A mouse C kappa-specific T cell clone indicates that DC-SIGN is an efficient target for antibody-mediated delivery of T cell epitopes for MHC class II presentation

In vaccine development, a major objective is to induce strong, specific T cell responses. This might be obtained by targeting antigen to cell surface molecules that efficiently channel the antigen into endocytic compartments for loading of MHC molecules. Antibodies have been used to deliver antigen; however, it is important to define optimal targets on antigen-presenting cells (APC) for efficient delivery. For this purpose, we have established a T cell readout that can be used to screen large numbers of different mAb for their ability to load MHC class II molecules. The novel human CD4+ T cell clone is specific for mouse Ig C kappa (40-48) and restricted by HLA-DR4 (DRA1,B1*0401). DR4 apparently presents both mouse and human C kappa 40-48, but there is no cross-reaction at the T cell level. B cells from DR4 transgenic mice spontaneously process and present the mouse C kappa peptide. The mouse C kappa -specific T cell readout was used to demonstrate that mouse mAb specific for human dendritic cell (DC)-specific ICAM-grabbing non-integrin (DC-SIGN), a novel DC-specific molecule, were 10- to 1000-fold more potent at inducing kappa-specific human CD4+ T cell proliferation compared to control mAb. Consistent with this finding, DC-SIGN-specific mAb were rapidly internalized upon binding and found in intracellular vesicles. These results strongly argue that DC-SIGN-specific mAb are channeled into the MHC class II presentation pathway. Thus, DC-SIGN could be an efficient target for antibody-mediated delivery of T cell epitopes in vaccine development.     

Vesicles bearing MHC class II molecules mediate transfer of antigen from antigen-presenting cells to CD4+ T cells

Previous studies have provided evidence that myelin basic protein (MBP)-specific rat T cells acquire antigen via transfer of preformed peptide/MHC class II complexes from splenic antigen-presenting cells (APC). The purpose of the present study was to determine how T cells acquire peptide/MHC class II complexes from APC in vitro. Our results show that a MHC class II+ T cell line, R1-trans, released MHC class II-bearing vesicles that directly stimulated MBP-specific CD4+ T cells. Vesicles expressing complexes of MHC class II and MBP were also specifically cytotoxic to MBP-specific T cells. Surviving T cells acquired MHC class II/antigen complexes from these vesicles by a mechanism that did not require protein synthesis but depended on specific TCR interactions with peptide/self MHC complexes. Furthermore, MBP/MHC class II-bearing vesicles enabled T cells to present MBP to other T cell responders. These studies provide evidence that APC release vesicles expressing preformed peptide/MHC class II complexes that interact with clonotypic TCR, allowing MHC class II acquisition by T cells. Vesicular transport of antigen/MHC class II complexes from professional APC to T cells may represent an important mechanism of communication among cells of the immune system.     

Peptide-induced proliferation and lymphokine production in human T cells in the absence of antigen-presenting cells: role of T-cell activation state and costimulatory signals.

The role of T-lymphocytes as antigen-presenting cells (APCs) for other T cells was investigated. Activated rabies-virus-specific human T-cell clones were shown to present peptide to class II major histocompatibility complex (MHC)-restricted T cells of a different fine specificity, resulting in lymphokine production and cell proliferation. Furthermore, purified and activated antigen-specific T cells could produce lymphokines and proliferate as a result of the addition of antigenic peptide in the absence of APC. The functional response of T cells to peptide in the absence of APC was amplified by the addition of phorbol ester (PMA) and was inhibited with antibodies specific to class II MHC or to the CD2 molecule. Experiments performed in single-cell suspension cultures using semisolid medium prepared with 1% agar demonstrate that T-cell proliferative and lymphokine responses to peptide both in the presence and absence of APC require the interaction of T-cell antigen receptor (TCR) molecules with class II MHC-peptide complexes on different cell surfaces (cell-cell contact). On the other hand, peptide self-presentation, which occurs by the binding of TCR with class II MHC-peptide complexes on the same cell surface (at the single-cell level), resulted in T-cell activation (i.e., high expression of surface CD2, CD25, and HLA-DR molecules), without proliferation or lymphokine secretion, a pattern observed in the induction of T-cell anergy by antigen. The results are discussed in terms of the role of class II MHC molecules on activated T-lymphocytes, which enable these cells to function as "professional APC" in the development of T-cell regulatory networks.     

T cell major histocompatibility complex class II molecules down-regulate CD4+ T cell clone responses following LAG-3 binding

T cell response to its antigen requires recognition by the T cell receptor together with a co-receptor molecule, either CD4 or CD8. Additional molecules have been identified that are capable of delivering the co-stimulatory signals provided by APC. Following T cell priming, a number of T cell activation antigens are expressed that may play a role in the inactivation phase of the T cell response. The lymphocyte activation gene (LAG)-3 protein and its counter-receptors, the major histocompatibility complex (MHC) class II molecules, are such activation antigens whose interaction may result in the down-regulation of the ongoing immune response. To investigate the role of LAG-3/class II molecule interaction, we produced a soluble form of LAG-3 by fusing the extracellular Ig domains of this membrane protein to the constant region of human IgG1 (LAG-31g). Here, we show a direct and specific binding of LAG-3Ig to class II molecules on the cell surface. In addition, we show that LAG-3/class II molecule interaction leads to the down-regulation of CD4⁺ Ag-specific T cell clone proliferation and cytokine secretion. This inhibitory effect is observed at the level of the effector cells and not the APC and is also found with anti-CD3 mAb, PHA + PMA or low-dose IL-2 driven stimulation in the absence of APC. These functional studies indicate that T cell MHC class II molecules down-regulate T cell proliferation following LAG-3 binding and suggest a role for LAG-3 in the control of the CD4⁺ T cell response.     

MHC class II loading of high or low affinity peptides directed by Ii/peptide fusion constructs: implications for T cell activation

CD4(+) T cells recognize peptides presented on the cell surface of antigen presenting cells in the MHC class II context. The biosynthesis and transport of MHC class II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. In this study we have genetically exchanged CLIP with peptides of either high or low affinity for the class II peptide binding groove and utilized the properties of Ii to manipulate MHC class II loading. An inducible promoter controlled expression of the Ii/peptide fusion constructs, and presentation at different expression levels was studied. Both peptides were excised from Ii and presented on MHC class II molecules as shown by liquid chromatography-tandem mass spectrometry, but the high affinity peptide was presented more efficiently than the low affinity peptide. Both peptides were efficient in eliciting T cell responses at high Ii/peptide concentration independent of the duration of T cell stimulation. The peptides were also able to elicit an IL-2 response at low expression levels; however, the kinetic differed as the T cells required longer duration of T cell contact to reach a significant T cell response. This probably reflects the number of class II/peptide complexes at the cell surface and is discussed.     

Immunoglobulin-specific T-B cell interaction. IV. B cell presentation of idiotypic determinant(s) of monoclonal anti-surface immunoglobulin antibody to idiotope-recognizing helper T clones

Previously, we have demonstrated T-B cell interactions mediated by T cell recognition of immunoglobulin (Ig) peptide/major histocompatibility complex (MHC) class II complexes derived by the B cell processing of endogenously synthesized Ig molecules. In this report Ig-specific T-B cell interaction mediated by B cell presentation of idiotopes (Id) of anti-sIg antibodies to Id-specific T cell clones has been studied in Ig kappa-1-congenic rat strains. A panel of August (RT-1c; Ig kappa-1a) rat T helper clones specific for Id of syngeneic anti-Ig kappa-1b C3B9 monoclonal antibody (mAb) has been developed to study IdC3B9 presentation by Ig kappa-1b-bearing B cells from congenic August.1b (RT-1c; Ig kappa-1b) rats. Five of seven IdC3B9-specific T clones responded even at very low concentrations (100-200 pg/ml) of C3B9 mAb presented by Ig kappa-1b+ B cells. In contrast, the presentation of intact C3B9 mAb by nonspecific antigen-presenting cells (macrophages, Ig kappa-1a+ B cells, etc.) to IdC3B9-specific T cells was of low efficiency. The IdC3B9-specific T cell response to idiotopes of anti-Ig kappa-1b C3B9 mAb was found to be restricted by RT-1B molecule and required the processing of intact C3B9 molecule. IdC3B9 epitope recognized by C31 and C5 clones was mapped to the heavy chain of C3B9 mAb. Thus, B cells are able to present peptides related to the V region of anti-sIg Ab, i.e. Id peptide/MHC class II complexes, to Id-recognizing T cells. IdC3B9-presenting B cells are specifically activated both to proliferation and Ig production upon interaction with IdC3B9-specific T clones. Based on the results of our studies on B cell presentation of Ig epitopes to T cells a hypothetical model of Ig peptide-driven T-B cell interaction has been proposed.     

Peptides related to the antigenic determinant block T cell recognition of the native protein as processed by antigen-presenting cells

A mouse T cell hybrid specific for pigeon cytochrome c in the context of I-Ek responds by secreting interleukin 2 when co-cultured with the native antigen and the B cell lymphoma, LK-35.2, or naive splenic B cells as antigen-presenting cells (APC). Cytochromes c and their corresponding C-terminal fragments which are not capable of stimulating the TPc9.1 cells, including the autologous mouse cytochrome c, block the T cells' response to pigeon cytochrome c. In contrast, nonstimulatory N-terminal peptides of cytochrome c, which share no homology with the antigenic peptide, do not block. Blocking is observed when the nonstimulatory cytochromes c or peptides are present in culture with the live APC and nonsaturating concentrations of pigeon cytochrome c. With tobacco hornworm moth cytochrome c as antigen, a protein for which the T cell has a higher functional affinity, the response of TPc9.1 cannot be blocked by the nonstimulatory cytochromes c or by peptides, even when limiting concentrations of the tobacco hornworm moth cytochrome c are used. When paraformaldehyde-fixed APC are employed, no native cytochrome c can stimulate the T cells, including the tobacco hornworm moth protein which with the live APC is effective at 50 to 100-fold lower concentrations than pigeon cytochrome c. However, with fixed APC the T cells are stimulated by the C-terminal fragments containing residues 81-104 of the pigeon protein or residues 81-103 of the tobacco hornworm moth protein as readily and with the same relative efficiencies as the native protein, presented by live APC. The nonstimulatory peptides, but not the native cytochromes c, block T cell activation by pigeon cytochrome c pulsed-fixed APC, indicating that the nonstimulatory peptides compete with the stimulatory pigeon cytochrome c peptides produced by the APC. This competition appears to be due to nonstimulatory peptides which associate at the APC surface and not to those acting from solution because the APC which have been incubated with pigeon cytochrome c and nonstimulatory peptides and washed free of excess antigen and peptides are not stimulatory to the T cell hybrid. It was concluded that the activation of a pigeon cytochrome c-specific T cell, which recognizes a peptide fragment of the native protein on the surface of an APC, can be blocked by an excess of nonstimulatory homologous peptides when these are also associated on the surface of the APC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Peptide loading of empty major histocompatibility complex molecules on RMA-S cells allows the induction of primary cytotoxic T lymphocyte responses.

The antigen processing-defective mutant cell line RMA-S expresses at the cell surface major histocompatibility complex (MHC) class I molecules devoid of peptide that can be efficiently loaded with exogenous immunogenic peptides. We now report that viral peptide-loaded RMA-S cells, unlike parental RMA cells, can induce primary cytotoxic T lymphocyte (CTL) responses in vitro, in a T helper cell-independent fashion. This was shown for an H-2Kb-binding peptide of Sendai virus nucleoprotein and an H-2Db-binding peptide of adenovirus type 5 E1A protein with responding spleen cells of C57BL/6 mice, the strain of origin of RMA and RMA-S cells. Primary Sendai peptide-induced CTL lyse both peptide-loaded and virus-infected cells. Pre-culture of RMA-S cells at low temperature (22 degrees - 26 degrees C), which increases the amount of empty MHC class I molecules at the cell surface, decreases the peptide concentrations required for the induction of primary CTL responses. Primary peptide-specific CTL responses induced by peptide-loaded RMA-S cells are CD4+ cell- and MHC class II+ cell-independent. CTL response induction is blocked by the presence of anti-CD8 monoclonal antibody during culture. Direct peptide binding studies confirm the efficient loading of empty MHC molecules on RMA-S cells with peptide and show 2.5-fold more peptide bound per RMA-S cell compared to RMA cells. An additional factor explaining the difference in primary response induction between RMA and RMA-S cells is related to the CD8 dependence of these responses. MHC class I molecules occupied with irrelevant peptides (a majority present on RMA, largely absent on RMA-S) may interfere in the interaction of the CD8 molecule with relevant MHC/peptide complexes. The results delineate a novel strategy of peptide based in vitro immunization to elicit CD8+ cytotoxic T cell responses.     

Differential expression of CLIP: MHC class II and conventional endogenous peptide: MHC class II complexes by thymic epithelial cells and peripheral antigen-presenting cells

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high-avidity interaction of T cell receptors with the endogenous peptide : MHC class II complexes expressed on bone marrow (BM)-derived antigen-presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of Eα52–68: I-A^b and class II-associated invariant chain peptide (CLIP): I-A^b complexes in thymic epithelial cells and in the bone-marrow derived splenic APC, i.e. B cells, was studied using YAe and 30-2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM-derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of Eα52–68:I-A^b complexes in thymic epithelial cells is approximately 5–10 % of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I-A^b complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.     

Antigen-independent acquisition of MHC class II molecules by human T lymphocytes

We report here that human T lymphocytes have the capacity of acquiring large amounts of MHC class II molecules from various types of antigen-presenting cells (APC) in an antigen-independent manner. The transfer of MHC class II molecules from APC to T cell required direct cell-to-cell contact and appeared to involve the interaction of numerous adhesion molecules between these cells. Depletion of cholesterol from the plasma membrane reduced the amount of MHC class II transferred onto the T cells. Most significantly, the newly acquired MHC class II molecules were capable of efficiently presenting antigen to T helper cells. These results suggest that T cells are able to interact with other T cells to regulate immune responses by presenting MHC peptide complexes that have been snatched away from nearby APC.     

Self peptide/MHC class I complexes have a negligible effect on the response of some CD8+ T cells to foreign antigen

MHC molecules loaded with self peptides do not trigger a T cell immune response but may deliver signals important for peripheral T cell survival and function. It is unclear if self peptide/MHC complexes on APC in addition can influence the T cell response to co-presented foreign ligands. To address this question, TAP-sufficient and TAP-deficient cells were loaded with ovalbumin peptide (pOVA) to generate APC that present pOVA/H-2Kb complexes in the context of high or low levels of self peptide-loaded MHC class I, respectively. The two cell types were then used to stimulate different CD8+ T cells specific for ovalbumin while the number of presented pOVA/H-2Kb complexes was independently assessed by staining with 25-D1, an antibody against pOVA/H-2Kb. In each case, T cell activation was independent of TAP expression by the APC and depended exclusively on the amount of 25-D1 staining. We conclude that the number of pOVA/Kb complexes and not their frequency relative to self peptide/MHC complexes determines the response of those T cells tested here. These results imply that the repertoire of self peptide/MHC class I complexes presented by APC has a negligible effect on the response of some CD8+ T cells to foreign ligands.     

Understanding the focused CD4 T cell response to antigen and pathogenic organisms

Immunodominance is a term that reflects the final, very limited peptide specificity of T cells that are elicited during an immune response. Recent experiments in our laboratory compel us to propose a new paradigm for the control of immunodominance in CD4 T cell responses, stating that immunodominance is peptide-intrinsic and is dictated by the off-rate of peptides from MHC class II molecules. Our studies have revealed that persistence of peptide:class II complexes both predicts and controls CD4 T cell immunodominance and that this parameter can be rationally manipulated to either promote or eliminate immune responses. Mechanistically, we have determined that DM editing in APC is a key event that is influenced by the kinetic stability of class II:peptide complexes and that differential persistence of complexes also impacts the expansion phase of the immune response. These studies have important implications for rational vaccine design and for understanding the immunological mechanisms that limit the specificity of CD4 T cell responses.     

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells

Summary: Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC), At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR). whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal I to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal I alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-l can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and [CAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.     

Antibody-mediated delivery of antigen to chemokine receptors on antigen-presenting cells results in enhanced CD4+ T cell responses

Here, we have investigated if targeting of T cell epitopes to chemokine receptors results in improved CD4+ T cell responses. Mouse monoclonal antibodies (mAb) with kappaL chains were targeted to various chemokine receptors expressed on human monocytes or immature dendritic cells (DC), and proliferation of cloned human, DR4-restricted CD4+ T cells specific for mouse Ckappa(40-48) was measured. When using monocytes as antigen-presenting cells, mAb specific for CCR1, CCR2, CCR5, and CXCR4 were 100-10,000-fold more efficient at inducing T cell proliferation when compared to isotype-matched control mAb on a per molecule basis. Targeting of immature DC was less effective and was only seen with anti-CCR1 and anti-CXCR4 mAb. Anti-chemokine receptors mAb required to be processed by the conventional endosomal MHC class II presentation pathway. The mAb did not induce signaling through the chemokine receptors as they failed to induce mobilization of cytosolic Ca2+ and actin polymerization. They also failed to induce APC maturation. The results strongly suggest that chemokine receptors channel antigen into the endocytic pathway for presentation on MHC class II molecules. Targeting T cell epitopes to chemokine receptors by recombinant antibody should be a useful vaccine strategy for the induction of strong CD4+ T cell responses.     

Clustering of MHC–peptide complexes prior to their engagement in the immunological synapse: lipid raft and tetraspan microdomains

Summary: Protein reorganization at the interface of a T cell and an antigen‐presenting cell (APC) plays an important role in T cell activation. Imaging techniques reveal that reorganization of particular receptor–ligand pairs gives rise to an intercellular junction, termed the immunological synapse. In this synapse antigenic peptides associated with major histocompatibility complex (MHC) molecules form multimolecular arrays on the APC side, engaging an equivalent number of clustered T cell receptors (TCRs) on the T cell. The accumulation of MHC molecules carrying cognate peptide in the APC–T cell interface was thought to depend on the specificity and presence of TCRs. Recent evidence, however, suggests that the APC is equipped to preorganize MHC–peptide complexes in the absence of T cells. To this end, MHC molecules become incorporated into two types of membrane microdomains: (i) cholesterol‐ and glycosphingolipid‐enriched domains, denoted lipid rafts, that preconcentrate MHC class II molecules; and (ii) microdomains made up of tetraspan proteins, such as CD9, CD63, CD81 or CD82, that mediate enrichment of MHC class II molecules loaded with a select set of peptides. It follows that the integrity, composition and dynamics of these microdomains are candidate determinants favoring activation or silencing of T cells.     

Antigen presentation for T cell interleukin-2 secretion is a late acquisition of neonatal B cells.

The ability of B lymphocytes to process and present antigen to helper T cells is essential to initiate T cell-B cell interactions in humoral immune responses. Here we describe the developmental acquisition of the antigen-presenting function of B cells as measured by the ability of B cells to stimulate a T cell hybrid to interleukin (IL)-2 secretion. Neonatal splenic B cells are not adult-like in their ability to process and present the model protein antigen pigeon cytochrome (Pc), which enters the B cell through fluid-phase pinocytosis, until 21 to 28 days of life. The ability of neonatal B cells to process and present antigen which enters the cell bound to surface Ig is not adult-like until 28 days of age. When neonatal B cells acquire antigen-presenting cell (APC) function, surface IgM facilitates antigen processing. The delayed acquisition of APC function cannot be accounted for solely by a deficiency in major histocompatibility complex MHC class II, ICAM-1, or LFA-1 as neonatal B cells express adult levels of these molecules by 7-14 days after birth. Moreover, the ability of neonatal B cells to present a peptide fragment of Pc which does not require processing is adult like by day 14. Furthermore, neonatal B cells are capable of binding, internalizing and degrading radiolabeled antigen, suggesting a more subtle level of regulation. In contrast to neonatal B cells, immature B cells in the adult bone marrow and adult B cells undergoing antigen-driven differentiation to memory B cells, as defined by the loss of the J11D marker, are competent to process and present antigen resulting in T cell IL-2 secretion. Thus, developing B cell subpopulations in the adult and in the neonate can be distinguished. Only neonatal B cells are deficient in their ability to stimulate T cells to IL-2 production.     

A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of α chain, β chain,and antigenic peptide,with immunogenicity in vitro and reduced affinity for bacterial superantigens

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogenous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 β chain. Co-transfection with DR α cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated β chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR α chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, β chain, and α chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated α chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.     

T cell stimulation in the absence of exogenous antigen: a T cell signal is induced by both MHC-dependent and -independent mechanisms

Mature T cells residing in peripheral lymphoid organs have frequent contact with antigen presenting cells (APC). Such contact may be required for T cell survival, but the degree to which signals in mature T cells are induced by TCR recognition of self peptide/MHC complexes is unclear. We have used induction of the early growth response gene 1 (Egr1) as an indicator of signal transduction in 3.L2 (I-Ek-restricted) T cells interacting with APC in the absence of exogenous antigen. The data show that Egr1 can be induced in 3.L2 T cells by TCR recognition of self peptides presented by I-Ek. However, a more transient induction of Egr1 can be induced in 3.L2 T cells interacting with dendritic cells derived from class II/beta2m double-deficient mice. Egr1 induction after T cell-APC contact was also observed in a freshly isolated polyclonal CD4 T cell population. The data suggest that self peptide/MHC recognition by the TCR induces a signal in T cells and that dendritic cells can also induce a more transient T cell signal by an MHC-independent mechanism.     

Discrimination of human CD4 T cell clones based on their reactivity with antigen-presenting T cells.

In this report, we describe the discrimination of human T cell clones based on their reactivity with activated T cells as antigen-presenting cells (APC). CD4+ T cell clones specific for peptide P30 of tetanus toxin (amino acids 947-967) and restricted to the DP4 molecule were established and tested for proliferation to peptide presented either by peripheral blood mononuclear cells (PBMC), Epstein-Barr virus (EBV)-transformed B cells or major histocompatibility complex (MHC) class II-expressing T cells. We found two sets of T cell clones: one set proliferated to peptide presentation by PBMC, EBV-transformed B cell lines (EBV-B cells) and MHC class II+ T cells (termed T-responder clones), while the other set of clones was only stimulated to proliferate, if the peptide was presented by PBMC or EBV-B cells, but not by T cells (T-nonresponder clones). Nevertheless, these T-nonresponder clones recognized P30 also on T cells, as revealed by Ca2+ influx. The discrimination of the clones was not due to different avidities of the T cell receptors (TcR) of individual clones for the MHC-peptide complex as T-responder and T-nonresponder clones had similar dose-response curves to P30 presented by fixed EBV-B cell lines. Addition of cytokines [interleukin (IL)-1, IL-2, IL-4 and interferon gamma] did not change the proliferative response of the clones, which was consistent throughout an observation period of greater than 4 months. T-nonresponder clones, exposed to P30 on MHC class II-expressing T cells, became not anergic, as they could be restimulated by P30 presented on EBV-B cells. The measurement of a panel of T cell activation markers and adhesion molecules on T-responder and T-nonresponder clones revealed a higher expression of the CD28 molecule on the T-nonresponder clones. The data suggest that freshly cloned T cells can be differentiated by peptide presentation on classical (PBMC, EBV-B cells) or non-classical APC (class II+ T cells), and that this discrimination is further underlined by different levels of adhesion molecules.     

T and B cell receptors discriminate major histocompatibility complex class II conformations influenced by the invariant chain.

Direct recognition of major histocompatibility complex (MHC) molecules may occur when T cells are positively selected in the thymus and also during recognition of non-self MHC molecules. Since peptide recognition and binding of particular monoclonal antibodies is strongly influenced by the invariant chain (Ii) of the class II molecule, we have asked whether Ii also affects recognition of non-self MHC molecules by T cells. We find that Ii binding alters MHC class II conformation as detected by a monoclonal antibody, and that this alteration is retained in cell surface MHC class II molecules after Ii dissociates. This altered conformation also affects recognition by allogeneic T cells. Normal T cells and T cell clones preferentially recognize MHC class II molecules that have been associated with Ii, suggesting that thymic selection may be influenced by MHC conformation independently of specific peptide binding.