Biomedical subjects

C A Janeway

Publications and source records attributed to C A Janeway.

At least 19 recordsLinked to original sources

PubMed1992

High levels of IL-2 alter signal transduction in cloned IL-4-producing CD4 T cells.

Cloned CD4 T cells of the Th2 type make IL-4 and related cytokines upon receptor cross-linking, whereas cloned CD4 T cells of the Th1 type make IL-2, IFN-gamma, and TNF-beta. These two types of CD4 T cell are also reported to use distinct mechanisms of signal transduction. It has been reported that Th1 cells flux Ca2+ upon receptor cross-linking, whereas Th2 cells do not. We have noted that when cloned Th2 cells are exposed to high levels (20 U/ml) of IL-2, they show an altered phenotype. Such cells are much more sensitive to activation by certain antireceptor antibodies, they flux calcium upon receptor ligation without additional cross-linking with anti-Ig antibodies, and they make much larger amounts of IL-4. In addition, the organization of their TCR is altered, with increased levels of the TCR-eta chain and an increase in the extent of association of CD4 with CD3 and CD45, changes similar to those found in Th1 cells. These results suggest that there is no fundamental difference in the signal transduction apparatus of Th1 and Th2 cells; rather, the IL-2 made by Th1 cells may create similar phenotypic changes in these cells and thus create the impression of altered signal transduction mechanisms. These results do show that exposure to high levels of IL-2 can profoundly affect signal transduction in T cells. Furthermore, we found that the Ca2+ signal caused by CD3 antibodies seemed to differ in character from that caused by TCR antibodies suggesting that the use of CD3 antibodies is not always a good model for activation through the TCR.

Biological Assay↗

PubMed1992

Truncation variants of peptides isolated from MHC class II molecules suggest sequence motifs.

T cells recognize foreign protein antigens in the form of peptide fragments bound tightly to the outer aspect of molecules encoded by the major histocompatibility complex (MHC). Most of the amino-acid differences that distinguish MHC allelic variants line the peptide-binding cleft, and different allelic forms of MHC molecules bind distinct peptides. It has been demonstrated that peptide-binding to MHC class I involves anchor residues in certain positions and that antigenic peptides associated with MHC class I exhibit allele-specific structural motifs. We have previously reported an analysis of MHC class II-associated peptide sequences. Here we extend this analysis and show that certain amino-acid residues occur at particular positions in the sequence of peptides binding to a given MHC class II molecule. These sequence motifs require the amino terminus to be shifted one or two positions to obtain alignment; such shifts occur naturally for a single peptide sequence without qualitatively altering CD4 T-cell recognition.

Amino Acid Sequence↗

PubMed1992

Breaking T cell tolerance with foreign and self co-immunogens. A study of autoimmune B and T cell epitopes of cytochrome c.

The initiation of autoimmune B cell and T cell responses by self Ag or by foreign pathogens (molecular mimics) is not well understood. In the present study, cytochrome c (cyt c) was used as a model autoantigen to investigate how self-proteins are involved in the priming of autoimmune T cell responses. Immunization with foreign cyt c has been extensively analyzed in previous studies as a model for both humoral and cellular immune responses. Mice do not, however, make antibody or T cell responses to immunization with self (mouse) cyt c. In addition, T cell tolerance can be broken by autoreactive B cells that are readily elicited by immunization with cross-reactive foreign cyt c. These immune B cells presumably bind self cyt c and process and present the self Ag to stimulate an autoreactive T cell response. Autoreactive T cell clones derived by this mechanism are all specific for determinants within amino acids 1-80 of the cyt c protein presented by I-Ek. No T cell responses were observed to the carboxyl terminal 81-104 fragment that dominates the response to foreign cyt c. All clones derived in this study are stimulated by a polypeptide encompassing amino acids 54-68 and utilized the V beta 8.2 TCR gene. In contrast, T cells stimulated by foreign cyt c did indeed respond to fragment 81-104 and appear to utilize alternate TCR genes. Our data demonstrate that B cells specific for linear determinants distributed along the entire length of the foreign cyt c molecule can provide the stimulus required for breaking T cell tolerance to self cyt c. The applications of this work to understanding the mechanisms of autoimmune disease are discussed.

Amino Acid Sequence↗

PubMed1992

An MHC interaction site maps to the amino-terminal half of the T cell receptor alpha chain variable domain.

We have used cloned T cell receptor (TCR) genes from closely related CD4 T cell lines to probe the interaction of the TCR with several specific major histocompatibility complex (MHC) class II ligands. Complementarity determining region 3 (CDR3) equivalents of both alpha and beta TCR chains are required for antigen-MHC recognition. Our data provide novel information about the rotational orientation of TCR-MHC contacts in that exchange of the amino terminal portion of the TCR alpha chain containing the putative CDR1 and CDR2 regions results in both gain and loss of MHC class II specificity by the resulting receptor. These two TCRs differ primarily in recognition of polymorphisms in the second hypervariable region of the MHC class II alpha chain. These results document the involvement of CDR1 and/or CDR2 of the TCR alpha chain in MHC recognition and suggest a rotational orientation of this TCR to its MHC ligand.

Amino Acid Sequence↗

PubMed1992

Monoclonal antibody detection of a major self peptide. MHC class II complex.

MHC class I and class II molecules transport foreign and self peptides to the cell surface and present them to T lymphocytes. Detection of these peptide:MHC complexes has thus far been limited to analysis of the response of a T cell. Previously, we showed that a mAb, Y-Ae, reacts with 10 to 15% of class II molecules on peripheral B lymphocytes and on cells in the thymus medulla but not thymus cortex in mice that express both I-Ab and I-Eb molecules. Elsewhere, we show that Y-Ae detects a self E alpha peptide bound to I-Ab molecules. Data presented here suggest that the antibody binds over the peptide binding groove of class II molecules, and, like a TCR, appears to recognize both the self peptide and polymorphic class II residues. In addition to B lymphocytes, the Y-Ae determinant is expressed at comparable levels on other APC, including macrophages and dendritic cells. Finally, the antibody does not react with invariant chain-associated class II complexes, thus providing direct evidence that invariant chain:class II complexes and peptide:class II complexes are mutually exclusive. These data provide further evidence that immunologic self is of limited complexity, and have important implications for T cell selection, self tolerance, and autoreactivity.

Animals↗

PubMed1992

Cells that present both specific ligand and costimulatory activity are the most efficient inducers of clonal expansion of normal CD4 T cells.

Clonal expansion of naive CD4 T cells is a necessary step in most adaptive immune responses. Two distinct signals are required for clonal expansion to occur, ligation of T-cell receptors by an antigenic peptide bound to self major histocompatibility complex-encoded class II molecules (signal 1) and a costimulatory signal derived from an antigen-presenting cell (signal 2). To study whether these two signals need to be delivered by a single cell in order to induce clonal expansion of normal CD4 T cells, we have used anti-CD3 bound to Fc receptors as a ligand for the T-cell receptor to deliver signal 1 to all CD4T cells, and we have inactivated signal 2 with a newly generated monoclonal antibody or by using Fc receptor-positive cells that lack the costimulator. Costimulation was delivered by cells whose Fc receptors were blocked with anti-Fc receptor monoclonal antibody. Our results indicate that delivery of ligand and costimulator on one cell is at least 30-fold more efficient than separate delivery. No significant clonal expansion was observed when signals 1 and 2 were delivered by different cells. We have also carried out experiments using fibroblast transfectants that can deliver either or both of these two signals. These studies show that separate delivery of these two signals is at least 80-fold less efficient than their combined delivery by one cell. These findings may explain why tissues can express autoantigens and contain active antigen-presenting cells without inducing autoimmunity.

Animals↗

PubMed1992

Heat-stable antigen is a costimulatory molecule for CD4 T cell growth.

Optimal induction of clonal expansion by normal CD4 T cells requires a ligand that can engage the T cell receptor as well as functionally defined costimulatory activity on the same antigen-presenting cell surface. While the presence of effective costimulation induces proliferation, T cell receptor ligation in its absence renders T cells inactive or anergic. The molecular basis of this costimulatory activity remains to be defined. Here we describe a monoclonal antibody that can block the costimulatory activity of splenic accessory cells. Treatment with this antibody not only blocks the proliferation of CD4 T cells to a T cell receptor ligand, but also induces T cell nonresponsiveness to subsequent stimulation. Sequence analysis of the antigen recognized by this antibody indicates that it recognizes a protein that is identical to heat-stable antigen. Gene transfer experiments directly demonstrate that this protein has costimulatory activity. Thus, heat-stable antigen meets the criteria for a costimulator of T cell clonal expansion.

Animals↗

PubMed1992

Physical association of CD4 with the T cell receptor.

The coreceptor hypothesis postulates that physical association of CD4 with the TCR is required for effective signaling for T cell activation. A variety of studies has suggested that the coreceptor function of CD4 allows responses to 10- to 100-fold lower levels of peptide:self MHC class II ligand. We test the hypothesis of CD4 physical association with the TCR in two different ways. First, we use a panel of soluble antibodies directed at different TCR epitopes to activate a cloned T cell line, and show that activation by antibodies directed at a particular TCR epitope can be inhibited by anti-CD4 antibodies binding to a certain CD4 epitope. These effects establish that the interaction of CD4 and the TCR occurs in a specific orientation. Second, we use the same system to provide evidence that the physical association of CD4 with the TCR is required for effective tyrosine phosphorylation of the TCR zeta-chain subunit, presumably reflecting delivery of p56lck (lck) to the TCR. Only anti-TCR antibodies that induce physical association of CD4 with the TCR as monitored by cocapping can induce efficient tyrosine-phosphorylation of the TCR zeta-chain, unless second antibodies are used to force CD4 and the TCR to associate. Furthermore, the phosphorylation of the TCR zeta-chain exactly parallesl physical association in time and drug sensitivity. We conclude from these studies that stimuli that drive physical association of CD4 and the TCR strongly favor T cell activation, supporting the coreceptor hypothesis of CD4 function.

Animals↗

PubMed1992

Isoform-specific associations of CD45 with accessory molecules in human T lymphocytes.

Association of CD45 with surface molecules was investigated in human T lymphocytes by co-capping. CD45 appeared to be associated with the CD3/T cell receptor complex and with CD4 or CD8 molecules in memory, but not in naive T cells, as previously reported in the mouse. Associations of CD45 isoforms with accessory molecules were then identified with seven anti-CD45R monoclonal antibodies (mAb). An isoform-specific association pattern was observed: CD2 co-capped with CD45 molecules recognized by UCHL1 mAb (CD45R0). LFA-1 with molecules bound by 2H4 mAb (CD45RA), and both CD4 and CD8 with molecules reacting with MCA.347 mAb (whose isoform specificity was not known). Further information on the CD45 isoform(s) associated to CD4 and CD8 was sought by assessing the isoform specificity of MCA.347. Cross-competition experiments showed that it reacts with an epitope clearly different from those recognized by 2H4 and UCHL1, and only partially overlapping the PD7/26 epitope (CD45RB). Moreover, the competition between MCA.347 and PD7/26 was maximal in naive T cells and minimal both in memory T cells and in a subset expressing CD11b, a marker of granular lymphocytes. Immunoprecipitation experiments showed that MCA.347 binds to CD45 molecules with a molecular mass of 220, 205 and 190 kDa, the 190-kDa molecules not being recognized by 2H4, PD7/26 or UCHL1. These data indicate that MCA.347 recognizes amino acid sequences different from those coded by the exon A or B of the gene, and not expressed by CD45R0, suggesting that it binds to sequences coded by the exon C. In conclusion, this work shows that in human T cells different CD45 isoforms are associated to different surface molecules: LFA-1 is associated to CD45RA, CD2 to CD45R0 and CD4 and CD8 presumably to CD45RC. This peculiar behavior of CD45 suggests that it may play a crucial role in lymphocyte activation, probably by modulating the signals delivered to the cell by different receptor systems.

Antibodies, Monoclonal↗

PubMed1992

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.

Animals↗

PubMed1992

Co-stimulation of murine CD4 T cell growth: cooperation between B7 and heat-stable antigen.

The B cell activation antigen B7/BB1 has been shown to co-stimulate growth of human T cells by binding the T cell molecule CD28. In mice, the heat-stable antigen (HSA) has also been shown to act as a co-stimulator for T cell growth. In this study, we have evaluated the contributions of B7 and HSA to the co-stimulatory activity of antigen-presenting cells (APC). Mouse B7 provides co-stimulatory activity for murine CD4 T cells in anti-CD3-induced proliferation. Human CTLA4Ig, a chimeric molecule comprising the extracellular region of CTLA-4 fused to an immunoglobulin C gamma fragment, binds to murine B7. We, therefore, use human CTLA4Ig and the hamster anti-HSA monoclonal antibody 20C9 to analyze the relative contributions of B7 and HSA to the co-stimulatory activity of murine spleen APC. Our data reveal that both murine B7 and HSA are expressed by dendritic cells and by low-density spleen B cells. Either CTLA4Ig alone or anti-HSA alone inhibited CD4 T cell proliferation to anti-CD3 by > 90%, while CTLA4Ig and anti-HSA together were far more efficient in inhibiting clonal expansion of CD4 T cells. These results demonstrate that functionally defined co-stimulation involves at least B7 and HSA and suggest that signals delivered by B7 and HSA synergize in promoting T cell growth.

Animals↗

PubMed1992

The immune system evolved to discriminate infectious nonself from noninfectious self.

Here, Charles Janeway argues that the requirement for two signals to initiate the adaptive immune response may reflect the evolutionary history of host defences. Early phases of host defence involve receptors and ligands that may have controlled immune responses prior to the development of clonally-distributed receptors encoded in rearranging genes. The former receptors persist in contemporary vertebrates both to trigger innate or nonclonal responses and to signal to lymphocytes that a particular antigen is associated with a microorganism.

Animals↗

PubMed1992

Extensive CD4 cross-linking inhibits T cell activation by anti-receptor antibody but not by antigen.

Anti-CD4 mAbs have been shown to inhibit T cell activation in a variety of ways. We have tested a panel of IgG and IgM anti-CD4 mAbs for their effects on the activation of a cloned T cell line by antigen presented by syngeneic accessory cells, by soluble anti-T cell receptor antibodies, and by mitogenic lectins. Both IgM and IgG mAbs to CD4 inhibit responses to mitogenic lectins. However, IgM, but not IgG, anti-CD4 antibodies inhibit T cell activation by mAbs specific for the TCR. This inhibitory activity appears to be due to the signaling effects of IgM mAbs, as cross-linked IgG antibodies mimic the behavior of the IgM anti-CD4 antibodies. Inhibition of T cell activation correlates with the ability of IgM and of cross-linked IgG anti-CD4 antibodies to induce tyrosine phosphorylation of the CD4-associated tyrosine kinase p56lck and an unknown substrate, pp32. Surprisingly, we find that the IgM anti-CD4 mAbs tested had no effect on the specific antigen recognition, despite their potent inhibitory effects on the other responses of the same cloned T cell line. These results suggest that multivalent CD4 interactions with ligands such as MHC class II molecules are inhibitory of T cell activation, but that this inhibition can be reversed when CD4 and the TCR bind the same ligand. We discuss the possible implications for positive intrathymic selection of these findings on signaling through CD4.

Animals↗

PubMed1992

The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation.

The T cell receptor is a multicomponent signalling machine. The three main components are the hypervariable alpha: beta heterodimer that confers specificity and its attendant invariant chains CD3 gamma, delta, and epsilon and the zeta:zeta or zeta:eta, the CD4 or CD8 coreceptor, and CD45. Each of these components is required for efficient signal transduction, and each has relevant enzymatic activity associated with it. The invariant part of the T cell receptor is associated with the tyrosine kinase p59fyn, the coreceptors are associated with the tyrosine kinase p56lck, and the cytoplasmic domain of CD45 has tyrosine-specific phosphatase activity. Moreover, there is strong evidence that these components interact in the plane of the membrane, and that these interactions are relevant for signal transduction. Finally, changes in the structure of CD45 that occur during differentiation of T cells alter the interactions of these three components of the signal transducing machinery, perhaps accounting for changes in signal transduction that accompany T-cell development in the thymus and the development of immunological memory cells.

Animals↗

PubMed1991

Sequence analysis of peptides bound to MHC class II molecules.

CD4 T cells recognize peptide fragments of foreign proteins bound to self class II molecules of the major histocompatibility complex (MHC). Naturally processed peptide fragments bound to MHC class II molecules are peptides of 13-17 amino acids which appear to be precessively truncated from the carboxy terminus, perhaps after binding to the MHC class II molecule. The finding of predominant self peptides has interesting implications for antigen processing and self-non-self discrimination.

Amino Acid Sequence↗

PubMed1991

On the complexity of self.

Self peptides bound to self major histocompatibility complex (MHC) molecules have been implicated both in positive and in negative selection of T cells during intrathymic development. We report here that the novel MHC-restricted monoclonal antibody Y-Ae detects the MHC class II bound form of a major self peptide. Y-Ae binds approximately 12% of the relevant MHC class II molecules on self antigen presenting cells. The peptide detected by Y-Ae is one of several major peptides eluted from the MHC molecule. These data suggest that self peptides presented by self MHC class II molecules at densities sufficient to signal a CD4 T cell are of very limited complexity. Furthermore, as Y-Ae stains antigen presenting cells that mediate negative selection but not thymic cortical epithelial cells that drive positive selection, differential expression of self peptide:self MHC class II complexes may be a key feature of intrathymic selection.

Amino Acid Sequence↗

PubMed1991

The use of the polymerase chain reaction to map CD4+ T cell epitopes.

CD4+ T cells recognize processed exogenous antigen in the form of peptides bound to syngeneic major histocompatibility complex class II molecules on antigen-presenting cells. We have developed a novel and convenient method to synthesize and map CD4+ T cell epitopes of cloned antigens using polymerase chain reaction (PCR)-directed construction of genes expressing recombinant protein fragments. Unique restriction sites incorporated into the PCR primers were employed for the unidirectional cloning of gene fragments into a bacterial expression vector that can be induced to high-level expression. The bacterial lysate could be used directly in T cell proliferation assays. Overlapping recombinant fragments spanning the entire protein were generated and tested. The length of the sequence containing the epitope was further reduced by utilizing PCR to generate 3' truncations. Finally, a small number of overlapping peptides spanning a sequence of 39 amino acids were synthesized to identify a thirteen-amino acid peptide epitope within chicken transferrin that stimulates the T helper cell clone D10.G4.1. PCR-directed construction of fragments of antigen allows for optimal design of strategies for the mapping and analysis of CD4+ T cell epitopes.

Amino Acid Sequence↗

PubMed1991

Regulatory responses in contact sensitivity: afferent suppressor T cells inhibit the activation of efferent suppressor T cells.

Two types of suppressor cells regulate the contact sensitivity (CS) response to picryl chloride (PCL). Afferent suppressor T cells (Ts-aff) inhibit the generation of CS responses to PCL, while efferent suppressor T cells (Ts-eff) inhibit the activity of Th 1 cells that mediate CS reaction. Intravenous injection of mice with TNP-substituted peritoneal exudate cells (TNP-PEC) induces Ts-eff cells that block the adoptive transfer of contact sensitivity. The induction of Ts-eff cells is prevented by the presence of Ts-aff cells, which in turn are induced by the injection of TNP-PEC coupled with antibodies of the IgG2a and IgG2b isotype (TNP-PEC-Ab). If an animal is injected with TNP-PEC prior to or simultaneously with TNP-PEC-Ab, it generates only Ts-aff cells, while if it is injected with TNP-PEC alone or TNP-PEC prior to TNP-PEC-Ab, it generates Ts-eff cells. Ts-aff cells effect only the generation of Ts-eff cells, as the addition of Ts-eff cells to assays for Ts-eff cells has no inhibitory effect on the suppressive effects of Ts-eff cells in adoptive transfer. Our experiments show that Ts-aff cells induced by TNP-PEC-Ab are phenotypically either Lyt 1+2- or Lyt 1-2+, but only the latter inhibit the generation of Ts-eff cells in vivo. The Ts-aff cells that inhibit Ts-eff activity adhere to the lectin Vicia villosa (VV), while Ts-eff cells are VV nonadherent. In addition, Ts-aff cells can prevent the generation of Ts-eff to linked haptens presented on the same PEC. It appears that a cascade of Ts cell interactions are involved in the regulation of CS responses.

Animals↗