Alteration of a model antigen by Au(III) leads to T cell sensitization to cryptic peptides

Certain metal ions are known to be potent sensitizers, but the self proteins modified by metal ions and the self peptides recognized by ‘metal-specific’ T cells are unknown. In humans and mice treatment with gold anti-rheumatic drugs, containing Au(I), may lead to allergic and autoimmune side effects. Human and murine T cells do not react to Au(I), however, but to the reactive metabolite Au(III). Here we show that alteration by Au(III) of a model antigen, bovine ribonuclease (RNase) A, results in T cell sensitization to cryptic peptides of this protein. Upon immunization of mice with Au(III)-pretreated RNase [RNase/Au(III)], CD4⁺ T cell hybridomas specific for RNase/Au(III) were obtained in addition to those recognizing the immunodominant peptide RNase 74–88; the latter also were obtained after immunization with native RNase. RNase/Au(III)-specific T cell hybridomas reacted against RNase/Au(III) and RNase denatured by S-sulfonation of cysteine residues, but not against native RNase, or RNase pretreated with Au(I), Al(III), Cu(II), Fe(II), Fe(III), Ni(II), Mn(II), or Zn(II). Using a panel of overlapping, synthetic RNase peptides which were devoid of gold or gold-induced modifications, epitope mapping revealed that RNase/Au(III)-specific T cell hybridomas recognized the cryptic peptides 7–21 and 94–108, respectively. Comparison of the proliferative response of bulk CD4⁺ T cells, prepared from splenocytes after immunization with either RNase/Au(III) or native RNase, revealed that Au(III) pretreatment of RNase led to a markedly enhanced response to the two cryptic peptides while it did not influence the response to the immunodominant peptide. The cryptic peptides were also presented after preincubation of bone marrow-derived macrophages with RNase and Au(I), but not with RNase alone, suggesting that oxidation of Au(I) to Au(III) and subsequent protein alteration by Au(III) can happen in mononuclear phagocytes. We conclude that Au(III) alteration of proteins alters antigen processing and, thus, leads to presentation of cryptic peptides. This mechanism may shed light on the development of allergic and autoimmune side effects of Au(I) anti-rheumatic drugs. In addition, it might provide a general mechanism of how metal ions act as T cell sensitizers.     

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.     

Carrier-reactive hapten-specific cytotoxic T lymphocyte clones originate from a highly preselected T cell repertoire: implications for chemical-induced self-reactivity

We have recently described trinitrophenyl (TNP)-specific cytotoxic T lymphocyte (CTL) clones from C57BL/6 mice specific for hapten-modified peptides bearing a TNP-lysine in a peripheral position, i.e. in position 7 of H-2K^b-bound octapeptides. CTL recognition of such determinants is always sequencedependent due to co-recognition of TNP as well as amino acid side chains of the carrier peptide. By the use of glycine-based designer peptides for primary induction of CTL in vitro, we have identified two sub-epitopes on individual position 7-haptenated peptides that form two TcR contact points and which can be independently recognized by cloned CTL. One of these sub-epitopes is represented by the hapten itself, the other by the amino acids tyrosine and lysine in positions 3 and 4 of the carrier peptide, respectively. Immunization with such TNP-modified peptides frequently results in the specific induction of CTL also reacting with the unmodified carrier peptides. DNA sequence analyses of the TcR revealed an extraordinary similarity of several independent TcR of CTL from individual mice and induced with different TNP-peptides. These receptor similarities clearly correlate with structural elements common to the immunizing peptides and suggest their origin from positive thymic selection of TcR on K^b-associated self-peptides bearing Tyr in position 3. Our data provide additional information concerning the topology of TcR binding to peptide/MHC complexes with, but also without, TNP. They also indicate a mechanism which might explain the potential of chemicals or drugs to induce autoimmune phenomena.     

Cross-reactive trinitrophenylated peptides as antigens for class II major histocompatibility complex-restricted T cells and inducers of contact sensitivity in mice. Limited T cell receptor repertoire

The induction of contact sensitivity in mice by hapten reagents such as trinitrochlorobenzene (TNCB) involves the activation of class II major histocompatibility complex (MHC)-restricted, hapten-specific, CD4⁺ T cells. Reports from different laboratories have indicated that the relevant antigenic epitopes in such reactions might include hapten-conjugated, MHC class II-associated peptides. This study for the first time directly demonstrates that hapten-peptides account for the majority of determinants recognized by trinitrophenyl (TNP)-specific CD4⁺ T lymphocytes. The sequences of those TNP carrier peptides do not have to be related to mouse proteins. Thus, we show that TNP-modified peptides derived from mouse IgG, pigeon cytochrome c or staphylococcal nuclease known to bind to I-A^b or from λ represser with specificity to I-A^d as well as TNP-proteins such as bovine serum albumin, ovalbumin or keyhole limpet hemocyanin all create class II-restricted hapten determinants for a number of TNP-specific T cell clones and hybridomas. All of these cells were induced with cells modified by trinitrobenzene sulfonic acid (TNBS). In addition, we present arguments indicating that individual TNP-specific helper T cells may cross-react with different TNP-peptides bound to identical class II molecules. Chemical treatment of antigen-presenting cells with TNCB or TNBS may thus result in a limited number of particularly repetitive immunodominant hapten epitopes. Immunodominant epitopes were also indicated by an overrepresentation of the TCR elements Vβ2 and Vα10 in I-A^b/TNP-specific T cells. Most importantly, however, we demonstrate that TNP attached to lysine 97 in the staphylococcal nuclease peptide 93–105 (i.e. a clearly “non-self” sequence) is able to prime mice for subsequent elicitation of contact sensitivity by TNCB in the absence of foreign protein. We take this to indicate that those TNP-peptide determinants defined by us as immuno-dominant are responsible for the induction of contact sensitivity to haptens.     

T cell recognition of penicillin G: Structural features determining antigenic specificity

Penicillin G (Pen G) and other beta-lactam antibiotics frequently induce allergic reactions constituting typical examples of human immune responses to haptens. In fact, penicillins represent a unique set of haptens with outstanding structural variability on the basis of an identical protein-reactive beta-lactam containing backbone. Although both cellular and humoral responses are involved in drug-induced allergies, little is known about the T cell reactivity to penicillins. To understand which structural features determine antigenic specificity, we isolated a panel of MHC-restricted, Pen G-reactive T cell clones from different penicillin-allergic patients and tested them for their capacity to proliferate in the presence of other penicillin derivatives. We found that the antigenic epitope consists of both the amide-linked side chain, which is different in every member of the penicillin family, as well as the thiazolidine ring common to all penicillin derivatives. We also demonstrated the presence of two different types of penicillin-specific T cells, one dependent, and the other independent of antigen processing by autologous antigen-presenting cells. Our data strongly suggest that penicillins form part of the epitopes contacting the antigen receptors of T cells.     

Characterization of processing requirements and metal cross-reactivities in T cell clones from patients with allergic contact dermatitis to nickel

Metal ions such as nickel, cobalt, copper and palladium are known to be potent sensitizers in humans, but the antigenic determinants created by these metals as well as the mechanisms of recognition by specific T cell clones are still not elucidated. In this paper, nickel-specific T lymphocyte clones were isolated from four patients exhibiting contact dermatitis to this metal. A panel of 42 independent T cell clones was studied. They were shown to recognize nickel in the context of major histocompatibility complex (MHC) class II molecules and to belong to the CD4 subset. Using fixed autologous Epstein-Barr virus-transformed B cells as antigen-presenting cells (APC), we could distinguish two distinct groups of T cell clones on the basis of processing requirements: 40% of the T cell clones were strictly processing dependent, whereas the remaining 60% could proliferate in response to nickel even in the presence of glutaraldehyde-fixed APC. Furthermore, we present arguments indicating that individual Ni-specific T cell clones cross-react with some transition metals (e.g. Cu or Pd), but not with others (e.g. Co, Cr and Pt), presented by identical MHC class II molecules. These results thus provide an explanation for the multiple metal-reactivities observed in vivo in human patients: they indicate that for Cu and Pd, these co-reactivities in vivo might be due to cross-reactivity at the clonal level. Our findings also suggest that this is not the case for cobalt allergy, which might result from co-sensitization of the patient to cobalt in addition to nickel.     

Human herpesvirus 6 and multiple sclerosis: a study of T cell cross-reactivity to viral and myelin basic protein antigens

Several reports have suggested an association of human herpesvirus 6 (HHV-6) with multiple sclerosis. Autoreactive T lymphocytes directed against myelin components seem to contribute to the pathogenesis of the disease. It has been suggested that molecular mimicry between viral and self-antigens might be one of the mechanisms that determine the onset of several autoimmune diseases. Following this hypothesis, the purpose of the present study was to evaluate if HHV-6 could play a role in activating T cells capable of cross-reaction with an important myelin component, the myelin basic protein. T cell lines were established from 22 multiple sclerosis patients and from 16 healthy controls, and their capability to react to both virus and myelin basic protein antigens was compared. The analysis of T cell cross-reactivity in patients and controls did not show significant differences in the HHV-6 ability to activate myelin basic protein-reactive T cells. Similarly, the evaluation of the humoral immune response to HHV-6 in patients and controls did not mirror any abnormality in the HHV-6 status in multiple sclerosis patients. Therefore, although the findings of activity in vitro of T cell lines with dual specificity are consistent with the hypothesis of molecular mimicry, the lack of differences in cross-reactivity between patients and controls do not support molecular mimicry as an important mechanism in the physiopathology of this disease.     

A sequence pattern for peptides presented to cytotoxic T lymphocytes by HLA B8 revealed by analysis of epitopes and eluted peptides

HLA B8-restricted cytotoxic T lymphocytes (CTL) specific for influenza A virus were generated and shown to recognize the nucleoprotein (NP). The dominant epitope was mapped using recombinant vaccinia viruses that expressed fragments of the NP and then synthetic peptides baseds on the NP amino acid sequence. The peptide 380-393 was first identified and further refined; it was shown that the glutamic acid at position 380 was essential for recognition by CTL and that the nonamer 380-388 was the optimum peptide. Six HLA B8-positive influenza immune donors that we have tested respond to this peptide as part of their influenza-specific CTL response. The amino acid sequence of the peptide epitope was compared to six other known virus peptides known to be restricted by HLA B8 and a sequence homology was identified, which predicted nonamer and octamer epitope sequences. Probable anchor residues were identified at peptide residues 3 (lysine/arginine), 5 (lysine/arginine) and 9 (leucine/isoleucine). Support for this pattern came from sequencing peptides eluted from purified HLA B8 molecules, where lysines were predominant at positions 3 and 5. One of the predicted epitope peptides was made and shown to be recognized by specific CTL. These and the two others were shown to compete with NP 380-388 for binding to HLA B8. A model was made of the HLA B8 molecule and negatively charged pockets predicted, which could accomodate the positively charged side chains of the peptide anchor residues.     

Cross-sensitization to haptens: formation of common haptenic metabolites, T cell recognition of cryptic peptides, and true T cell cross-reactivity

To analyze T cell cross-reactivity to para-compounds, we established CD4(+) T cell hybridomas from mice immunized with adducts of self-globin and one of three different para-compounds: p-aminophenol, p-phenylenediamine, or Bandrowski's base. Some of the hybridomas obtained reacted not only to the immunizing antigen, but also to metabolically related para-compounds, bound to the same protein, thus suggesting formation of common metabolites. Other hybridomas cross-reacted to globin adducts of metabolically unrelated para-compounds, which denotes them as truly cross-reactive cells whose TCR failed to distinguish among the different haptens. One of these hybridomas also reacted against a non-haptenated, cryptic peptide of hemoglobin but not to the full-length native protein. As this hybridoma reacted even more strongly to the respective peptide after it was haptenated, recognition of the native, cryptic peptide was apparently due to true cross-reactivity. To conclude, true T cell cross-reactivity to haptens does occur, as well as the formation of a common reactive metabolite, and T cell recognition of cryptic self-peptides may underlie cross-sensitization to chemicals.     

Differential activation of human autoreactive T cell clones by altered peptide ligands derived from myelin basic protein peptide (87–99)

We have examined the functional consequences induced by interaction of DR2a-restricted myelin basic protein (MBP) (87–99)-specific T cell clones (TCC) with altered peptide ligands (APL) derived from MBP peptide (87–99). The immunodominant MBP peptide (87–99) has been implicated as a candidate antigen in multiple sclerosis (MS) by several lines of evidence. In the present study, we have defined the T cell receptor (TCR) contact residues for DR2a-restricted, (87–99)-specific T helper type 1 T cells to design APL suitable to modify the functions of such T cells potentially relevant for the pathogenesis of MS. We show that neutral (L-alanine substitutions) or conservative exchanges of the primary and secondary TCR contact residues lead to various alterations of T cell function, ranging from differences in interleukin-2 receptor up-regulation to anergy induction and TCR antagonism. The potential usefulness of APL as an immunomodulating therapy for DR2⁺ MS patients is discussed.     

Agonist-induced T cell receptor down-regulation: molecular requirements and dissociation from T cell activation

T cell receptor (TCR) down-regulation is a consequence of specific receptor engagement and plays an important role in modulating the T cell response. We have investigated the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the induction of TCR down-regulation. We report that the mutation of S126 in the CD3-γ chain that is known to inhibit phorbol-12-myristate 13-acetate-induced TCR down-regulation does not affect down-regulation induced by a specific agonist. In addition, agonist-induced TCR down-regulation is not affected by blockade or depletion of PKC, neither by blockade or lack of PTK, while the same treatments efficiently interfere with T cell activation. These results demonstrate that TCR down-regulation is induced by early events which follow specific engagement by an agonist and can be dissociated from those required for full T cell activation.     

Hierarchy in the ability of T cell epitopes to induce peripheral tolerance to antigens from myelin

Nasal administration of peptide antigens has been shown to induce T cell tolerance. We have investigated the potential for peptide therapy of the autoimmune response to myelin antigens in experimental autoimmune encephalomyelitis (EAE). Three major encephalitogenic epitopes were studied for their ability to induce nasal tolerance to myelin antigens. These included epitopes Ac1 – 9 and 89 – 101 of myelin basic protein (MBP) and 139 – 151 from proteolipid protein (PLP). Peptide Ac1 – 9 from MBP effectively suppressed responses to both MBP epitopes, following immunization with whole myelin (linked suppression). The N-terminal epitope failed, however, to modify the response to epitope 139 – 151 of PLP. The second MBP epitope (89 – 101) was poorly tolerogenic for the immune response to any naturally processed myelin epitope. By contrast, PLP[139 – 151] was able to induce bystander suppression of T cells responsive to both itself and the two epitopes from MBP. Furthermore, this epitope suppressed EAE induced with peptides derived from MBP and was capable of treating ongoing disease. The mechanism of bystander suppression, mediated by PLP[139 – 151], did not correlate with an overt switch from the T helper 1 to the T helper 2 phenotype. These results demonstrate how a complex autoimmune disease may be controlled by treatment with a single peptide epitope and reveal a hierarchy in the suppressive properties of different myelin antigens.     

Coronin-1 expression in T lymphocytes: insights into protein function during T cell development and activation

Coronin has been described as an actin-binding protein of Dictyostelium discoideum, and it has been demonstrated to play a role in cell migration, cytokinesis and phagocytosis. Coronin-related proteins are found in many eukaryotic species, including Coronin-1 in mammals whose expression is enriched in the hematopoietic tissues. Here, we characterize Coronin-1 gene and protein expression in mouse embryonic and adult T lymphocytes. Coronin-1 is expressed throughout T cell ontogeny and in peripheral alphabeta T cells. Expression varies along thymic cell development, with maximum levels observed in embryonic early thymocytes and, in the adults, the selected TCRalphabeta(+) single-positive thymocytes. Subcellular localization analysis indicates that Coronin-1 is in equilibrium between the cytosol and the cell cortex, where it accumulates in F-actin-rich membrane protrusions induced by polarized activation of TCR-CD3-stimulated T cells. These data are consistent with a role of Coronin-1 in T cell differentiation/activation events involving membrane dynamisms and the cortical actin cytoskeleton.     

Oral tolerance to haptens: intestinal epithelial cells from 2,4-dinitrochlorobenzene-fed mice inhibit hapten-specific T cell activation in vitro

The mechanisms underlying the induction of immunological tolerance after feeding soluble exogenous antigens, including proteins and haptens, are still unclear. Using a model of oral tolerance to the contact-sensitizing hapten 2,4-dinitrochlorobenzene (DNCB), we have compared the ability of intestinal epithelial cells and of Peyer's patch APC to present DNCB in vitro or ex vivo after oral feeding, to specific peripheral lymph node T cells from DNCB-sensitized mice. In contrast to Peyer's patch APC, which induce efficient hapten-specific T cell activation upon exposure to the hapten either in vitro or in vivo, mature MHC class-II-positive intestinal epithelial cells were unable to induce T cell activation in either case. Interestingly, enterocytes from DNCB-fed mice exerted a dramatic inhibitory effect on the proliferative response of hapten-primed T cells in response to dinitrobenzene sulfonate presented by syngeneic spleen cells. This inhibitory effect, which was also observed with supernatant of intestinal epithelial cells from DNCB-fed mice, could be reversed by neutralizing anti-transforming growth factor (TGF)-β antibodies. In addition, pre-incubation of hapten-sensitized T cells with enterocytes from DNCB-fed mice induced T cell anergy, which could be reversed by exogenous interleukin-2 or interleukin-4. These data demonstrate that intestinal epithelial cells activated in vivo by oral administration of DNCB are able to block proliferation of activated T cells through secretion of immunosuppressive cytokines such as TGF-β. It is proposed that intestinal epithelial cells may play a significant role in oral tolerance by limiting T cell-mediated hypersensitivity responses.     

Fibronectin induces phosphorylation of a 120-kDa protein and synergizes with the T cell receptor to activate cytotoxic T cell clones

Fibronectin (FN) has been shown to act as a costimulator in both CD4⁺ and CD8⁺ T cell activation through the T cell receptor (TcR). Consistent with previous studies, we found that FN is able to both enhance the maximal amount of TcR-triggered degranulation and lower the threshold for activation. The density of immobilized anti-CD3 or anti-TcR required to induce degranulation and tyrosine phosphorylation of cellular proteins by several cytotoxic T lymphocyte clones is quantitatively about tenfold lower in the presence of FN. We further demonstrate that FN alone stimulates transient tyrosine phosphorylation of a 120-kDa protein (pp120) in CD8⁺ T cells and when FN is coimmobilized with substimulatory amounts of anti-CD3 or anti-TcR there is a synergistic response, resulting in prolonged and enhanced phosphorylation of pp120. To determine if FN acts as a costimulator in CD8⁺ cells solely through mediating adhesion events or if it also transduces signals in T cells we conducted remote stimulation experiments. Degranulation was induced when FN and sub-stimulatory anti-CD3 were presented on separate surfaces, indicating that FN induces independent transmembrane signals capable of augmenting TcR-induced signals resulting in a functional response. Both FN plus TcR-induced tyrosine phosphorylation of pp120 and degranulation are inhibited by RGD-containing peptides, implying that an RGD-dependent FN receptor is mediating phosphorylation of pp120 and enhancing TcR-mediated degranulation.     

Longitudinal analysis of Mycobacterium tuberculosis 19-kDa antigen-specific T cells in patients with pulmonary tuberculosis: association with disease activity and cross-reactivity to a peptide from HIVenv gp120

CD8(+) T cells play a central role in immune protection against infection with Mycobacterium tuberculosis. One of the target epitopes for anti-M. tuberculosis directed CD8(+) T cells is the HLA-A2-restricted 19-kDa lipoprotein peptide VLTDGNPPEV. T cell clones directed against this epitope recognized not only the nominal peptide ligand, but also a closely related peptide (VPTDPNPPEV) from the HIV envelope gp120 (HIV(env) gp120) protein characterized by IFN-gamma release. This cross-reactivity was confirmed in ex vivo in M. tuberculosis 19-kDa tetramer-sorted T cells from patients with tuberculosis and in HIVgp120 tetramer-reactive T cells sorted from HIV(+) patients. M. tuberculosis 19-kDa antigen-reactive T cells were present in HLA-A2(+) patients (10/10) with HIV infection with no evidence of M. tuberculosis infection, but they are absent in peripheral blood lymphocytes from healthy HLA-A2(+) individuals (10/10). M. tuberculosis 19-kDa antigen-reactive T cells were elevated in acute pulmonary tuberculosis, declined with response to therapy (7/10 patients) and resided in the terminally differentiated CD8(+) T cell subset. CD8(+) cross-reactive T cells recognizing HIV(env) or M. tuberculosis 19-kDa antigens may contribute to pathogenesis in individuals co-infected with both pathogens and may also present a marker for active tuberculosis.     

Induction of central T cell tolerance: recombinant antibodies deliver peptides for deletion of antigen-specific (CD4+)8+ thymocytes

In order to prevent or ameliorate autoimmune disease, it would be desirable to induce central tolerance to peripheral self-antigens. We have investigated whether recombinant antibodies (Ab) that deliver T cell epitopes to antigen-presenting cells (APC) in the thymus can be used to induce thymocyte deletion. Troybodies are recombinant Ab with V regions specific for APC surface molecules that have T cell epitopes genetically introduced in their C domains. When MHC class II-specific Troybodies with the lambda2(315)T cell epitope were injected into lambda2(315)-specific TCR transgenic mice, a profound deletion of (CD4+)8+ thymocytes was observed. MHC class II-specific Troybodies were 10-100-fold more efficient than non-targeting peptide Ab, and 500-fold more efficient than synthetic peptide at inducing deletion. Similar findings were observed when MHC class II-specific Troybodies with the OVA(323-339) T cell epitope were injected into OVA-specific TCR transgenic mice. Although deletion was transient after a single injection, newborn mice repeatedly injected with MHC class II-specific Troybodies for 4 weeks, had reduced antigen-specific T cells in peripheral lymphoid tissues and reduced T cell responses. These experiments suggest that Troybodies constructed to target specifically thymic APC could be useful tools for induction and maintenance of central T cell tolerance in autoimmune diseases.     

Antigen presentation by T cells: T cell receptor ligation promotes antigen acquisition from professional antigen-presenting cells

The purpose of this study was to determine whether the clonotypic specificity of the T cell receptor influences the specificity of T cell-mediated antigen presentation. We have previously shown that myelin basic protein (MBP)-specific Lewis rat GP2.E5/R1 (R1) T cells cultured with antigen, irradiated syngeneic splenocytes (IrrSPL) and tolerogenic monoclonal antibody become highly effective antigen-presenting cells (APC). In the current studies, we investigated the transfer of specific (MBP) and unrelated (conalbumin) antigens from antigen-pulsed SPL to R1 T cells. R1 T cells cultured with IrrSPL that were pulsed simultaneously with both MBP and conalbumin acquired and presented both antigens to the appropriate T cell responders in a secondary assay. These results suggested a physical transfer of major histocompatibility complex (MHC)/peptide complexes from professional APC to R1 T cells. Transfer of conalbumin from professional APC to R1 T cells required specific recognition of MBP and was optimal when both conalbumin and MBP were presented on the same group of professional APC. Antigens transfer did not occur when allogeneic SPL were used as APC. The anti-I-A mAb OX6 inhibited antigen transfer but only when added during the initiation of culture. OX6 also inhibited antigen acquisition by R1-trans, a variant of the R1 T cell line which constitutively synthesizes high levels of I-A, from MBP-pulsed IrrSPL but blockade of I-A did not inhibit antigen acquisition when soluble MBP was added directly to the culture. Despite constitutive synthesis of I-A, R1-trans T cells did not acquire guinea pig MBP from pulsed allogeneic APC. These studies demonstrate that although T cells of a particular specificity can present unrelated antigens, the cognate interaction of the T cell antigen receptor with the appropriate antigen/self-MHC complex strongly promotes acquisition of these complexes from professional APC.     

Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus

B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA. We have shown that recombinant HVEM and a panel of different monoclonal antibodies specifically bind murine BTLA on both B and T cells and that some antibodies inhibit anti-CD3ε-induced T cell proliferation in vitro, but only when constrained appropriately with a putatively cross-linking reagent. The antibodies had no significant effect on in vitro T cell proliferation in a mixed lymphocyte reaction (MLR) assay nor on in vitro DO11.10 antigen-induced T cell proliferation. None of these antibodies, nor HVEM-Fc, had any significant effect on in vitro B cell proliferation induced by anti-immunoglobulin M antibodies (±anti-CD40) or lipopolysaccharide. We further elucidated the requirements for inhibition of in vitro T cell proliferation using a beads-based system to demonstrate that the antibodies that inhibited T cell proliferation in vitro were required to be presented to the T cell in a cis, and not trans, format relative to the anti-CD3ε stimulus. We also found that antibodies that inhibited T cell proliferation in vitro had no significant effect on the antibody captured interleukin-2 associated with the in vivo activation of DO11.10 T cells transferred to syngeneic recipient BALB/c mice. These data suggest that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation.     

Oxidative inactivation of CD45 protein tyrosine phosphatase may contribute to T lymphocyte dysfunction in the elderly

The decline in T lymphocyte function during ageing has been linked to changes in intracellular signalling pathways. Oxidative damage has long been thought to be involved in the ageing process and we investigated a link between ageing, oxidation and T cell signalling focusing on the membrane phosphatase CD45. We investigated the relative sensitivity of CD45 to oxidative inactivation and then compared the phosphatase activity of CD45 in blood lymphocytes from elderly and young volunteers and related this to intracellular levels of the antioxidant glutathione. The CD45 phosphatase was particularly sensitive to oxidative inactivation compared to total Jurkat T cell PTP activity. The IC50 with H(2)O(2) was 3 mM for CD45 at which concentration there was a minimal decrease in global PTP activity. In normal peripheral blood CD4(+) T cells the IC50 was much lower at 54 microM. In a group of elderly healthy individuals, whose T cell responses to mitogen were depressed, PB lymphocyte CD45 phosphatase activity was decreased by about 60% compared to young controls. There was no difference in intracellular levels of glutathione. The loss of CD45 activity in lymphocytes from the elderly may underlie poor T cell function associated with ageing. The relative sensitivity of CD45 to oxidative damage may result from its location in the plasma membrane, where it might be more accessible to extracellular oxidants.