Bidirectional membrane molecule transfer between dendritic and T cells

The acquisition of dendritic cell (DC) molecules by T cells has been previously reported. However, it remains unclear whether the transfer is only mono- or bidirectional. In this study, we incubated CMFDA-labeled ovalbumin (OVA)-pulsed DC2.4 (DC2.4(OVA)) cells with Dil-labeled OT II CD4(+) T cells and analyzed the potential bidirectional molecule transfer. We also assessed the distribution of internalized membrane using two engineered DC2.4/Ia(b)GFP and MF4/TCRCFP DC lines. Our findings showed that membrane molecule transfer is bidirectional. CD4(+) T cells acquired Ia(b), CD11c, CD40, and CD80 from DC2.4(OVA) cells, and conversely DC2.4(OVA) cells took up CD4, CD25, CD69, and T cell receptor from T cells. The internalized molecules acquired by T cells and DCs mostly localized in endosomes and lysosomes, respectively. Taken together, this study demonstrated a novel phenomenon of bidirectional membrane molecule transfer between DCs and T cells.     

T cells induce extended class II MHC compartments in dendritic cells in a Toll-like receptor-dependent manner

Interaction of Ag-loaded dendritic cells with Ag-specific CD4 T cells induces the formation of long tubular class II MHC-positive compartments that polarize toward the T cell. We show involvement of a Toll-like receptor-mediated signal in this unusual form of intracellular class II MHC trafficking. First, wild-type dendritic cells loaded with LPS-free Ag failed to show formation of class II-positive tubules upon Ag-specific T cell engagement, but did so upon supplementation of the Ag with low concentrations of LPS. Second, Ag-loaded myeloid differentiation factor 88 -deficient dendritic cells failed to form these tubules upon interaction with T cells, regardless of the presence of LPS. Finally, inclusion of a cell-permeable peptide that blocks TNFR-associated factor 6 function, downstream of myeloid differentiation factor 88, blocked T cell-dependent tubulation. A Toll-like receptor-dependent signal is thus required to allow Ag-loaded dendritic cells to respond to T cell contact by formation of extended endosomal compartments. This activation does not result in massive translocation of class II MHC molecules to the cell surface.     

Tubulation of Endosomal Structures in Human Dendritic Cells by Toll-like Receptor Ligation and Lymphocyte Contact Accompanies Antigen Cross-presentation

Mouse dendritic cells (DCs) can rapidly extend their Class II MHC-positive late endosomal compartments into tubular structures, induced by Toll-like receptor (TLR) triggering. Within antigen-presenting DCs, tubular endosomes polarize toward antigen-specific CD4⁺ T cells, which are considered beneficial for their activation. Here we describe that also in human DCs, TLR triggering induces tubular late endosomes, labeled by fluorescent LDL. TLR triggering was insufficient for induced tubulation of transferrin-positive endosomal recycling compartments (ERCs) in human monocyte-derived DCs. We studied endosomal remodeling in human DCs in co-cultures of DCs with CD8⁺ T cells. Tubulation of ERCs within human DCs requires antigen-specific CD8⁺ T cell interaction. Tubular remodeling of endosomes occurs within 30 min of T cell contact and involves ligation of HLA-A2 and ICAM-1 by T cell-expressed T cell receptor and LFA-1, respectively. Disintegration of microtubules or inhibition of endosomal recycling abolished tubular ERCs, which coincided with reduced antigen-dependent CD8⁺ T cell activation. Based on these data, we propose that remodeling of transferrin-positive ERCs in human DCs involves both innate and T cell-derived signals.     

MARCH1 down-regulation in IL-10-activated B cells increases MHC class II expression

IL-10 is vastly studied for its anti-inflammatory properties on most immune cells. However, it has been reported that IL-10 activates B cells, up-regulates their MHC class II molecules and prevents apoptosis. As MARCH1 was shown to be responsible for the intracellular sequestration of MHC class II molecules in dendritic cells and monocytes in response to IL-10, we set out to clarify the role of this ubiquitin ligase in B cells. Here, we demonstrate in mice that splenic follicular B cells represent the major cell population that up-regulate MHC II molecules in the presence of IL-10. Activation of these cells through TLR4, CD40 or the IL-10 receptor caused the down-regulation of MARCH1 mRNA. Accordingly, B cells from MARCH1-deficient mice do not up-regulate I-A(b) in response to IL-10. In all, our results demonstrate that IL-10 can have opposite effects on MARCH1 regulation in different cell types.     

The Ia.2 epitope defines a subset of lipid raft-resident MHC class II molecules crucial to effective antigen presentation

Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a β-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.     

Induction of differential T-cell epitope by plain- and liposome-coupled antigen

The T-cell receptors of CD4(+) T lymphocytes recognize immunogenic peptide sequences bound within the groove of MHC class II molecules, and the peptides that bind to these molecules are known to share common structural motifs. For example, OVA(323-339), an I-A(d)-binding peptide, involves a motif of the I-A(d) peptide-binding groove. In the present study, OVA peptides of up to 26-mer were sequentially synthesized and screened, and two additional I-A(d) binding OVA peptides, OVA(20-43) and OVA(264-286), were found to stimulate CD4(+) T cells of OVA-immune BALB/c mice. OVA(20-43) involved structural motifs of the I-A(d) peptide-binding groove, while OVA(264-286) did not. The ability of these three I-A(d) binding OVA peptides to induce antigen-specific cytokine production was compared among CD4(+) T cells of mice immunized either with alum-adsorbed OVA (OVA-alum) or OVA chemically coupled to the surface of liposome (OVA-liposome). CD4(+) T cells of mice immunized with OVA-alum produced more cytokines when stimulated with OVA(264-286) than with OVA(323-339), while CD4(+) T cells of mice immunized with OVA-liposome conjugates produced more cytokines when stimulated with OVA(323-339) than with OVA(264-286). OVA(20-43) induced production of comparable levels of cytokines in mice immunized either with OVA-alum or OVA-liposome. Confocal laser scanning microscopic analysis demonstrated that chemically coupled OVA and liposomes were colocalized in APCs until OVA received processing. Three-dimensional structural analysis demonstrated that both OVA(264-286) and OVA(323-339) were present on the surface of OVA, but OVA(20-43) was not. These results suggested that the chemical coupling of OVA to liposome affected antigen processing in APCs and thus resulted in the induction of differential T-cell epitopes as compared with those induced by plain OVA.     

Detection of low-avidity CD4+ T cells using recombinant artificial APC: following the antiovalbumin immune response

Subtle differences oppose CD4+ to CD8+ T cell physiologies that lead to different arrays of effector functions. Interestingly, this dichotomy has also unexpected practical consequences such as the inefficacy of many MHC class II tetramers in detecting specific CD4+ T cells. As a mean to study the CD4+ anti-OVA response in H-2(d) and H-2(b) genetic backgrounds, we developed I-A(d)- and I-A(b)-OVA recombinant MHC monomers and tetramers. We were able to show that in this particular system, despite normal biological activity, MHC class II tetramers failed to stain specific T cells. This failure was shown to be associated with a lack of cooperation between binding sites within the tetramer as measured by surface plasmon resonance. This limited cooperativeness translated into a low "functional avidity" and very transient binding of the tetramers to T cells. To overcome this biophysical barrier, recombinant artificial APC that display MHC molecules in a lipid bilayer were developed. The plasticity and size of the MHC-bearing fluorescent liposomes allowed binding to Ag-specific T cells and the detection of low numbers of anti-OVA T cells following immunization. The same liposomes were able, at 37 degrees C, to induce the full reorganization of the T cell signaling molecules and the formation of an immunological synapse. Artificial APC will allow T cell detection and the dissection of the molecular events of T cell activation and will help us understand the fundamental differences between CD4+ and CD8+ T cells.     

Distinct MHC Class II Molecules Are Associated on the Dendritic Cell Surface in Cholesterol-dependent Membrane Microdomains

Very small amounts of MHC class II-peptide complexes expressed on the surface of antigen-presenting cells (APCs) are capable of stimulating antigen-specific CD4 T cells. There is intense interest to elucidate the molecular mechanisms by which these small amounts of MHC-II can cluster, cross-link T cell receptors, and promote T cell proliferation. We now demonstrate that a significant fraction of the total pool of MHC-II molecules on the surface of dendritic cells is physically associated in macromolecular aggregates. These MHC-II/MHC-II interactions have been probed by co-immunoprecipitation analysis of the MHC-II I-A molecule with the related I-E molecule. These molecular associations are maintained in gentle detergents but are disrupted in harsh detergents such as Triton X-100. MHC-II I-A/I-E interactions are disrupted when plasma membrane cholesterol is extracted using methyl β-cyclodextrin, suggesting that lipid raft microdomains are important mediators of these MHC-II interactions. Although it has been proposed that tetraspanin proteins regulate molecular clustering, aggregation, and co-immunoprecipitation in APCs, genetic deletion of the tetraspanin family members CD9 or CD81 had no effect on MHC-II I-A/I-E binding. These data demonstrate that the presence of distinct forms of MHC-II with plasma membrane lipid rafts is required for MHC-II aggregation in APCs and provides a molecular mechanism allowing dendritic cells expressing small amounts of MHC-II-peptide complexes to cross-link and stimulate CD4 T cells.     

CD1 and major histocompatibility complex II molecules follow a different course during dendritic cell maturation

The maturation of dendritic cells is accompanied by the redistribution of major histocompatibility complex (MHC) class II molecules from the lysosomal MHC class II compartment to the plasma membrane to mediate presentation of peptide antigens. Besides MHC molecules, dendritic cells also express CD1 molecules that mediate presentation of lipid antigens. Herein, we show that in human monocyte-derived dendritic cells, unlike MHC class II, the steady-state distribution of lysosomal CD1b and CD1c isoforms was unperturbed in response to lipopolysaccharide-induced maturation. However, the lysosomes in these cells underwent a dramatic reorganization into electron dense tubules with altered lysosomal protein composition. These structures matured into novel and morphologically unique compartments, here termed mature dendritic cell lysosomes (MDL). Furthermore, we show that upon activation mature dendritic cells do not lose their ability of efficient clathrin-mediated endocytosis as demonstrated for CD1b and transferrin receptor molecules. Thus, the constitutive endocytosis of CD1b molecules and the differential sorting of MHC class II from lysosomes separate peptide- and lipid antigen-presenting molecules during dendritic cell maturation.     

Immunoproteomic discovery of novel T cell antigens from the obligate intracellular pathogen Chlamydia

Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-A(b)) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.     

Co-dominant restriction by a mixed-haplotype I-A molecule (alpha k beta b) for the lysozyme peptide 52-61 in H-2k x H-2b F1 mice

Helper (CD4+) T lymphocytes recognize protein Ag as peptides associated to MHC class II molecules. The polymorphism of class II alpha- and beta-chains has a major influence on the nature of the peptides presented to CD4+ T lymphocytes. For instance, T cell responses in H-2k and H-2b mice are directed at different epitopes of the hen egg lysozyme (HEL) molecule. The current studies were undertaken with the aim of defining the role of mixed haplotype I-A (alpha k beta b and alpha b beta k) molecules in T cell responses to HEL in (H-2k x H-2b)F1 mice, as well as the nature of the immunogenic peptides of HEL recognized in the context of I-A alpha k beta b and I-A alpha b beta k. A series of HEL-reactive T cell lines and hybridomas derived from MHC class II heterozygous (C57BL/6 x C3H F1) mice were established. Their responsiveness to HEL and synthetic HEL peptides was analyzed with the use of L cells transfected with either I-A alpha k beta b or I-A alpha b beta k as APC. Out of 28 clonal T cell hybridomas tested, 13 (46%) only responded to HEL presented by I-A alpha k beta b, 11 (40%) by I-A alpha b beta k (and to a minor extent I-A alpha k beta k), only 4 (14%) were primarily restricted by I-Ak, and none by I-Ab. All the I-A alpha k beta b-restricted T cell hybridomas responded to the HEL peptide 46-61 and to its shorter fragment 52-61, even at concentrations as low as 0.3 nM. As this determinant has been previously defined as immunodominant for I-Ak but not for I-Ab mice, these results suggest a role for the I-A alpha k chain in the selection and immunodominance of HEL 52-61 in H-2k mice. The fine specificity of I-A alpha k beta b-restricted T cell hybridomas for a series of different HEL peptides around the sequence 52 to 61 suggests that peptide 52-61 binds to I-A alpha k beta b with higher affinity than to I-A alpha k beta k. The peptides recognized in the context of I-A alpha b beta k and I-A alpha k beta k were not identified.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules

CD1 family members are antigen-presenting molecules capable of presenting bacterial or synthetic glycolipids to T cells. Here we show that a subset of human CD1d molecules are associated with major histocompatibility complex (MHC) class II molecules, both on the cell surface and in the late endosomal/lysosomal compartments where class II molecules transiently accumulate during transport. The interaction is initiated in the endoplasmic reticulum with class II-invariant chain complexes and appears to be maintained throughout the class II trafficking pathway. A truncated form of CD1d which lacks its cytoplasmic YXXZ internalization motif is transported to late endosomal/lysosomal compartments in the presence of class II molecules. Furthermore, the same CD1d deletion mutant is targeted to lysosomal compartments in HeLa cells expressing class II molecules and invariant chain by transfection. The deletion mutant was also found in lysosomal compartments in HeLa cells expressing only the p33 form of the invariant chain. These data suggest that the intracellular trafficking pathway of CD1d may be altered by class II molecules and invariant chain induced during inflammation.     

Peptide-binding motifs for the I-Ad MHC class II molecule: alternate pH-dependent binding behavior

The ability of peptides to form stable complexes with MHC class II molecules expressed in the host determines their ability to recruit CD4 T cells during an immune response. In this study, we sought to define the features of the antigenic peptides that control their kinetic stability with I-A(d) because of the diversity of peptides that this molecule is known to present. Peptide dissociation assays indicated that each pocket of I-A(d) displays exquisite sensitivity to side chain structure, size, and charge. Most surprising were results related to the P1 pocket, which has been difficult to define by conventional competition assays. Our studies revealed a considerable degree of specificity in the P1 pocket but also an unexpected degree of structural flexibility. Amino acids with neutral side chains such as Met and the alternatively negatively charged Glu are both highly favored at P1. Interestingly, these two options at the P1 pocket in I-A(d) display dramatically different pH-dependent interactions with the class II molecule. These findings are discussed in the context of a structural model to explain these data and in light of the immunological implications of pH-dependent behavior of class II-peptide complexes in acidic endosomal compartments, where DM-catalyzed loading of class II molecules takes place, and at the neutral pH of the APC cell surface, where class II-peptide complexes promote activation of CD4 T cells.     

Transfer of T cell surface molecules to dendritic cells upon CD4+ T cell priming involves two distinct mechanisms

Activation of CD4(+) T cells by APCs occurs by multiple Ag recognition events including the exchange of costimulatory signals and cytokines. Additionally, the T cells acquire APC-derived surface molecules. Herein, we describe for the first time the transfer of human and murine T cell surface receptors to APCs after Ag-specific interaction. This transfer occurs in two qualitatively different phases. The first group of molecules (e.g., CD2) derived from the T cell surface was transferred rapidly after 2 h of interaction, was strongly bound on the DC surface (acid wash-resistant), was strictly dependent on dendritic cell-T cell contact, and transferred independently of T cell activation. The second group, including the CD3/TCR complex, CD27, and OX40, was of intracellular origin, transferred later after 10-16 h in a cell-cell contact-independent fashion, was noncovalently bound, and was strictly dependent on Ag-specific T cell activation. Functionally, murine dendritic cells that received TCR molecules from OVA-specific CD4(+) T cells after Ag-specific interaction were less efficient in priming naive CD4(+) T cells of the same specificity without losing their ability for CD8(+) T cell stimulation, indicating that the transferred TCR molecules mask the Ag-bearing MHC II molecules, thereby reducing their accessibility to following Ag-specific CD4(+) T cells. While the first group of transferred T cell surface molecules might facilitate the detachment of the CD4(+) T cell from the dendritic cell during the early scanning phases, the second group could play an important immunomodulatory role in intraclonal competition of T cells for APC access, making the physical presence of CD4(+) T cells unnecessary.     

Cancer Associated Aberrant Protein O-Glycosylation Can Modify Antigen Processing and Immune Response

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/− glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.     

Phosphatidylserine receptor-mediated recognition of archaeosome adjuvant promotes endocytosis and MHC class I cross-presentation of the entrapped antigen by phagosome-to-cytosol transport and classical processing

Archaeal isopranoid glycerolipid vesicles (archaeosomes) serve as strong adjuvants for cell-mediated responses to entrapped Ag. We analyzed the processing pathway of OVA entrapped in archaeosomes composed of Methanobrevibacter smithii lipids, high in archaetidylserine (OVA-archaeosomes). In vitro, OVA-archaeosomes stimulated spleen cells from OVA-TCR-transgenic mice, D011.10 (CD4(+) cells expressing OVA(323-339) TCR) or OT1 (>90% CD8(+) OVA(257-264) cells), indicating both MHC class I and II presentations. In vivo, when naive (Thy1.2(+)) CFSE-labeled OT1 cells were transferred into OVA-archaeosome-immunized Thy 1.1(+) recipient mice, there was profound accumulation and cycling of donor-specific cells, and differentiation of H-2K(b)Ova(257-264) CD8(+) T cells into CD44(high)CD62L(low) effectors. Both macrophages and dendritic cells (DCs) efficiently cross-presented OVA-archaeosomes on MHC class I. Blocking phagocytosis by phosphatidylserine-specific receptor agonists strongly inhibited MHC class I presentation of OVA-archaeosomes, whereas blocking mannose receptors or FcRs lacked effect, indicating specific recognition of the archaetidylserine head group of M. smithii lipids by APCs. In addition, inhibitors of endosomal acidification blocked MHC class I processing of OVA-archaeosomes, whereas endosomal protease inhibitors lacked effect, suggesting acidification-dependent phagosome-to-cytosol diversion. Proteasomal inhibitors blocked OVA-archaeosome MHC class I presentation, confirming cytosolic processing. Both in vitro and in vivo, OVA-archaeosome MHC class I presentation required TAP. Ag-free archaeosomes also activated DC costimulation and cytokine production, without overt inflammation. Phosphatidylserine-specific receptor-mediated endocytosis is a mechanism of apoptotic cell clearance and DCs cross-present Ags sampled from apoptotic cells. Our results reveal the novel ability of archaeosomes to exploit this mechanism for cytosolic MHC class I Ag processing, and provide an effective particulate vaccination strategy.     

In situ analysis reveals physical interactions between CD11b+ dendritic cells and antigen-specific CD4 T cells after subcutaneous injection of antigen

In situ staining techniques were used to visualize physical interactions between dendritic cell subsets and naive Ag-specific CD4 T cells in the lymph node. Before injection of Ag, CD8(+) dendritic cells and naive OVA-specific CD4 T cells were uniformly distributed throughout the T cell-rich paracortex, whereas CD11b(+) dendritic cells were located mainly in the outer edges of the paracortex near the B cell-rich follicles. Many OVA-specific CD4 T cells were in contact with CD8(+) dendritic cells in the absence of OVA. Within 24 h after s.c. injection of soluble OVA, the OVA-specific CD4 T cells redistributed to the outer paracortex and interacted with CD11b(+), but not CD8(+) dendritic cells. This behavior correlated with the uptake of OVA and the presence of peptide-MHC complexes on the surface of CD11b(+) dendritic cells, and subsequent IL-2 production by the Ag-specific CD4 T cells. These results are consistent with the possibility that CD11b(+) dendritic cells play a central role in the activation of CD4 T cells in response to s.c. Ag.     

Self-specific MHC class II-restricted CD4-CD8- T cells that escape deletion and lack regulatory activity

In the presence of the I-Ealpha protein, transgenic (Tg) mice expressing the 1H3.1 alphabeta TCR that is specific for the Ealpha52-68:I-A(b) complex display drastic intrathymic deletion. Although peripheral T cells from these mice remained unresponsive to the Ealpha52-68:I-A(b) complex, they contained a subpopulation able to specifically react to this complex in the presence of exogenous IL-2, indicating that some 1H3.1 alphabeta TCR Tg T cells have escaped clonal deletion and efficiently populated the periphery. IL-2-dependent, Ealpha52-68:I-A(b) complex-responsive T cells were CD4-CD8- and expressed the 1H3.1 alphabeta TCR. Such T cells could develop intrathymically, did not show sign of regulatory/suppressor activity, displayed a typical naive phenotype, and seemed to persist in vivo over time. CD4-CD8- TCR Tg T cells were also detected when the surface density of the deleting ligand was increased on MHC class II+ cells. In addition, the development of CD4-CD8- 1H3.1 alphabeta TCR Tg T cells could be supported by I-A(b) molecules. These observations indicate that CD4 surface expression neither specifies, nor is required for, the thymic export of mature thymocytes expressing a MHC class II-restricted alphabeta TCR. The data also show that, although the avidity of the interaction involved in intrathymic deletion is significantly lower than that involved in mature T cell activation, its range can be large enough to be influenced by the presence or absence of coreceptors. Finally, the margin created by the absence of CD4 coreceptor was substantial because it could accommodate various amounts of the deleting ligand on thymic stromal cells.     

H2-Mbeta 1 and H2-Mbeta 2 heterodimers equally promote clip removal in I-A(q) molecules from autoimmune-prone DBA/1 mice

Antigen-presenting cells degrade endocytosed antigens, e.g. collagen type II, into peptides that are bound and presented to arthritogenic CD4(+) helper T cells by major histocompatibility complex (MHC) class II molecules. Efficient loading of many MHC class II alleles with peptides requires the assistance of H2-M (HLA-DM in humans), a heterodimeric MHC class II-like molecule that facilitates CLIP removal from MHC class II molecules and aids to shape the peptide repertoire presented by MHC class II to CD4(+) T cells. In contrast to the HLA-DM region in humans, the beta-chain locus is duplicated in mice, with the H2-Mb1 beta-chain distal to H2-Mb2 and the H2-Ma alpha-chain gene. H2-M alleles appear to be associated with the development of autoimmune diseases. Recent data showed that Mbeta1 and Mbeta2 isoforms are differentially expressed in isolated macrophages and B cells, respectively. The tissue expression and functional role of these heterodimers in promoting CLIP removal and peptide selection have not been addressed. We utilized the human T2 cell line, which lacks part of chromosome 6 encompassing the MHC class II and DM genes, to construct transgenic cell lines expressing the MHC class II heterodimer I-A(q) alone or in the presence of H2-Malphabeta1 or H2-Malphabeta2 heterodimers. Both H2-M isoforms facilitate the exchange of CLIP for cognate peptides on I-A(q) molecules from arthritis-susceptible DBA/1 mice and induce a conformational change in I-A(q) molecules. Moreover, I-A(q) cell-surface expression is not absolutely dependent on H2-M molecules. These data suggest that I-A(q) exhibits a high affinity for CLIP since virtually all I-A(q) molecules on T2 cells were found to be associated with CLIP in the absence of both H2-M isoforms.