Biomedical subjects

M M Davis

Publications and source records attributed to M M Davis.

At least 109 records · Page 6Linked to original sources

PubMed1994

A peptide antigen antagonist prevents the differentiation of T cell receptor transgenic thymocytes.

The developmental fate of an immature T cell is determined in the thymus. Depending on the specificity of its TCR, a thymocyte receives signals to either die or differentiate. We have used fetal thymic organ cultures derived from TCR transgenic mice to examine the role of MHC/peptide ligands in T cell selection. Single amino acid substituted peptide analogues of the Ag recognized by the transgenic TCR were examined for their ability to enhance or interfere with positive selection. We have identified a nonstimulatory peptide analogue that interferes with the differentiation of transgenic CD4+8+ thymocytes into CD4+8- cells. We also show that this peptide, substituted in a TCR contact residue, is a competitive antagonist for activation of the T cell hybridoma expressing the same TCR. These observations demonstrate a novel mechanism for tolerance induction in the thymus.

Amino Acid Sequence↗

PubMed1994

Induction of autoimmune disease in mice by germline alteration of the T cell receptor gene expression.

Germline expression of rearranged TCR alpha-chain transgenes with the Ig H chain enhancer reproducibly elicits T cell-mediated autoimmune disease in the thyroid gland, gastric mucosa, Langerhans islets, salivary gland, ovaries, and testes in selected strains of normal mice. Multiple organs are destroyed in a single transgenic mouse and the same organ in transgenic strains with different MHC background, suggesting the transgene expression can elicit self-reactive T cell clones having different Ag specificities and MHC restrictions. Construction of this autoimmune-inducing TCR alpha EH transgene does not require particular V alpha J alpha gene segments or Ag specificities. Moreover, the autoimmune disease can be adoptively transferred to syngeneic normal mice by T cells expressing endogenous TCR alpha-chains. Taken together, these results indicate that the TCR alpha EH transgene expression does not suppress endogenous alpha-chain gene rearrangement and may trigger the expansion/activation of various self-reactive T cells expressing endogenous TCR alpha- and beta-chains. Furthermore, it appears that the transgene-induced autoimmune T cells are not deleted in the normal thymus or rendered anergic upon contact with the normal target self Ag, but can be controlled by a T cell-dependent mechanism, since transfer of the transgenic bone marrow cells to histocompatible SCID mice produces the same autoimmune disease as in the donors, and the autoimmune development in the SCID mice is effectively prevented by co-transfer of syngeneic nontransgenic T cells. This novel autoimmune model produced by genetic manipulation of the T cell lineage, not the target self Ag or the environment of T cell differentiation/selection, should be useful for elucidating the immunologic and genetic basis of autoimmune disease.

Age Factors↗

PubMed1994

The nature of major histocompatibility complex recognition by gamma delta T cells.

Despite intensive efforts, the general rules for gamma delta T cell recognition remain undefined. Here, we take advantage of the detailed knowledge of the molecular structure and biosynthetic pathways of major histocompatibility complex (MHC) molecules to analyze the recognition properties of the gamma delta T cell clones LBK5 (specific for the class II MHC, IEk) and G8 (specific for the nonclassical class I MHC, TL10b). We find that the activation of these clones requires neither class I nor class II antigen-processing and that peptides do not confer specificity. Epitope mapping also shows that the topology of gamma delta T cell receptor interaction with the MHC is distinct from that of alpha beta T cells. These results suggest that the molecular nature of gamma delta T cell recognition is fundamentally different than that of alpha beta T cells.

Amino Acid Sequence↗

PubMed1994

CDR3 length in antigen-specific immune receptors.

In both immunoglobulins (Ig) and T cell receptors (TCR), the rearrangement of V, D, and J region sequence elements during lymphocyte maturation creates an enormous degree of diversity in an area referred to as the complementarity determining region 3 (CDR3) loop. Variations in the particular V, D, and J elements used, precise points of recombination, and random nucleotide addition all lead to extensive length and sequence heterogeneity. CDR3 loops are often critical for antigen binding in Igs and appear to provide the principal peptide binding residues in TCRs. To better understand the physical and selective constraints on these sequences, we have compiled information on CDR3 size variation for Ig H, L (kappa and lambda) and TCR alpha, beta, gamma, and delta. Ig H and TCR delta CDR3s are the most variable in size and are significantly longer than L and gamma chains, respectively. In contrast, TCR alpha and beta chain distributions are highly constrained, with nearly identical average CDR3 lengths, and their length distributions are not altered by thymic selection. Perhaps most significantly, these CDR3 length profiles suggest that gamma/delta TCRs are more similar to Igs than to alpha/beta TCRs in their putative ligand binding region, and thus gamma/delta and alpha/beta T cells may have fundamentally different recognition properties.

Amino Acid Sequence↗

PubMed1994

Requirement of a second signal from antigen presenting cells in the clonal deletion of immature T cells.

The role of antigen presenting cells (APC) in T cell clonal deletion was investigated by culturing murine thymic lymphocytes with the superantigen staphylococcal enterotoxin B (SEB) in the absence or presence of APC. As the APC, we used B lymphoma cell lines A20.2J and BAL17.2, both expressing MHC class II antigens at high levels. SEB reactive V beta 8+ cells were deleted only when A20.2J cells were used as APC. By using thymocytes from transgenic mice carrying a TCR beta chain transgene, it was further shown that the deletion occurred at the CD4+CD8+ stage. The other cell line, BAL17.2, failed to induce clonal deletion, although this cell line was able to stimulate the proliferative response of SEB-primed T cells. The activity of A20.2J cells to induce clonal deletion was completely abolished by fixation with paraformaldehyde, whereas the same treatment kept the ability of this cell line to induce the proliferative response of non-primed as well as SEB-primed T cells. It was further shown that the deletion was abolished by the addition of anti-MHC class II but not anti-B7 mAb in the culture. These results provided explicit evidence that a signal(s) from APC, which is distinct from that required for primary or secondary proliferative response of mature peripheral T cells, is involved in clonal deletion of thymic immature T cells.

Animals↗

PubMed1993

Sequence of the cDNA encoding Ras GTPase-activating protein from rat.

We cloned and sequenced a 3296-bp cDNA encoding the rat Ras GTPase-activating protein (GAP). Comparison of the nucleotide (nt) and deduced amino acid (aa) sequences to those of previously described GAP cDNAs revealed greater than 90% identity. Homology is highest between rat and mouse GAP both at the nt (96% identity) and deduced aa levels (98% identity).

Amino Acid Sequence↗

PubMed1993

pH affects both the mechanism and the specificity of peptide binding to a class II major histocompatibility complex molecule.

We have compared the contribution of electrostatic forces in the binding of antigenic peptides to the class II MHC molecule, IEk, at weakly acidic (pH 5.4) and neutral (pH 7.5) pH values. The binding of specific moth cytochrome c (MCC) and hemoglobin (Hb) peptides to IEk is very sensitive to ionic strength at pH 7.5 but not at pH 5.4, indicating that the mechanism of peptide binding is pH-dependent. Substitution of the C-terminal Lys in MCC for an Ala residue selectively destroyed peptide binding at neutral pH and increased the dissociation rate at least 30-fold, implicating this residue in the pH-dependent electrostatic interaction. The presence of a C-terminal Lys in many of the peptides that are restricted to IEk suggests that this electrostatic interaction is widely used to bind peptides to this MHC molecule. We also probed the electrostatic environment of the peptide binding groove adjacent to the N-terminus of the bound peptide by rapid-diffusion fluorescence energy transfer using a terbium-labeled MCC peptide. In this region of the peptide binding groove, more negative charge is present at pH 7.5 than at pH 5.4. These findings indicate the importance of MHC carboxylates to the mechanism and specificity of peptide binding. The biological importance of having two distinct mechanisms of peptide binding at different pH may be that it acts to broaden the spectrum of antigenic peptides that can be presented to T-cells.

Amino Acid Sequence↗

PubMed1993

Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli.

Class II major histocompatibility complex molecules play a major role in the immune response by binding peptide fragments of exogenous antigens and displaying them on the surfaces of antigen-presenting cells, where they can be recognized by T cells. To facilitate structural and functional studies of these molecules, we have produced truncated alpha and beta chains of the murine class II molecule I-Ek in Escherichia coli (Ec-I-Ek) and have developed conditions to fold them in the presence of specific peptides with yields of complex approaching 2%. Reconstitution is specific since only unlabeled peptide known to bind I-Ek compete with biotinylated peptide, as assessed by ELISA. Complexes of the refolded heterodimer (Ec-I-Ek) with either of two different peptide antigens remain associated during nonreducing SDS/PAGE. Immobilized Ec-I-Ek-peptide complexes stimulate lymphokine production by three T-cell clones in an antigen-specific manner with a dose-response relation comparable to previously described soluble I-Ek molecules produced in CHO cells. These results demonstrate that folding of Ek alpha and Ek beta polypeptides does not require any other protein to produce the biologically relevant conformation and that carbohydrate modification of this class II molecule is not necessary for alpha beta T-cell recognition.

Amino Acid Sequence↗

PubMed1993

Activation and differentiation requirements of primary T cells in vitro.

The progression of T cells from a quiescent or resting state to fully activated, proliferating cells is a crucial step in the initiation of an immune response. We have developed an in vitro system to study the requirements for triggering or hindering this pathway by using naive T cells derived from T-cell antigen receptor alpha beta transgenic animals and peptide-major histocompatibility (MHC) complexes coated on plates. Whereas previously stimulated T cells require only peptide-MHC complexes to produce interleukin 2 (IL-2), naive cells require at least one additional signal, which can be provided by either an anti-CD28 antibody or the protein kinase C stimulant phorbol 12-myristate 13-acetate. In contrast, the anti-CD28 antibody augments IL-2 production by primed T cells but is not required, and phorbol 12-myristate 13-acetate has no discernable effect. Thus we find that native T cells have significantly more stringent requirements for IL-2 production than primed cells and that this fits well with previous observations in other in vitro systems as well as in vivo models of autoimmunity. We also find that peptide-MHC complex stimulation of naive T cells, together with exogenous IL-2, is sufficient to convert these cells to primed T cells in vitro in 2 days, as assayed both by surface marker analysis and stimulation requirements. Taken together with the above results, this suggests that the activation of primary T cells requires at least two signals and that IL-2 produced by naive T cells in vivo may act in an autocrine fashion to allow them to proliferate and differentiate.

Animals↗

PubMed1993

T cell receptor interaction with peptide/major histocompatibility complex (MHC) and superantigen/MHC ligands is dominated by antigen.

While recent evidence strongly suggests that the third complementarity determining regions (CDR3s) of T cell receptors (TCRs) directly contact antigenic peptides bound to major histocompatibility complex (MHC) molecules, the nature of other TCR contact(s) is less clear. Here we probe the extent to which different antigens can affect this interaction by comparing the responses of T cells bearing structurally related TCRs to cytochrome c peptides and staphylococcal enterotoxin A (SEA) presented by 13 mutant antigen-presenting cell (APC) lines. Each APC expresses a class II MHC molecule (I-Ek) with a single substitution of an amino acid residue predicted to be located on the MHC alpha helices and to point "up" towards the TCR. We find that very limited changes (even a single amino acid) in either a CDR3 loop of the TCR or in a contact residue of the antigenic peptide can have a profound effect on relatively distant TCR/MHC interactions. The extent of these effects can be as great as that observed between T cells bearing entirely different TCRs and recognizing different peptides. We also find that superantigen presentation entails a distinct mode of TCR/MHC interaction compared with peptide presentation. These data suggest that TCR/MHC contacts can be made in a variety of ways between the same TCR and MHC, with the final configuration apparently dominated by the antigen. These observations suggest a molecular basis for recent reports in which either peptide analogues or superantigens trigger distinct pathways of T cell activation.

Amino Acid Sequence↗

PubMed1993

Two-dimensional nuclear magnetic resonance analysis of a labeled peptide bound to a class II major histocompatibility complex molecule.

The formation of peptide/major histocompatibility complex (MHC) complexes and their subsequent recognition by T cells is a pivotal event in the initiation of an immune response. While X-ray crystal structures are now available for class I MHC/peptide complexes, little detailed structural information is known about the class II MHC equivalent, and there are no solution structure data for either. A 16 amino acid residue moth cytochrome c peptide (residues 88 to 103) was 13C-labeled for two-dimensional isotope-edited NMR analysis. The peptide was labeled either selectively in the methyl groups of alanine residues or uniformly at every carbon position, and bound to unlabeled soluble mouse I-Ek class II MHC molecules. Although alpha-helical in the native cytochrome c protein and with no uniform structure in solution, the peptide is bound to the I-Ek molecule with the alpha-carbon atoms of the 11 C-terminal residues held in the binding groove. This indicates that the class II MHC peptide binding site is somewhat larger than that of class I MHC molecules (> or = 11 amino acid residues versus 8 to 10 amino acid residues), consistent with recent data on eluted peptides. Despite the large size of the complex (approximately 70 kDa), nuclear Overhauser effects are clearly detectable between peptide side-chains and the MHC molecule. Indications of the buried or exposed nature of particular side-chains within the bound peptide are derived from the NMR data and these are used together with information from previous biological studies to propose a crude model of the interaction of the peptide with the groove of the MHC molecule. We find no evidence for a conformational change in the peptide/MHC complex in the spectra at pH 5.0 versus pH 7.0, despite a 40-fold faster on-rate for the peptide at the lower pH value.

Amino Acid Sequence↗

PubMed1993

Transfer of putative complementarity-determining region loops of T cell receptor V domains confers toxin reactivity but not peptide/MHC specificity.

We have used multiple-amino acid replacement mutagenesis to examine the roles of the TCR homologues of Ig complementarity-determining regions (CDR) and framework sequences in Ag-MHC and Staphylococcus aureus enterotoxin reactivity. In the three cases examined, transplantation of Ig CDR3 homologues between I-Ek-restricted TCR that recognize distinct peptides did not result in transfer of peptide reactivity. Thus the structural context of the CDR3 loops, e.g., both neighboring CDR and the V beta structure, must play a crucial, albeit supporting, role in ligand recognition. The extreme lability of this context was also shown by the fact that transplantation of the CDR1, -2, and -3 loops from the beta chain of 5C.C7 onto a V beta 1 framework failed to transfer MHC-peptide specificity even when the TCR-alpha chains were identical. In contrast, superantigen reactivity was readily transferred in several cases, with CDR2 transplants conferring strong staphylococcal enterotoxin B and A reactivity and CDR1 transplants yielding weak reactivities. This suggests that bacterial (and perhaps other) superantigens bind to many of the same regions of the TCR V beta that are believed to interact with MHC molecules. These regions of V beta may be ideal targets for superantigen binding precisely because they interact with MHC molecules and thus may be relatively conserved.

Animals↗

PubMed1993

Regulation of T cell production in T cell receptor transgenic mice.

The thymus produces many more cells than it releases into the periphery. According to generally accepted models of T cell development most of this loss occurs in the thymic cortex, among CD4+8+ thymocytes. An interesting situation arises in the case of T cell receptor (TcR) transgenic mice in which all cells can potentially be positively selected, leading to a theoretical increase of about 30-fold in the survival rate of CD4+8+ cells and in their transition to mature CD4+8- or CD4-8+ thymocytes. This in turn should lead to a 30-fold increase in the size of the thymic medulla, in the emigration rate and in the size of the peripheral T cell pool. Increases in medullary or peripheral pool sizes of this magnitude are not seen in TcR transgenic mice. The question was therefore asked whether some form of homeostatic process regulated the size of the mature T cell pool and at what level it might operate. In this report we demonstrate that the increased rate of double-positive to single-positive transition in the TcR transgenic mice is directly reflected in an increased emigration rate, and that the medulla seems to be relatively efficient regardless of the number of cells passing through it. However, the potential increases in emigrant numbers in TcR transgenic mice are offset by the reduced size of the CD4+8+ thymocyte pool. It would appear then that regulation of T cell production, if it occurs, probably does so through regulation of the size of the CD4+8+ thymocyte pool. Mechanisms for regulation of this kind are not yet known.

Animals↗

PubMed1993

How alpha beta T-cell receptors 'see' peptide/MHC complexes.

Recent results have added new information to our understanding of alpha beta T-cell receptor mediated recognition. In particular, we find that the V(D)J junction or 'CDR3' portion of TCR alpha and beta seem most important in contacting peptides bound to MHC molecules, consistent with previous predictions. Surprisingly, these same CDR3-peptide contacts also appear to have a major influence on the TCR-MHC molecule interactions as well.

Amino Acid Sequence↗

PubMed1993

Topology and affinity of T-cell receptor mediated recognition of peptide-MHC complexes.

Significant progress has been made on several long-standing issues regarding T-cell receptor mediated recognition of antigen-MHC complexes. For one, early data suggest that the affinity of the T-cell receptor for the peptide-MHC complex is extremely low, with a KD of approximately 10(-4)-10(-5)M, much weaker than most antibody-antigen interactions. The fact that this affinity is lower than that of some T-cell adhesion molecules for their ligands could have important implications for immune surveillance. A second area of interest is the topology of T-cell receptor recognition; evidence of direct contact between the third complementarity determining region of the T-cell receptor and peptide determinants has been obtained. In addition, the orientation of the T-cell receptor with respect to several antigen-MHC complexes has been predicted. They suggest that whereas most or all peptides seem to bind in the same orientation in both class I and class II MHC molecules, the orientation of the T-cell receptor over the peptide-MHC complex may not be fixed.

Animals↗

PubMed1992

The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice.

To study the factors that determine whether CD4+ T cells produce interleukin 4 (IL-4) or interferon gamma (IFN-gamma) upon stimulation we used a system allowing naive T cells to be primed in vitro by specific antigen. Dense CD4+ T cells were purified from mice that expressed transgenes encoding a T cell receptor specific for pigeon cytochrome C peptide 88-104 in association with I-Ek. These T cells produced very limited amounts of IL-4 and IFN-gamma upon immediate challenge with 88-104 and antigen-presenting cells (APC). However, after an initial "priming" culture in which they were incubated for 4 d in the presence of 88-104, APC, and 1,000 U/ml IL-4, the T cells acquired the capacity to produce substantial amounts of IL-4 upon rechallenge but made very little IFN-gamma. Cells primed in the absence of IL-4 produced IFN-gamma upon rechallenge but virtually no IL-4. The inhibitory effect of IL-4 on IFN-gamma production did not appear to be mediated by the induction of IL-10 production since IL-10 addition to initial cultures did not suppress priming for IFN-gamma production, nor did anti-IL-10 block the inhibitory effect of IL-4. IFN-gamma itself did not increase priming for IFN-gamma production, nor did anti-IFN-gamma reduce such priming. IFN-gamma did, however, diminish priming for IL-4 production when limiting amounts of IL-4 (100 U/ml) were used in the initial culture. The dominant effect of IL-4 in determining the lymphokine-producing phenotype of primed cells was observed with dendritic cells (DC), activated B cells, and I-Ek-transfected fibroblasts as APC. However, the different APC did vary in their potency, with DC being superior to activated B cells, which were superior to transfected fibroblasts.

Amino Acid Sequence↗

PubMed1992

Analysis of a T cell receptor gene as a target of the somatic hypermutation mechanism.

In an effort to identify cis-acting elements required for targeting of the somatic hypermutation process in mice, we examined whether a T cell receptor (TCR) transgene under the control of the immunoglobulin (Ig) heavy (H) chain intron enhancer would be mutated in antigen-stimulated B cells. Hybridomas were established from splenic B cells of mice carrying two copies of the TCR transgene after hyperimmunization with phosphorylcholine keyhole limpet hemocyanin. Northern analysis revealed that all of the transgene-containing hybridomas expressed the TCR mRNA. Multiple somatic point mutations were found in seven of eight endogenous Ig VH genes examined. In contrast, 29 of 32 TCR genes examined contained no mutations. One potential mutation was seen in each of the three other TCR genes. Our data indicate that although the TCR transgene is expressed in B cells, it is not efficiently targeted by the mutator mechanism. Furthermore, the presence of an Ig H chain enhancer is itself not sufficient for targeting of the somatic hypermutation mechanism.

Animals↗

PubMed1992

Mapping T-cell receptor-peptide contacts by variant peptide immunization of single-chain transgenics.

To test models of T-cell recognition, mice transgenic for T-cell receptor alpha or beta chain have been immunized with variant peptides that force changes in the resulting T-cell response. In particular, charge substitutions on the peptide often elicit reciprocal charges in the junctional (CDR3) sequences of T-cell receptor V alpha or V beta chains, indicating direct T-cell receptor-peptide contact, and allowing derivation of a topology for the T-cell receptor-MHC interaction. At one position on the peptide, variants transformed a homogeneous V beta response into a very heterogeneous one.

Amino Acid Sequence↗