Streptococcus pyogenes type 5 M protein is an antigen, not a superantigen, for human T cells

M proteins are coiled-coil dimers expressed on group A streptococcal cell surfaces. They have an importantrole in host antistreptococcal immunity and in poststreptococcal autoimmune sequelae. Controversy has arisen regardingwhether type 5 M proteins are superantigenic for human T cells. To investigate this, we have produced and tested M5 inthe form of two novel recombinant proteins. We found no evidence of superantigenicity using either recombinant whole M5protein (rM5) or recombinant pep M5 protein (rpepM5) to activate peripheral blood mononuclear cells (PBMC) from healthyadult volunteers. Short-term, rM5-specific T-cell lines from different subjects were uniformly self-APC restricted andshowed no consistent pattern of TCR Vβ usage. A synthetic peptide of M5 residues 217–237 was found tocontain epitope(s) recognized by some rM5-specific human T cells. PBMC responses to rM5 and rpepM5 in 3- and 7-dayproliferation assays were characteristic of antigenic rather than superantigenic stimulation. We conclude that type5 M protein activates human T cells as a conventional antigen.     

MHC class II-associated invariant chain peptide replacement by T cell epitopes: engineered invariant chain as a vehicle for directed and enhanced MHC class II antigen processing and presentation

Proteolysis of the invariant chain (Ii) leads to the generation of abundant MHC class II-associated invariant chain peptides (CLIP), which bind in the MHC class II binding groove via supermotifs in a manner similar to that of antigenic peptides. We have engineered an Ii vector with the capacity to express any antigenic peptide of interest instead of CLIP, for T cell stimulation. When peripheral blood mononuclear cells (PBMC) were pulsed with Ii hybrids encoding T cell epitopes of tetanus toxin or acetylcholine receptor, stimulation of T cells was dramatically enhanced compared to stimulation after priming with either the native or recombinant proteins. Site-specific insertion of antigenic sequences into the CLIP region promoted enhanced antigenicity of Ii hybrids which were shown to be processed intracellularly in a chloroquine-sensitive compartment. Naturally processed T helper epitopes were visualized directly on the surface of PBMC and identified as analogs of CLIP associated with MHC class II molecules. This novel Ii vector provides a flexible and efficient system for the delivery of defined peptide epitopes to T cells which might be useful in the development of specific vaccines and in the study of intracellular processing.     

Podocytes present antigen to activate specific T cell immune responses in inflammatory renal disease

Infiltration of activated T cells into renal tissue plays an essential role in inflammatory nephropathy. However, the mechanism enabling the renal recruitment and activation of T cells remains elusive. Here we report that inflammatory cytokine-promoted antigen presentation by podocytes is a key for recruiting and activating specific T cells. Our results showed that diabetes-associated inflammatory cytokines IFNγ and IL-17 all upregulated expression of MHC-I, MHC-II, CD80 and CD86 on the podocyte surface. Both IFNγ and IL-17 stimulated the uptake and processing of ovalbumin (OVA) by mouse podocytes, resulting in presentation of OVA antigen peptide on the cell surface. OVA antigen presentation by podocytes was also validated using human podocytes. Furthermore, OVA antigen-presenting mouse podocytes were able to activate OT-I mouse T cell proliferation and inflammatory cytokine secretion, which in turn caused podocyte injury and apoptosis. Finally, OT-I mice subjected to direct renal injection of OVA plus IFNγ/IL-17 but not OVA alone exhibited OVA antigen presentation by podocytes and developed nephropathy in 4 weeks. In conclusion, antigen presentation by podocytes under inflammatory conditions plays an important role in activating T cell immune responses and facilitating immune-mediated glomerular disease development. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Deficient antigen processing of a protein quaternary structure can be overcome by receptor-mediated uptake

Human chorionic gonadotropin (hCG) is a dimer of non-covalently associated alpha (hCG-α) and beta (hCG-β) subunits. This molecule was used to study whether receptor-mediated uptake influences the presentation of a protein quaternary structure. Unprimed splenocytes and a B cell lymphoma were capable of presenting only the free (hCG-α) but not the combined (hCG) α subunit to hCG-α T cell hybridomas, while hCG-α-primed lymph node cells (LNC) responded to both hCG-α and hCG. As antigen (Ag)-specific antigen-presenting cells (APC) present in the hCG-α-primed LNC population may be potentially effective for presenting hCG, we investigated the role of specific Ag capture, through mIg and FcγR, in the processing and presentation of hCG and hCG-a to HAG 5, a T cell hybridoma directed against the immunodominant region (amino acids 61-81) of hCG-α. Results showed that only B cells bearing membrane immunoglobulin capable of recognizing hCG-α and hCG, and present in hCG-α-primed mice, were extremely effective in presenting the free as well as the combined a subunit. The effect of FcR-mediated uptake was analyzed using a B cell line transfected with the FcγRII-B2 gene to present immune complexes of either hCG-α or hCG. We found that hCG-α and hCG were presented equally well, whatever the Ag-binding site of each antibody to hCG or its a subunit. Using HBG 6, an hCG-β Tcell hybridoma, we performed similar experiments with the FcγRII-B2 cell line and determined that the potentiation of hCG presentation to HBG 6 was similar to that observed with HAG 5. Then kinetic experiments were performed to examine the effect of Ag uptake through FcR on processing. Results demonstrated that the uptake pathway drastically influenced the expression of α T cell determinants in the αβ dimer. In addition, treatment with cycloheximide, a protein synthesis inhibitor, only impaired the ability of APC to present specifically captured Ag. Thus, the processing pathway for specifically captured Ag might be different from the pathway used to process nonspecifically captured Ag. This observation might explain why receptor-enhanced uptake bypasses the inefficient processing of the hCG quaternary structure and enables similar efficiency in the presentation of α and β T cell specificities. These findings provide new insight into the antigenicity of oligomeric molecules, which is modified whether antigen capture is specific or not.     

Indirect presentation in the thymus limits naive and regulatory T‐cell differentiation by promoting deletion of self‐reactive thymocytes

Acquisition of T‐cell central tolerance involves distinct pathways of self‐antigen presentation to thymocytes. One pathway termed indirect presentation requires a self‐antigen transfer step from thymic epithelial cells (TECs) to bone marrow‐derived cells before the self‐antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context‐dependent, potentially due to variation in self‐antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane‐bound transgenic self‐antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow‐derived antigen‐presenting cells, insHEL‐mediated thymocyte deletion was abolished, whereas thyroHEL‐mediated deletion occurred at a later stage of thymocyte development and Foxp3⁺ regulatory T‐cell differentiation increased. Indirect presentation increased the strength of T‐cell receptor signalling that both self‐antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self‐reactive thymocytes.     

Targeting TARP, a novel breast and prostate tumor-associated antigen, with T cell receptor-like human recombinant antibodies

MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptide-specific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRgamma alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes.     

Non-covalent presentation of sulfamethoxazole to human CD4+ T cells is independent of distinct human leucocyte antigen-bound peptides

BACKGROUND: It has been shown that drugs comprise a group of non-peptide antigens that can be recognized by human T cells in the context of HLA class II and that this recognition is involved in allergic reactions. Recent studies have demonstrated a MHC-restricted but processing- and metabolism-independent pathway for the presentation of allergenic drugs such as lidocaine and sulfamethoxazole (SMX) to drug-specific T cells. However, there is little information so far on the precise molecular mechanisms of this non-covalent drug presentation. OBJECTIVE: The aim of this study was to evaluate the requirements for a specific peptide occupying the groove of the MHC class II molecule for the efficient presentation of non-covalently bound drugs to CD4+ T cells. METHODS: We analysed the effect of coincubation or prepulse of antigen presenting cells (APC) with different peptides on the proliferative responses of SMX-specific CD4+ T cell clones. In a second series of experiments, we eluted HLA-bound peptides from the surface of antigen presenting cells by mild acid treatment. Successful removal of peptides was tested directly using labelled peptides and functionally by monitoring activation and proliferation of peptide-specific T cell clones. Finally, the presentation of SMX to SMX-specific T cell clones before and after elution of MHC class II bound peptides was tested. RESULTS: We found that neither peptide coincubation nor peptide prepulse of APC altered the proliferative response of SMX-specific T cells. APC treated with the acid for a short time retained cell viability, MHC class II expression and antigen presenting cell function. However, defined peptides could be eluted from surface MHC class II molecules nearly quantitatively. Nevertheless, the chemically non-reactive drug SMX could still be presented to specific T cells independent of the presence of distinct self-peptides. CONCLUSION: Our data suggest that small molecules like drugs can bind to a multitude of HLA-bound peptides or that, similar to superantigens, they might bind directly to HLA.     

Peptides of 23 residues or greater are required to stimulate a high affinity class II-restricted T cell response

Helper T cells recognize fragments of antigen bound to the class II molecules on the surface of antigen-presenting cells. Naturally processed antigenic fragments have been isolated from the class II molecules and shown to be heterogeneous in length, ranging from 13 to 25 residues, and to vary at both the N and C termini. A 15-residue peptide in an extended conformation is predicted to fit in an open peptide-binding cleft of the class II molecules. Thus, the longer peptides observed bound to class II presumably have regions which reside outside the cleft. It is not known if the additional length contributes significantly to T cell activation. We have carried out a systematic analysis of the antigenicity of peptides of increasing length beyond the minimally defined T cell antigenic peptide. Here we show that the full functional activities of peptides representing the major antigenic determinant of the protein antigen, cytochrome c, minimally require that the peptides be 23 amino acids long. The long peptides do not require processing and are presented by purified class II molecules incorporated into synthetic membranes, indicating that such peptides associate directly with class II and require no additional cellular machinery for presentation. We also show that a hybrid peptide, 51 residues in length, containing a 29-residue cytochrome c peptide and a “promiscuous” peptide of tetanus toxoid, is more antigenic than the 23-residue peptide alone and significantly, does not require processing. Thus, the additional peptide length, although not predicted to bind in the peptide-binding groove of the MHC class II molecule, has a significant impact on the ability of the peptides to stimulate T cell responses maximally.     

Lowering the affinity between antigen and the B cell receptor can enhance antigen presentation

The B cell receptor (BCR) enables antigen-specific B cells to bind, internalize and target antigens for processing into small peptide fragments. These epitopes are then expressed on the plasma membrane in association with MHC class II molecules for recognition by CD4+ T cells. The affinity of the interaction between the BCR and antigen plays an important part in determining T cell epitope generation. In this report we provide evidence that the efficiency of antigen presentation by specific B cells does not need to be directly proportional to antigen/BCR affinity. We show that increased presentation can result from lowering the affinity of the antigen/BCR interaction. This finding suggests a novel mechanism by which B cells can recruit T cell help and obtain survival signals. Activation of these cells may have consequences for the generation of the B cell repertoire.     

Idiotype-induced T cell stimulation requires antigen presentation in association with HLA-DR molecules

An important and yet unresolved question concerns the mode of T cell recognition of idiotypic epitopes on immunoglobulin molecules in humans. Results from murine and human studies show that some idiotype-specific T cells recognize conformational epitopes on immunoglobulin, and such T cells are not MHC-restricted. In the present study T cell stimulation induced by idiotypic determinants on the autologous monoclonal IgG (M-components) from patients with monoclonal gammopathies was studied. In parallel, T cell stimulation in response to a conventional antigen, purified protein derivative, was also examined. It is shown that, as with conventional antigen, idiotype-induced T cell stimulation requires the presence of antigen-presenting cells (APC; monocytes and/or B cells), and is MHC class II (DR)-restricted. B cells, but not monocytes, can present idiotypic determinants to T cells at very low antigen concentrations, while monocytes do so only when antigen is present at high concentrations. Antigen processing and presentation is abrogated by treatment of APC with chloroquine. In conclusion, our study demonstrates that human idiotype-specific T cells recognize processed idiotypic determinants presented by MHC class II (HLA-DR) molecules on APC, and that B cells require about 1000-fold less antigen than monocytes.     

A novel T‐cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice

We used a newly generated T‐cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10‐mer) or SSVWNATTA (9‐mer) in complex with class I MHC antigen H‐2D^b, were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8⁺ T cells can detect 10‐ and 9‐mer length variants similarly. This result varies from the idea that a given T‐cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10‐mer was presented on the surface of uninfected and infected CD8α⁺CD11c⁺ dendritic cells, which suggests the use of direct and cross‐presentation pathways. In contrast, CD11b⁺CD11c^? cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell‐type specific mechanisms of antigen presentation in vivo.     

Targeting antigen to CD19 on B cells efficiently activates T cells

CD19 is a B cell-surface molecule that participates as an important regulatory signaling complex for antigen bound at the surface by Ig. Triggering of CD19 through its linkage with CD21 amplifies signals transduced through the Src family kinases and modulates B cell differentiation in response to antigen. This study examines the kinetics of antigen uptake and processing of antigen directly targeted to the CD19 protein on purified B cells. We have demonstrated that the antigen internalized within minutes through CD19 forms a cap at the B cell surface and can be found within lysosomes in the cytoplasm in 90 min. B cells acquiring antigen via CD19 express elevated levels of B7-1 and B7-2 co-stimulatory molecules. Moreover, antigen-anti-CD19 complexes administered intravenously bind B cells in vivo and activate antigen-specific T cells more efficiently than non-specific uptake and in a manner similar to antigen taken up through surface IgM on B cells. This work illustrates an important and previously unrecognized mechanism for targeting proteins to B lymphocytes for antigen presentation and activation of CD4 T cells.     

MHC class I restricted T cell responses tolisteria monocytogenes, an intracellular bacterial pathogen

Studies of the murine immune response to infection with the intracellular bacterial pathogenListeria monocytogenes have provided a wealth of information about innate and acquired immune defenses in the setting of an infectious disease. Our studies have focused on the MHC class I restricted, CD8⁺ T cell responses of Balb/c mice toL. monocytogenes infection. Four peptides that derive from proteins thatL. monocytogenes secretes into the cytosol of infected cells are presented to cytotoxic T lymphocyte (CTL) by the H2-K^d major histocompatibility complex (MHC) class I molecule. We have found that bacterially secreted proteins are rapidly degraded in the host cell cytosol by proteasomes that utilize, at least in part, the N-end rule to determine the rate of degradation. The MHC class I antigen processing pathway is remarkably efficient at generating peptides that bind to MHC class I molecules. The magnitude of in vivo T cell responses, however, is influenced to only a small degree by the amount of antigen or the efficiency of antigen presentation. Measurements of in vivo T cell expansion followingL. monocytogenes infection indicate that differences in the sizes of peptide-specific T cell responses are more likely owing to differences in the repertoire of naive T cells than to differences in peptide presentation. This notion is supported by our additional finding that dominant T cell populations express a more diverse T cell receptor (TCR) repertoire than do subdominant T cell populations.     

Influence of B cell receptor ligation and TCR affinity on T-B collaboration in vitro

Co-culture of purified T and B cells obtained from cytochrome c-specific TCR- and hen egg lysozyme (HEL)-specific Ig-transgenic mice was used to examine the role of B cell receptor (BCR) ligation and TCR affinity on the efficiency of T-B cell collaboration. The results showed that BCR ligation of naive B cells with HEL was not required for effective presentation of high-affinity antigen to T cells, although it did enhance activation and division of both T and B cells. Anergic B cells were also effective at presentation of high-affinity antigen and proliferated more than naive B cells in response to T cell help, due to prior exposure to antigen in vivo. Despite the fact that induction of CD86 on anergic B cells following BCR ligation was suboptimal, these cells supported T cell activation and survival in culture as efficiently as naive B cells exposed to HEL. In contrast, when the low-affinity antigen mls-3^a served as the T cell stimulus, BCR ligation was essential to elicit a detectable T cell response. Thus the in vitro model demonstrates that co-stimulation is not an absolute requirement for effective antigen presentation and delivery of T cell help to B cells. Rather, the cooperative effects of BCR ligation and TCR affinity determine the relative requirement for co-stimulation.     

Viral and bacterial minigene products are presented by MHC class I molecules with similar efficiencies

MHC class I molecules present short peptides, usually 8-10 amino acids in length, to CD8⁺ T cells. These peptides are typically generated from full-length endogenously synthesized proteins degraded by the antigen processing machinery of the target cell. However, exogenous proteins, whether originating from intracellular bacteria or parasites or via phagocytosis during cross-presentation, can also be processed for presentation by MHC class I molecules. It is currently not known whether endogenously synthesized proteins and proteins acquired from exogenous sources follow the same presentation pathway. One clue that the processing pathways followed by endogenous and exogenous proteins may not be identical is the vastly different presentation efficiencies reported for viral versus bacterial antigens. Because class I antigen processing involves multiple steps, we sought to determine where in the processing pathway these differences in efficiency occur. To accomplish this, we expressed identical minimal peptide determinants from viral and bacterial vectors using a minigene expression system and determined the rate of peptide-MHC generation per molecule of minigene product synthesized. We found that peptides expressed from either the viral or bacterial vector were presented with virtually identical efficiencies. These results suggest that differences in the processing pathways followed by endogenous versus exogenous proteins most likely occur at a point prior to where free peptide is liberated from full-length protein.     

Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induce IgE-mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam-specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401-binding motif and covalently modified with Pen G via a lysine σ-amino group were found to induce proliferation of Pen G-specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101-binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.     

Peripheral tolerance in T cell receptor-transgenic mice: Evidence for T cell anergy

T cell tolerance can be induced in adult mice by injection of soluble antigenic peptide. The underlying mechanism has been difficult to establish in normal mice due to the low precursor frequency of T cells specific for any given antigen. Therefore, we examined peripheral tolerance in mice transgenic for a T cell receptor specific for a cytochrome c peptide bound to I-E^k. Antigen-specific hyporesponsiveness could be induced in the transgenic mice. We followed the transgene-bearing T cells with a clonotypic monoclonal antibody and found similar numbers of clonotypic T cells in tolerized and control mice. To prevent de novo differentiation of T cells we analyzed thymectomized mice in which antigen-specific hyporesponsiveness was induced. Our analysis of thymectomized transgenic mice showed that antigen-specific T cell hyporesponsiveness following injection of peptide intravenously is not caused by gross elimination of T cells. These data provide evidence for the role of anergy in peripheral tolerance.     

CD40-CD40 ligand interactions stimulate B cell antigen processing

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell-dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen-specific T cell hybrid by B cells co-cultured with insect cells expressing recombinant CD40L or with a CD40-specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross-linking the surface immunoglobulin, and processing of antigen taken up by fluid-phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40-induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA-1 or CD23. CD40 ligation does not affect the biosynthesis of the class II E^k molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen-dependent T cell-B cell interactions.     

In vivo induction of CD4+ T cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant

Macrophages, dendritic cells or B lymphocytes have been shownto play a major role in the presentation of soluble antigensto CD4⁺ T cells. In contrast, the capacity of these cells topresent particulate antigens such as bacterial or parasiticantigens to T cells remains controversial. To investigate thisquestion, well defined particulate antigens were prepared bycovalent linkage of proteins or peptides to 1 µm in diametersynthetic microspheres. The T cell immunogenicity of such particulateantigens was analyzed in vitro and in vivo. In vitro, a solubleprotein such as hen egg lysozyme (HEL) coupled to beads stimulateda strong proliferative T cell response of lymph node cells fromHEL-primed mice or of specific T cell hybridomas. HEL coupledto beads was presented to the specific T cell hybridomas bysplenocytes or by peritoneal macrophages, but not by lymphomaB cells. Immunization of mice with several different proteinantigens or with a synthetic peptide covalently linked to beadsinduced strong CD4⁺ T cell responses in the absence of adjuvant.The strong in vivo immunogenicity of proteins coupled to beadsdid not result from a non-specific adjuvant effect of beadssince covalent linkage of the antigen to beads was strictlyrequired to induce T cell responses in the absence of adjuvant.In vivo treatment by carrageenan showed that macrophages arerequired for the in vivo stimulation of T cell responses bythese particulate antigens. Thus, these results demonstratedthe role of phagocytic cells, especially macrophages, for invivo presentation of particulate antigens. These particulateantigens represent an interesting approach for the developmentof new vaccines, and for the in vivo analysis of the role ofvarious antigen presenting cells in T cell activation and differentiation.