Strong Influence of the Processing of the Antigen on Negative Effects on T Cell Activation by Regions Outside the Determinant Area of Bovine αs1-Casein

α_s1-Casein can elicit a proliferative response in responding T cell clone 3D20 cells (specific for I-A^b plus fragment 136–151), even when using fixed splenic antigen-presenting cells (APC) not carrying antigen processing ability. The order of potency of each tested antigen for fixed APC was the determinant peptide (136–151) > the long peptide (136–195) > the intact protein (199 residues), indicating that regions outside the determinant area negatively affected the stimulatory potency of the antigens. On the other hand, the order for normal splenic APC was the short peptide > the intact protein > the long peptide. This shows that negative effects by regions outside the determinant area were strongly influenced by the antigen processing.     

The molecular basis of the requirement for antigen processing of pigeon cytochrome c prior to T cell activation

Antigen-induced activation of T lymphocytes that co-recognize Ia molecules has been shown to require an antigen-processing step by the presenting cell before T cell stimulation can occur. In this report, we demonstrate that antigen presentation of pigeon cytochrome c to an E kappa beta:E kappa alpha-restricted T cell hybridoma, 2C2, is inhibited by pretreatment of the antigen-presenting cells (APC) either with chloroquine or with fixation by paraformaldehyde. The chloroquine effect was partially reversible after 22 hr; the paraformaldehyde effect was not. In contrast, these treatments had little or no effect on the presentation of the carboxy-terminal cyanogen bromide cleavage fragment of pigeon cytochrome c, residues 81 to 104. There was at least a 50-fold greater potency of the fragment, as compared to that of the intact molecule, when paraformaldehyde-fixed APC were used. In addition, the fixed cells did not present synthetic fragments of the cytochrome c that were nonstimulatory when presented by unfixed cells. This observation showed that the loss of potency, demonstrated previously for analogs of pigeon cytochrome c with single amino acid substitutions at positions such as 99, was not a consequence of an alteration in the rate of antigen-processing. This result is consistent with our earlier hypothesis that these residues are contact amino acids with the antigen-specific T cell receptor or the Ia molecule. The major goal of these experiments was to define the molecular transition that occurred as a result of antigen processing. To achieve this end, we tested a variety of pigeon cytochrome c molecules and fragments for their ability to be presented by paraformaldehyde-fixed APC. Apocytochrome c, the denatured form of the molecule with the heme removed, could not be presented by the fixed cells, nor could the fragment 60-104, derived by acid cleavage of the tryptophan at position 59. Both molecules stimulated an IL 2 response from the T cell hybridoma when unfixed APC were utilized, demonstrating that the conditions used to prepare these two molecules did not destroy their antigenic determinant. In contrast, carboxy-terminal fragments, both native and synthetic, ranging in size from 16 to 39 amino acids, were capable of stimulating in the presence of paraformaldehyde-fixed APC. In particular, the partial-digest cyanogen bromide fragment, residues 66 to 104, was only twofold less potent than the pigeon fragment 81-104.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro processing of insulin for recognition by murine T cells results in the generation of A chains with free CysSH.

Studies on the processing of insulin as an Ag for the presentation to MHC class II-restricted T cells revealed that the amino acid residues 1-14 of the insulin A chain are recognized by insulin-specific T cells. An A1-14 peptide containing three cys-residues that were protected by S-sulfonate groups still needed processing by APC for efficient presentation similar to native insulin. We suspected that reductive deblocking or opening of disulfide bonds that generates CysSH-residues may be an essential processing step for these Ag. Due to the instability of SH-groups it was not possible to test A chain peptides with free SH-groups in the usual way for processing-independent presentation by fixed APC. However, under acidic conditions (pH 5) during APC pulsing with the Ag we could demonstrate that the freshly reduced A1-14 fragment as well as reduced insulin are able to bind to Ia Ag and to stimulate appropriate T cells without further processing. Various substitutions of cys-residues by Ser within this peptide revealed that only CysA7 is critical for Ia binding and/or T cell recognition. In intact insulin, this residue links the A chain containing the T cell epitope to the B chain. Therefore, we propose that insulin processing is not dependent on proteolysis or on the generation of a conformational determinant but on the separation of A and B chains resulting in A chains whose cys-residues are converted into CysSH.     

T lymphocyte recognition of insolubilized peptide antigen

To study the role of antigen-presenting cells (APC) in T lymphocyte responses, the stimulation requirements of a murine T cell hybridoma specific for the peptide antigen human fibrinopeptide B (hFPB)/I-Ak was examined. The fine specificity of T cell recognition of this peptide was determined by using several hFPB homologs and analogs, which indicated that the intact 14-amino acid peptide must remain intact to preserve the antigenic determinant, and that the carboxyl terminal Arg14 was important for T cell responses. Of particular interest was the finding that APC-associated hFPB failed to stimulate the T cells, and that activation was only observed with soluble peptide or by brief hFPB treatment of the T cells and APC mixed together. In addition, hFPB covalently bound to agarose beads was able to cause T cell activation, provided that I-Ak+ APC were also present in the culture. A number of control experiments were performed that showed that hFPB was not released from the bead and that the antigenic peptide involved in T cell responses remained bound to the beads. These results indicate that the form of the hFPB peptide antigen recognized by this T cell can be provided separately from APC.     

Identification of MHC class II-associated peptides that promote the presentation of toxic shock syndrome toxin-1 to T cells

Previous studies have shown that the DM-deficient cell line, T2-I-A(b), is very inefficient at presenting toxic shock syndrome toxin 1 (TSST-1) to T cells, suggesting that I-A(b)-associated peptides play an essential role in the presentation of this superantigen. Consistent with this, the loading of an I-A(b)-binding peptide, staphylococcal enterotoxin B 121-136, onto T2-I-A(b) cells enhanced TSST-1 presentation >1000-fold. However, despite extensive screening, no other peptides have been identified that significantly promote TSST-1 presentation. In addition, the peptide effect on TSST-1 presentation has been demonstrated only in the context of the tumor cell line T2-I-A(b). Here we show that peptides that do not promote TSST-1 presentation can be converted into "promoting" peptides by the progressive truncation of C-terminal residues. These studies result in the identification of two peptides derived from IgGV heavy chain and I-Ealpha proteins that are extremely strong promoters of TSST-1 presentation (47,500- and 12,000-fold, respectively). We have also developed a system to examine the role of MHC class II-associated peptides in superantigen presentation using splenic APC taken directly ex vivo. The data confirmed that the length of the MHC class II-bound peptide plays a critical role in the presentation of TSST-1 by splenic APC and showed that different subpopulations of APC are equally peptide dependent in TSST-1 presentation. Finally, we demonstrated that the presentation of staphylococcal enterotoxin A, like TSST-1, is peptide dependent, whereas staphylococcal enterotoxin B presentation is peptide independent.     

Antigenic properties of synthetic fragments of the heavy chain of influenza virus A hemagglutinin

The antigenic properties of the synthetic peptides corresponding to the regions 122-133, 136-147, 154-164 of the virus A/Aichi/2/68 hemagglutinin heavy chain have been studied. The 122-133 and 136-147 peptides comprise together almost whole antigenic determinant A, while the 154-164 peptide is a part of determinant B. Rabbits immunized by the peptides conjugated with carrier-protein BSA gave the high immune response to hapten-peptides. Each antiserum reacted only with homologous conjugate. All the antipeptide serums reacted with the virus A/Aichi/2/68 fixed on the base. Conjugate of the 136-147 peptide reacted with the rabbit antiserum against the virus A/Aichi/2/68 rendering the direct evidence to location of at least one hemagglutinin antigenic determinant in the region 136-147.     

Functional and biochemical parameters of peptide antigen presentation

To understand the mechanism by which peptide antigens are processed and presented to T cells, we examined the T-cell response to the 13-amino-acid peptide alpha-melanocyte-stimulating hormone (alpha-MSH). To determine the fine specificity of T-cell recognition, T cells specific for alpha-MSH, and genetically restricted by I-Ab/d, were challenged with different alpha-MSH analogs and homologs. It was found that intact alpha-MSH, including the blocked amino and carboxy termini of the native molecule, was required for T-cell responsiveness. Antigen-presenting cells (APC) could be briefly pulsed with alpha-MSH and then present the alpha-MSH antigenic determinant to T cells, indicating that the relevant antigen was retained by the APC. APC stimulatory capacity was dramatically reduced by aldehyde treatment of the APC, or by pulsing the APC with alpha-MSH at low temperature. Efficient alpha-MSH pulsing was also impaired by treatment of the APC with the carboxylic ionophore, monensin, but not by the lysosomotropic agents chloroquine and methylamine. In addition, isolated APC plasma membranes added to the T cells in the presence of soluble alpha-MSH were not stimulatory. However, plasma membranes isolated from APC that had been previously pulsed with alpha-MSH retained stimulatory activity for T-cell responses. The only detectable alpha-MSH contained in these pulsed APC membranes was in an acid-stable complex of higher molecular weight than native peptide. The amount of alpha-MSH detected in the cellular membrane fraction isolated by density gradient sedimentation was also reduced by treatments that reduced the APC stimulatory capacity, such as pulsing at low temperature or in the presence of monensin. Taken together, these results suggest that processing of alpha-MSH is unlike that heretofore described for other peptide antigens and seems to involve APC handling to form the stimulatory moiety presented on the APC surface.     

Influence of the H-2u haplotype on immune function in F1 hybrid mice. I. Antigen presentation

Previously, we reported that myelin basic protein (MBP) peptide 1-37 contains an encephalitogenic epitope for PL/J mice, and MBP peptide 89-169 is encephalitogenic for SJL/J mice. (SJPL)F1 hybrid mice do not respond to immunization with these peptides in a co-dominant manner because the encephalitogenic response to peptide 1-37 dominates. To examine this phenomenon more closely, we tested the ability of MBP-primed parental or F1 T cells to respond to MBP or MBP peptides in the context of PL, SJL, or F1 antigen-presenting cells (APC). It was found that the F1 T cells responded to either the protein or the peptides when these were presented in the context of F1 or PL APC. However, F1 T cells would not respond to MBP in the context of SJL APC, although the latter cells were functionally intact. This effect was not antigen-specific because SJL APC would not present ovalbumin or PPD to primed F1 T cells. F1 T cells from mice immune to the strongly antigenic bacterium Listeria monocytogenes responded to bacterial antigens presented by SJL APC, although at a significantly lower level compared to the results obtained when these antigens were presented by F1 or PL APC. This finding implied that unbalanced antigen presentation was a quantitative rather than a qualitative phenomenon. When F1 hybrid mice from other strain combinations were tested, a similar effect was observed whenever one of the parental strains was PL/J. This effect was mapped to the MHC in MHC-congenic B10 mice.     

Genetic fusion of chemokines to a self tumor antigen induces protective, T-cell dependent antitumor immunity

We converted a model, syngeneic, nonimmunogenic tumor antigen into a vaccine by fusing it with a proinflammatory chemokine. Two chemokines, interferon inducible protein 10 and monocyte chemotactic protein 3, were fused to lymphoma Ig variable regions (sFv). The sFv-chemokine fusion proteins elicited chemotactic responses in vitro and induced inflammatory responses in vivo. Furthermore, in two independent models, vaccination with DNA constructs encoding the corresponding fusions generated superior protection against a large tumor challenge (20 times the minimum lethal dose), as compared with the best available protein vaccines. Immunity was not elicited by controls, including fusions with irrelevant sFv; fusions with a truncated chemokine that lacked receptor binding and chemotactic activity; mixtures of free chemokine and sFv proteins; or naked DNA plasmid vaccines encoding unlinked sFv and chemokine. The requirement for linkage of conformationally intact sFv and functionally active chemokine strongly suggested that the mechanism underlying these effects was the novel targeting of antigen presenting cells (APC) for chemokine receptor-mediated uptake of antigen, rather than the simple recruitment of APC to tumor by the chemokine. Finally, in addition to superior potency, these fusions were distinguished from lymphoma Ig fusions with granulocyte-macrophage colony-stimulating factor or other cytokines by their induction of critical effector T cells.     

Dissection of the functions of antigen-presenting cells in the induction of T cell activation

The functions of antigen-presenting cells (APC) in the initiation of T cell activation was examined by culturing antigen-bearing guinea pig macrophages (M phi) with T cells obtained from antigen-primed animals. Although such antigen-bearing M phi stimulated primed syngeneic T cell DNA synthesis, as assessed by tritiated thymidine incorporation, paraformaldehyde fixation (0.15% for 1 min at 37 degrees C) abolished this capacity. Analysis with acridine orange staining indicated that fixed antigen-bearing M phi could not trigger primed syngeneic T cells to progress from the G0 to the G1 phase of the cell cycle. The addition of control non-antigen-bearing syngeneic or allogeneic M phi but not interleukin 1 or 2 to cultures of T cells and fixed APC permitted a proliferative response. Although the interaction between fixed antigen-bearing M phi and responding T cells was genetically restricted, there was no similar restriction for the supplemental control M phi. In fact, completely Ia-negative endothelial cells (EC) and fibroblasts (FB) could restore antigen responsiveness to cultures of fixed antigen-bearing M phi and syngeneic responding T cells, although they could not directly present antigen. Moreover, metabolically intact accessory cells, including Ia-negative EC and FB, could take up and process antigen to an immunogenic moiety, which fixed Ia-positive M phi could present to primed T cells. These data indicate that recognition of the antigen-Ia complex on an APC is necessary but not sufficient to trigger proliferation of freshly obtained primed T cells. The results additionally support the conclusion that APC carry out at least two separate functions necessary for the initiation of antigen-induced T cell activation. Not only must the APC display the antigen-Ia complex, but it must also convey another required effect. This influence, which apparently involved the establishment of cell to cell contact, was neither Ia nor antigen dependent and could only be provided by a metabolically intact cell. By contrast, genetically restricted antigen presentation could be accomplished by a fixed Ia-positive cell. Only when both the antigen-Ia complex and the influence of an intact accessory cell were provided by the same or different accessory cell were T cells triggered to enter the cell cycle.     

Contribution of antigen processing to the recognition of a synthetic peptide antigen by specific T cell hybridomas

Most antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the alpha-helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I-Ad. Hybridoma A.1.1 responds to EYK(EYA)4 as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5 sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the alpha-helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC molecules.     

Preserved MHC Class II Antigen Processing in Monocytes from HIV-Infected Individuals

Background

MHC-II restricted CD4+ T cells are dependent on antigen presenting cells (APC) for their activation. APC dysfunction in HIV-infected individuals could accelerate or exacerbate CD4+ T cell dysfunction and may contribute to increased levels of immunodeficiency seen in some patients regardless of their CD4+ T cell numbers. Here we test the hypothesis that APC from HIV-infected individuals have diminished antigen processing and presentation capacity.

Methodology/Principal Findings

Monocytes (MN) were purified by immuno-magnetic bead isolation techniques from HLA-DR1.01+ or DR15.01+ HIV-infected and uninfected individuals. MN were analyzed for surface MHC-II expression and for antigen processing and presentation capacity after overnight incubation with soluble antigen or peptide and HLA-DR matched T cell hybridomas. Surface expression of HLA-DR was 20% reduced (p<0.03) on MN from HIV-infected individuals. In spite of this, there was no significant difference in antigen processing and presentation by MN from 14 HIV-infected donors (8 HLA-DR1.01+ and 6 HLA-DR15.01+) compared to 24 HIV-uninfected HLA-matched subjects.

Conclusions/Significance

We demonstrated that MHC class II antigen processing and presentation is preserved in MN from HIV-infected individuals. This further supports the concept that this aspect of APC function does not further contribute to CD4+ T cell dysfunction in HIV disease.     

Adherent Ia+ murine cell lines with characteristics of dendritic cells. II. Characteristics of I region-restricted antigen presentation

The ability of an adherent Ia+, interleukin 1+ (IL-1) tumor cell line (P388AD) to present turkey gamma-globulin (TGG) to primed T lymphocytes was demonstrated and compared with normal antigen-presenting cells (APC) found in mouse spleen. P388AD tumor cells presented TGG to long-term cultures of TGG-reactive T cells (LTTC) and to lymph node-derived T cells which were enriched on nylon wool columns and subsequently depleted of endogenous antigen-presenting cells with anti-Ia antisera and complement. MHC-restricted antigen presentation by P388AD was observed when long-term cultures of TGG-reactive T cells were used as the responding T-cell population. Furthermore, antisera directed against I-region determinants expressed on the P388AD tumor cells inhibited TGG-specific T-cell proliferation in a dose-related fashion, suggesting a functional role for the tumor cell-associated Ia molecules. The kinetics of antigen presentation to LTTC by P388AD were similar to the kinetics observed for splenic APC, although the magnitude of the proliferative response to LTTC to TGG was generally lower when antigen (Ag) was presented by the tumor cells compared to splenic antigen-presenting cells (APC). However, the magnitude of T-cell proliferation of immune lymph node (LN) T cells was comparable when Ag was presented on tumor cells or splenic APC. Several experiments suggested that Ag uptake and/or processing may be less effective in P388AD tumor cells as compared to normal splenic APC. A nonadherent Ia+, IL-1- tumor cell line (P388NA), which was isolated from the same parental tumor as P388AD, was also tested for the ability to present Ag to primed T lymphocytes and Ag-reactive LTTC. In contrast, to P388AD, the nonadherent tumor cell failed to present TGG under identical culture conditions even though Ia molecules were expressed on the tumor cells and Ag uptake had occurred. However, the defect in Ag presentation by P388NA could be corrected if an exogenous source of purified interleukin 1 was supplied to the cultures. A unique opportunity thus exists with both the P388AD and P388NA tumor cell lines to decipher some of the molecular interactions leading to T-cell proliferation during antigen presentation.     

Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity

At the interface between T cell and antigen-presenting cell (APC), peptide antigen presented by MHC (pMHC) binds to the T cell receptor (TCR) and initiates signaling. The mechanism of TCR signal initiation, or triggering, remains unclear. An interesting aspect of this puzzle is that although soluble agonist pMHCs cannot trigger TCR even at high concentrations, the same ligands trigger TCR very efficiently on the surface of APCs. Here, using lipid bilayers or plastic-based artificial APCs with defined components, we identify the critical APC-associated factors that confer agonist pMHCs with such potency. We found that CD4⁺ T cells are triggered by very low numbers of monomeric agonist pMHCs anchored on fluid lipid bilayers or fixed plastic surfaces, in the absence of any other APC surface molecules. Importantly, on bilayers, plastic surfaces, or real APCs, endogenous pMHCs did not enhance TCR triggering. TCR triggering, however, critically depended upon the adhesiveness of the surface and an intact T cell actin cytoskeleton. Based on these observations, we propose the receptor deformation model of TCR triggering to explain the remarkable sensitivity and specificity of TCR triggering.     

Antigen Presenting B Cells Facilitate CD4 T Cell Cooperation Resulting in Enhanced Generation of Effector and Memory CD4 T Cells

We show that the in vivo generation of cytokine-producing CD4 T cells specific for a given major histocompatibility class-II (MHCII)-binding peptide of hen egg lysozyme (HEL) is facilitated when mice are immunized with splenic antigen presenting cells (APC) pulsed with this HEL peptide and another peptide that binds a different MHCII molecule. This enhanced generation of peptide-specific effector CD4 T cells requires that the same splenic APC be pulsed with both peptides. Pulsed B cells, but not pulsed dendritic cells (DCs), can mediate CD4 T cell cooperation, which can be blocked by disrupting OX40-OX40L (CD134-CD252) interactions. In addition, the generation of HEL peptide-specific CD4 T cell memory is greater when mice are primed with B cells pulsed with the two peptides than with B cells pulsed with the HEL- peptide alone. Based on our findings, we suggest CD4 T cell cooperation is important for vaccine design, underlies the phenomenon of “epitope-spreading” seen in autoimmunity, and that the efficacy of B cell-depletion in the treatment of human cell-mediated autoimmune disease is due to the abrogation of the interactions between autoimmune CD4 T cells that facilitates their activation.     

Characterization of legumain

The mammalian legumain, also called asparaginyl endopeptidase (AEP), is critically involved in the processing of bacterial antigens for MHC class II presentation. In order to investigate the substrate specificity of AEP in the P1' position, we created a peptide library and digested it with purified pig kidney AEP. Digestion was less efficient only when proline was in the P1' position. Maximum AEP activity was found in lysosomal fractions of different types of antigen presenting cells (APC). When the multiple sclerosis-associated autoantigen myelin basic protein (MBP) was digested with AEP, the immunodominant epitope 83-99 was destroyed. Myoglobin as an alternative substrate was AEP resistant. These results suggest an important, but not necessarily critical role for AEP in lysosomal antigen degradation.     

Studies on the capacity of B cells as well as T cells to serve as accessory cells for the activation of herpes simplex virus-specific cytolytic T cells

Experiments were conducted in an effort to determine the ability of B and T lymphocytes to serve as APC for the activation of HSV-primed splenic T cells to become class I-restricted, HSV-specific CTL. The results showed that both freshly isolated splenic B cells as well as LPS and dextran sulfate (L/D)-activated B cells were effective at stimulating the generation of CTL during a 5-day in vitro culture. There was no requirement for the addition of exogenous IL-2 to the culture and, since murine B cells do not appear to express either membrane or secreted IL-1, this lymphokine appears to either not be required for the activation of virus-specific CTL or to be provided by the T cells themselves. When normal B cells were separated into fractions enriched for resting vs activated cells and then tested for their ability to stimulate the generation of HSV-specific CTL, it was found that while the activated B cells were quite effective at stimulating the generation of CTL, resting B cells were ineffective at carrying out this function. In contrast to normal B cells, normal T cells were unable to act as APC. However, Con A-activated T lymphoblasts were equivalent to L/D B cells in their ability to mediate the generation of CTL activity. L/D B cells that had been pulsed with HSV and then incubated at 37 degrees C for greater than 1 h could be fixed with paraformaldehyde and were still able to function as APC. The finding that L/D B cells, that had been fixed at 1 h or less after exposure to HSV, were unable to function as APC suggested that either active Ag "processing" steps may be required for the presentation of Ag in the context of class I molecules or that there is a requirement for the synthesis of viral protein Ag before presentation.     

A therapeutic peptide in lupus alters autophagic processes and stability of MHCII molecules in MRL/lpr B cells

The P140 phosphopeptide encompassing residues 131–151 of the spliceosomal U1-70K snRNP protein

displays protective properties in lupus patients and MRL/lpr mice. It increases peripheral blood lymphocyte apoptosis via a mechanism involving γδ T cells. After intravenous administration, P140 accumulates in the lungs and spleen. It binds both the HSC70/Hsp73 chaperone and MHC class II (MHCII) molecules, which colocalize in splenic MRL/lpr B cells. Expression of HSC70 and MHCII, which is increased in MRL/lpr splenic B

cells, is diminished after P140 administration. P140 impairs refolding properties of HSC70 and alters expression of stable MHCII molecules in B lymphocytes. In MRL/lpr B cells, P140 increases the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulation of autophagic flux. Our study reveals a very unique property of P140 peptide that alters the autophagy pathway leading to a defect of endogenous (auto)antigen processing in MRL/lpr antigen-presenting B cells and a decrease of T cell priming and signaling.     

Silencing of immunodominant epitopes by contiguous sequences in complex synthetic peptides.

We have previously shown that the T cell response to the synthetic peptide cI12-26:NP365-380 (covalently linked epitopes of lambda repressor (cI) and influenza A nucleoprotein (NP) polypeptides) requires amino acid sequences located in the junctional region between the cI12-26 and NP365-380 epitopes in the H-2d and H-2k haplotypes. In this study, we show that the dominant epitope of cI12-26:NP365-380 in H-2b mice is also located within the junctional region of the peptide, indicating that the same amino acid sequence is immunodominant in three different H-2 haplotypes. Based on results using fixed APC, there was no qualitative difference in epitope recognition due to antigen processing. In addition, antigen presentation by APC expressing mutant I-A molecules constructed by hemiexon shuffling of regions of the molecule containing primarily beta sheet or alpha helix showed that many different substitutions were permissive for at least one of the T hybridomas. More importantly, however, when the junctional sequences are covalently linked in composite synthetic peptides containing additional previously defined T cell epitopes, antigenicity of the immunodominant junctional region was silenced and a new epitope assumed immunodominance. Thus, immunodominance does not correlate with the primary amino acid sequence of the potential epitope. Instead, the immunodominant epitope is determined by complex interactions among the epitopes, which most likely depend on the structural conformation of the composite peptide.     

A therapeutic peptide in lupus alters autophagic processes and stability of MHCII molecules in MRL/lpr B cells

The P140 phosphopeptide encompassing residues 131-151 of the spliceosomal U1-70K snRNP protein displays protective properties in lupus patients and MRL/lpr mice. It increases peripheral blood lymphocyte apoptosis via a mechanism involving γδ T cells. After intravenous administration, P140 accumulates in the lungs and spleen. It binds both the HSC70/Hsp73 chaperone and MHC class II (MHCII) molecules, which colocalize in splenic MRL/lpr B cells. Expression of HSC70 and MHCII, which is increased in MRL/lpr splenic B cells, is diminished after P140 administration. P140 impairs refolding properties of HSC70 and alters expression of stable MHCII molecules in B lymphocytes. In MRL/lpr B cells, P140 increases the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulation of autophagic flux. Our study reveals a very unique property of P140 peptide that alters the autophagy pathway leading to a defect of endogenous (auto)antigen processing in MRL/lpr antigen-presenting B cells and a decrease of T cell priming and signaling.