae by disrupting the cytoskeleton of polymorphonuclear leukocytes and macrophages (12, 33, 40, 68). Thus, the consequence of this translocation process is that pathogenic yersiniae survive and proliferate at extracellular sites in the infected host (77).Our laboratory has described and analyzed in detail the potential of YopE to be a carrier molecule for heterologous antigen delivery by Yersinia (74). YopE is a translocated GTPase-activating molecule that can downregulate Rho activity, leading to actin filament disruption and inhibition of phagocytosis by macrophages (10, 69, 87). The N-terminal 18 amino acids (aa) of YopE fused to a large protein fragment of the p60 antigen from the intracellular pathogen Listeria monocytogenes were sufficient for T3SS-dependent secretion to the extracellular environment of Yersinia-infected target cells (74). In contrast, fusion of p60 to the N-terminal 138 aa of YopE resulted in translocation of the chimeric protein into the cytosol of host cells. As expected, T-cell activation assays revealed that the cytosolic delivery of this hybrid protein was a prerequisite to induce a p60-specific MHC class I-restricted CD8 T-cell response (74). Because translocated p60 easily enters the endogenous cytosolic antigen presentation pathway, it is processed like an endogenously synthesized antigen. In a more recent publication, we used Y. pseudotuberculosis expressing YopE fused to listeriolysin O (LLO) from Listeria to assess the influence of secreted versus translocated antigen display on in vitro antigen presentation and in vivo T-cell priming in the oral mouse infection model (72). We constructed two different plasmid-encoded hybrid YopE/LLO proteins. By engaging the above-mentioned well-defined secretion and translocation domains of YopE (79) fused to aa 51 to 363 of LLO, chimeric YopE was expressed either in secreted or in translocated form (72). Biochemical fractionation of Yersinia-infected macrophage-like P338D₁ cells clearly revealed that YopE from aa 1 to 38 (YopE_1-138)/LLO was translocated to the cytosol of host cells, whereas YopE_1-18/LLO lacking the translocation domain was efficiently secreted to the culture medium but was not detected in the P338D₁ cell lysate containing cytosolic proteins. Thus, it was theoretically expected that the well-secreted version of chimeric YopE/LLO could have the potential to enter the exogenous MHC class II antigen presentation pathway for proper CD4 T-cell priming. Strikingly, results from in vitro antigen presentation assays and also the enumeration of LLO-specific CD4 T cells from infected mice indicated a superior efficacy of translocated over secreted LLO for MHC class II antigen presentation and CD4 T-cell induction, respectively (72).This study addresses the requirements for processing and MHC class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the T3SS of Yersinia pseudotuberculosis. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. However, a distinct MHC class II antigen presentation pathway was identified for YopE fusion proteins originating from the cytosol. Presentation of cytoplasm-derived chimeric YopE requires acidification of the phagolysosome and is sensitive to inhibitors of autophagy.     

Major Histocompatibility Complex Class II Dextramers: New Tools for the Detection of antigen‐Specific,CD4 T Cells in Basic and Clinical Research

The advent of major histocompatibility complex (MHC) tetramer technology has been a major contribution to T cell immunology, because tetramer reagents permit detection of antigen‐specific T cells at the single‐cell level in heterogeneous populations by flow cytometry. However, unlike MHC class I tetramers, the utility of MHC class II tetramers has been less frequently reported. MHC class II tetramers can be used successfully to enumerate the frequencies of antigen‐specific CD4 T cells in cells activated in vitro, but their use for ex vivo analyses continues to be a problem, due in part to their activation dependency for binding with T cells. To circumvent this problem, we recently reported the creation of a new generation of reagents called MHC class II dextramers, which were found to be superior to their counterparts. In this review, we discuss the utility of class II dextramers vis‐a‐vis tetramers, with respect to their specificity and sensitivity, including potential applications and limitations.     

Immunological Response of Major Histocompatibility Complex Class II-Deficient (Aβ°) Mice Infected by the Parasite Schistosoma mansoni

We have characterized the immunological behaviour of major histocompitibility complex (MHC) Class II molecule-deficient (Aβ°) mice after infection by Schistosoma mansoni . In Aβ° mice, morbidity developed dramatically 7 weeks after infection leading to death, despite the absence of an increase in parasite burden or of eggs trapped in the liver. Histological examination of the liver revealed the absence of a classical granulomatous reaction. Antibodies were produced only against schistosomulum antigens. Specific antibodies against adult worm (SWAP) or egg antigen (SEA) were not detected. Cytokine production (IFN-γ and IL-4) was absent after in vitro restimulation of splenic cells from infected Aβ° mice with parasite antigens. Adoptive transfer of primed splenic cells (total, purified CD4⁺ or CD8⁺ T cells) failed to improve survival or to induce a granulomatous reaction in infected Aβ° mice. Survival, cellular and humoral responses in CD8⁺ T-cell-depleted Aβ° mice or MHC° mice (lacking MHC class I and II molecules) were similar to nondepleted Aβ° mice, suggesting that anti-schistosomula antibody production was thymo-independent. Our results demonstrate a high degree of susceptibility of Aβ° mice to infection and corroborate the importance of CD4⁺ T cells in the initiation of the granulomatous response. However, our results do not show evidence for the involvement of CD8⁺ T cells in response to S. mansoni infection.     

Intrathymic Tolerance Induction: Determination of Tolerance to Class II Major Histocompatibility Complex Antigens in Maturing T Lymphocytes by a Bone Marrow-Derived Non-Lymphoid Thymus Cell

Two new experimental approaches were established to analyse the influence of the thymus on tolerance induction to major histocompatibility complex (MHC) antigens: The aim of the first experiment was to perform successful transplantation of adult allogeneic thymus tissue into nude mice, an attempt that has been unsuccessful in the past. Tolerance for the MHC genotype of a prospective thymus graft recipient (A) was induced in mice of strain B by injection of (A X B) splenocytes during the neonatal period. Adult thymic tissue obtained from these allogeneic donors (B) were grafted into the nude mice of strain A. The allogeneic thymus was accepted by the nude mice and immunoreconstitution was achieved. Subsequently the recipients developed tolerance to the MHC antigens of the allogeneic thymus donor as proved by mixed lymphocyte cultures and the acceptance of skin grafts. The second experiment was designed to determine which Ia-positive thymic compartment participates in conferring tolerance to MHC antigens in maturing T lymphocytes. Chimaeric thymus grafts were created by transplantation of neonatal thymus (A) into allogeneic nude mice (B) for a period of 8 weeks. The graft was populated with host bone marrow-derived Ia antigen-positive cells. The chimaeric thymuses consisting of type A epithelium but populated with both type A and B lymphocytes and non-lymphoid cells (i.e. Ia-positive macrophages and dendritic cells), were newly transplanted into nude mice of strain A. The engraftment lead to immunological reconstitution and the nude mice acquired tolerance to the MHC antigens expressed by the allogeneic Ia-positive cells populating the chimaeric graft. Irradiation of the chimaeric thymus prior to transplantation allowed transplantation of chimaeric thymus devoid of living thymocytes but still populated with functionally intact Ia-positive non-lymphoid cells. Transplantation of irradiated chimaeric thymuses resulted in immunoreconstitution and induced exactly the same allotolerance pattern as described above. The results demonstrate that not thymus epithelial cells but a bone-marrow-derived non-lymphoid thymus cell, most likely the Ia-antigen-positive thymic macrophage of dendritic cell, is responsible for the induction of tolerance to MHC antigens in developing T lymphocytes.     

Mycobacterium tuberculosis Synergizes with ATP To Induce Release of Microvesicles and Exosomes Containing Major Histocompatibility Complex Class II Molecules Capable of Antigen Presentation

Major histocompatibility complex class II (MHC-II) molecules are released by murine macrophages upon lipopolysaccharide (LPS) stimulation and ATP signaling through the P2X7 receptor. These studies show that infection of macrophages with Mycobacterium tuberculosis or M. bovis strain BCG enhances MHC-II release in synergy with ATP. Shed MHC-II was contained in two distinct organelles, exosomes and plasma membrane-derived microvesicles, which were both able to present exogenous antigenic peptide to T hybridoma cells. Furthermore, microvesicles from mycobacterium-infected macrophages were able to directly present M. tuberculosis antigen (Ag) 85B(241-256)-I-A^b complexes that were generated by the processing of M. tuberculosis Ag 85B in infected cells to both M. tuberculosis-specific T hybridoma cells and naïve P25 M. tuberculosis T-cell receptor (TCR)-transgenic T cells. In the presence of prefixed macrophages, exosomes from mycobacterium-infected macrophages provided weak stimulation to M. tuberculosis-specific T hybridoma cells but not naïve P25 T cells. Thus, infection with M. tuberculosis primes macrophages for the increased release of exosomes and microvesicles bearing M. tuberculosis peptide-MHC-II complexes that may generate antimicrobial T-cell responses.Exosomes are 50- to 80-nm membrane vesicles that are released by many cell types, including reticulocytes, B cells, and dendritic cells (DCs) (16, 17, 33-35, 40, 42, 49, 53). Invagination of the limiting membrane of late endosomes leads to the formation of intraluminal vesicles in multivesicular endosomes. The intraluminal vesicles are secreted as exosomes upon the fusion of multivesicular endosomes with the plasma membrane.Exosomes from B cells contain major histocompatibility complex class II (MHC-II) molecules and can stimulate CD4⁺ T-cell responses in vitro (40), although they may be more capable of activating primed T cells than naïve CD4⁺ T cells (27). The activation of naïve CD4⁺ T cells by DC exosomes occurs via an indirect pathway in which the exosomes and their constituent peptide-MHC-II molecules are presented in the context of intact antigen (Ag)-presenting cells (APCs) (e.g., DCs that may be MHC-II negative but must bear the costimulatory molecules CD80 and CD86 [48]). The presence of ICAM-1 on exosomes is important for naïve T-cell priming (43).While the shedding of exosomes can be constitutive (27, 40), it can also be significantly enhanced by the stimulation of certain receptors, e.g., Toll-like receptors (TLRs) and the P2X7 purinergic receptor (P2X7R), which trigger inflammatory responses (37, 38). P2X7R can be activated by ATP, which is released into the extracellular milieu following cell death or injury (50). P2X7R signaling induces the assembly of inflammasome signaling complexes (10), which drive the proteolytic activation of caspase-1 and the maturation of interleukin 1b (IL-1b). Another P2X7R-induced response is the rapid extracellular release of MHC-II molecules (38), which was previously observed within 15 min of the addition of ATP and resulted in the release of ∼15% of the total MHC-II pool in macrophages within 90 min (38). Released MHC-II molecules were contained in two membrane fractions: larger (100- to 1,000-nm) plasma membrane-derived microvesicles and smaller (50- to 80-nm) exosomes. The ATP-stimulated release of MHC-II was markedly reduced in macrophages isolated from NLRP3 knockout or ASC knockout mice. Thus, P2X7R activation of the NLRP3 inflammasome induces the biogenesis and release of MHC-II-containing membranes. The precedent of synergy between lipopolysaccharide (LPS) and ATP suggests that MHC-II shedding might be enhanced in the context of bacterial infection, but this hypothesis has not been explored.Mycobacterium tuberculosis is a major human pathogen that infects one-third of the world population. M. tuberculosis and the related organism Mycobacterium bovis strain BCG infect host cells and regulate host cell functions by signaling through innate immune receptors, including TLR2. Cells infected with M. tuberculosis also secrete exosomes containing mycobacterial molecules that function as PAMPs (pathogen-associated molecular patterns) (2-5, 42) and can stimulate proinflammatory responses via TLR2, TLR4, and MyD88 (4, 5). The dissemination of PAMPs by exosomes released from infected cells may induce innate immune responses by a greater number of cells than are directly infected, magnifying host responses. M. tuberculosis and other mycobacteria can activate the ASC/NLRP3/caspase-1 inflammasome in macrophages via a mechanism dependent on the mycobacterial RD1 locus (encoding components of the ESX-1 secretion system, including the ESAT-6 protein) (9, 20, 26). The ability of M. tuberculosis to stimulate inflammasome activity is dependent on increased K⁺ efflux and occurs in macrophages from P2X7 receptor knockout mice (20). Thus, P2X7 receptor activation and M. tuberculosis infection may elicit similar signaling pathways that converge on the NLRP3 inflammasome and, possibly, on the inflammasome-dependent release of MHC-II membranes.In the current study, we demonstrate that infection of macrophages with mycobacteria elicits the shedding of MHC-II-containing membranes. Furthermore, M. tuberculosis increases the ATP-triggered release of exosomes and microvesicles containing MHC-II. In addition, we demonstrate that MHC-II in membranes released from mycobacterium-infected macrophages can present Ag to T cells. These findings suggest that exosomes and microvesicles from mycobacterium-infected cells may broadcast the stimulation of both innate and adaptive immune receptors beyond the directly infected host cells, contributing to the genesis of CD4 T-cell responses to mycobacterial pathogens such as M. tuberculosis.     

On the Heterologous Interaction between β2-Microglobulin and the Heavy Chain of Rat Major Histocompatibility Complex Class 1 Antigens

The heterologous interaction between β₂-microglobulin (β₂m) and rat major histocompatibility complex (MHC) (RT1) antigens was measured in a two-step binding assay consisting of binding of radiolabelled β₂-m to RT1 antigens and immunoprecipitation of β₂m-RT1 antigen complexes with RT1 antisera. The effects of varying the concentrations of the three reactants involved were studied. The molecular events taking place in the two steps were analysed by gel chromatography. The β₂m-RT1 antigen complex had the apparent size of albumin and reacted completely with specific alloantisera. RT1 antigens prepared from Wistar/Furth (RT1^u) and Brown Norway (RT1ⁿ), respectively, both effectively bound heterologous β₂m. The times for association and dissociation, respectively, at 37°C, were of the same order, but dissociation was slightly slower. Association was markedly temperature-dependent and was considerably slower at low temperatures. All these processes were slower for RT1^u than for RT1^u antigens. The association constant for the interaction between RT1^u antigens and ¹²⁵I-human β₂m was estimated by Scatchard analysis to be about 10⁹ M^-1. Contribution to the heterologous interaction by products from various rat MHC subloci (A, B, and C) was investigated by the introduction of sublocus-specific antisera in step 2. The reaction apparently involved neither class 2 antigens (sublocus B) nor the presumed rat Qa homologue (sublocus C). Classical class 1 antigens (suhlocus A) clearly contributed to the binding. However, a monoclonal antibody against products from rat MHC class 1 genes only precipitated less than half of the RTI antigen-complexed β₂m. Thus, at least two RT1^u class 1 alloantigen molecules seem to participate in the reaction. This, in turn, indicates that the rat genome may contain multiple class 1 genes, an is the case for most other mammals investigated.     

Human Interferon-γ Enhances the Expression of Class I and Class II Major Histocompatibility Complex Products in Neoplastic Cells More Effectively than Interferon-α and Interferon-β

Human interferon-γ was more effective than interferon-β or -α in stimulating production of immunoassociated antigens; HLA-A, -B, and -C; and β₂-microglobulin in human M14 and Namalva cells. The comparison was made on the basis of antiviral units, and the stimulation could be abolished by treatment of the interferon-γ preparation with pH 2 or anti-interferon-γ serum.