Search PubMed⌕ Search

Biomedical subjects

P M Allen

Publications and source records attributed to P M Allen.

At least 19 recordsLinked to original sources
PubMed2006

Development of a novel strategy for engineering high-affinity proteins by yeast display.

Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.

Cell Differentiation↗
PubMed2001

Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis.

Neutrophils are prominent participants in the joint inflammation of human rheumatoid arthritis (RA) patients, but the extent of their role in the inductive phase of joint inflammation is unknown. In the K/BxN mouse RA model, transfer of autoreactive Ig from the K/BxN mouse into mice induces a rapid and profound joint-specific inflammatory response reminiscent of human RA. We observed that after K/BxN serum transfer, the earliest clinical signs of inflammation in the ankle joint correlated with the presence of neutrophils in the synovial regions of recipient mouse ankle joints. In this study, we investigated the role of neutrophils in the early inflammatory response to transferred arthritogenic serum from the K/BxN transgenic mouse. Mice were treated with a neutrophil-depleting mAb before and following transfer of arthritogenic serum and scored for clinical indications of inflammation and severity of swelling in ankle joints and front paws. In the absence of neutrophils, mice were completely resistant to the inflammatory effects of K/BxN serum. Importantly, depletion of neutrophils in diseased recipient mice up to 5 days after serum transfer reversed the inflammatory reaction in the joints. Transfer of serum into mice deficient in the generation of nitrogen or oxygen radicals (inducible NO synthase 2 or gp91(phox) genes, respectively) gave normal inflammatory responses, indicating that neither pathway is essential for disease induction. These studies have identified a critical role for neutrophils in initiating and maintaining inflammatory processes in the joint.

Animals↗
PubMed2001

Two MHC surface amino acid differences distinguish foreign peptide recognition from autoantigen specificity.

KRN T cells can recognize two self MHC alleles with differing biological consequences. They respond to the foreign peptide RN(42--56) bound to I-A(k) or alternatively initiate autoimmune arthritis by interacting with a self Ag, GPI(282--294), on I-A(g7). Five surface amino acid differences between the two MHC molecules collectively alter which peptide side chains are recognized by the KRN TCR. In this study, it is shown that mutation of only two of these residues, alpha 65 and beta 78, in I-A(k) to their I-A(g7) counterparts is sufficient to allow recognition of the TCR contacts from GPI(282--294). To provide a detailed mechanism for the specificity change, the distinct contributions of each of these two mutations to the global effect on peptide specificity were analyzed. The alpha65 mutation is shown to broaden the spectrum of amino acids permissible at P8 of the peptide. In contrast, the beta 78 mutation alone blocks KRN TCR interaction with I-A(k) and requires the simultaneous presence of the alpha 65 mutation to preserve recognition. In the presence of the alpha 65 mutation, the beta 78 residue broadens peptide recognition at P3 and prevents recognition of the P8 L in RN(42--56), thus producing the observed specificity shift. These results localize the functionally relevant differences between the surfaces of two self-restricted MHC molecules to two residues that have counterbalanced positive and negative contributions to interaction with a single TCR. They highlight how subtle structural distinctions attributable to single amino acids can stand at the interface between foreign Ag responsiveness and pathogenic autoreactivity.

Amino Acid Sequence↗
PubMed2001

Structural and functional consequences of altering a peptide MHC anchor residue.

To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-E(k) complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 A resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, approximately 10 A distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.

Amino Acid Sequence↗
PubMed2001

Environmental control of immunological synapse formation and duration.

The coordination of T-cell migration and antigen recognition is crucial for an effective immune response. We have proposed that this coordination is achieved by formation of an immunological synapse between the T cell and the antigen-presenting cell (APC). Our view contrasts with the serial encounter model also proposed in this issue of Trends in Immunology, which is based on transient T cell-APC interactions when surrounded by collagen. Here, we propose a model that reconciles immunological synapse formation and serial encounters based on environmental control of immunological synapse formation.

Animals↗
PubMed2001

Facility of accommodation in myopia.

PURPOSE: Myopes have been shown to have abnormal accommodative characteristics. This study investigated the characteristics of accommodation facility in myopic and emmetropic students. METHODS: Distance and near positive and negative accommodation response time components of facility were measured over a 1 min period using a -2.00 D/zero lens pair for distance responses and a +/-2.00 D lens pair for near responses. 79 students (37 myopes and 42 emmetropes) aged 18-27 years acted as subjects. Subjects were masked, and the results were analysed in a masked fashion. RESULTS: Mean distance facility was significantly lower (9.7 cycles per minute (cpm)) in the myopic group compared with the mean distance facility in the emmetropic group (15.6 cpm; p < 0.005). There was no significant difference in the near facilities of the two groups (11.5 cpm in myopes vs 12.9 cpm in emmetropes). Positive accommodation response time for distance vision was greater than 4 s in 45% of myopes and in 9% of emmetropes. CONCLUSIONS: Our findings confirm that myopes tend to have abnormal accommodation responses to blur. Distance facility, but not near facility of accommodation is more frequently reduced in myopes than in emmetropes.

Accommodation, Ocular↗
PubMed2001

The immunological synapse.

The adaptive immune response is initiated by the interaction of T cell antigen receptors with major histocompatibility complex molecule-peptide complexes in the nanometer scale gap between a T cell and an antigen-presenting cell, referred to as an immunological synapse. In this review we focus on the concept of immunological synapse formation as it relates to membrane structure, T cell polarity, signaling pathways, and the antigen-presenting cell. Membrane domains provide an organizational principle for compartmentalization within the immunological synapse. T cell polarization by chemokines increases T cell sensitivity to antigen. The current model is that signaling and formation of the immunological synapse are tightly interwoven in mature T cells. We also extend this model to natural killer cell activation, where the inhibitory NK synapse provides a striking example in which inhibition of signaling leaves the synapse in its nascent, inverted state. The APC may also play an active role in immunological synapse formation, particularly for activation of naïve T cells.

Animals↗
PubMed2000

Ligand-specific selection of MHC class II-restricted thymocytes in fetal thymic organ culture.

Positive and negative selection of thymocytes is determined by the specificity of the TCR and signaling through its associated molecules. We have studied selection of thymocytes bearing a MHC class II-restricted TCR using fetal thymic organ culture. This system allows the addition of peptides to the already diverse panoply of endogenous peptide ligands and is useful for analyzing ligand-specific negative selection of CD4 single positive (CD4SP) thymocytes. The data reveal that the ability of a given ligand to mediate negative selection is related to its dissociation rate from the TCR. We find that negative selection is very sensitive, and only the weakest ligand that we can identify fails to induce negative selection. None of the numerous peptides tested were able to induce an increase in CD4SP thymocytes. In addition, the ligands that induce negative selection of CD4SP thymocytes also cause an increase in numbers of CD8SP thymocytes bearing high levels of the class II-restricted TCR. Although these cells have a cell surface phenotype consistent with positive selection, they most likely represent cells in the process of negative selection. Further analysis reveals that these cells are not induced by these ligands in intact adult animals and that their induction is probably only revealed in the organ culture system.

Amino Acid Substitution↗
PubMed2000

Molecular basis for recognition of an arthritic peptide and a foreign epitope on distinct MHC molecules by a single TCR.

KRN TCR transgenic T cells recognize two self-MHC molecules: a foreign peptide, bovine RNase 42-56, on I-Ak and an autoantigen, glucose-6-phosphate isomerase 282-294, on I-Ag7. Because the latter recognition event initiates a disease closely resembling human rheumatoid arthritis, we investigated the structural basis of this pathogenic TCR's dual specificity. While peptide recognition is altered to a minor degree between the MHC molecules, we show that the receptor's cross-reactivity critically depends upon a TCR contact residue completely conserved in the foreign and self peptides. Further, the altered recognition of peptide derives from discrete differences on the MHC recognition surfaces and not the disparate binding grooves. This work provides a detailed structural comparison of an autoreactive TCR's interactions with naturally occurring peptides on distinct MHC molecules. The capacity to interact with multiple self-MHCs in this manner increases the number of potentially pathogenic self-interactions available to a T cell.

Amino Acid Sequence↗
PubMed2000

Genetically separable determinants of hair keratin gene expression.

The nude locus encodes Whn, a transcription factor of the forkhead/winged-helix class. Mutations in Whn cause failure of differentiation of thymic epithelium with a corresponding lack of intrathymic T-cell development; in the skin, differentiation of follicular keratinocytes is disturbed resulting, in the formation of fragile hair shafts. Here, we describe the identification and characterization of a novel nude allele, nu(StL). nu(StL) encodes a truncated Whn transcription factor protein, designated Whn(StL), lacking the activation domain but retaining the characteristic DNA binding domain. In contrast, the previously described Whn(nu) mutant protein lacks both domains. nu(StL)/nu(StL) mice show an alymphoid thymic rudiment and lack of peripheral T cells, similar to nu/nu mice. In the skin, impaired expression of hair keratin genes mHa1, mHa2, mHa3 and mHa4, mHb3, mHb4, mHb5, and mHb6 is observed in a pattern that parallels that of nu/nu mice: both mutant alleles behave as hypomorphs with respect to the expression of these hair keratin genes. However, a significant difference between these two alleles exists for mHa5 expression, which is reduced in nu(StL)/nu(StL) but not in nu/nu mice. We show that the mutant Whn protein in nu/nu mice cannot enter the nucleus, whereas the mutant Whn protein in nu(StL)/nu(StL) mice is present in the nucleus. The antimorphic characteristic of the activation-deficient Whn(StL) protein with respect to mHa5 expression is therefore most likely caused by its non-productive interaction with other proteins at cis-regulatory regions of the mHa5 gene. Our results indicate that the molecular consequences of mutations of the Whn gene can be different and demonstrate an unexpected complexity of transcriptional control mechanisms of hair keratin genes.

Alleles↗
PubMed2000

Hb(64-76) epitope binds in different registers and lengths to I-Ek and I-Ak.

The nature of peptide binding to MHC molecules is intrinsically degenerate, in what, one given MHC molecule can accommodate numerous peptides which are structurally diverse, and one given peptide can bind to different alleles. The structure of the MHC class II molecules allows peptides to extend out of the binding groove at both ends and these residues can potentially influence the stability and persistence of peptide/class II complexes. We have previously shown that both I-E(k) and I-A(k)-restricted T cell hybridomas could be generated against the Hb(64-76) epitope. In this study, we characterized the binding register of the Hb(64-76) epitope to I-A(k), and showed that it was shifted by one residue in comparison to its binding to I-E(k), and did not use a dominant anchor residue at P1. This conclusion was further supported by the modeling of the Hb(64-76) epitope bound to I-A(k), which revealed that all of its putative anchor residues fit into their corresponding pockets. We identified the naturally processed Hb epitopes presented by both I-E(k) and I-A(k), and found that they consisted of different species. Those associated with I-A(k) being 20-22 residues long, whereas, those found to I-E(k) contained 14-16 residues. These findings suggested that the lack of a dominant P1 anchor could be compensated by the selection of longer peptides. Overall, these studies revealed the Hb(64-76) epitope bound to I-E(k) and I-A(k) in distinct registers and lengths, demonstrating the plasticity MHC molecules have in generating distinct TCR ligands from the same amino acid sequence.

Antigen Presentation↗
PubMed2000

Eradication of established tumors by CD8+ T cell adoptive immunotherapy.

We generated the DUC18 T cell receptor transgenic mouse expressing an H-2Kd -restricted transgenic T cell receptor specific for the syngeneic CMS5 fibrosarcoma rejection antigen mutated ERK2(136-144). DUC18 mice were capable of specifically eliminating lethal CMS5 tumor challenges, and transfer of DUC18 splenocytes to naive nontransgenic recipients conferred protection from subsequent and established CMS5 tumor burdens. Eradication of established tumor burdens by adoptive transfer of DUC18 splenocytes was dose and time dependent. Transferred tumor-specific T cells remained functional in vivo and capable of rejecting small tumors even in the presence of large, established tumor burdens. These findings highlight the kinetic battle between tumor growth and the production of a tumor-specific response and have critical implications for effective adoptive immunotherapy.

Animals↗
PubMed1999

Partially phosphorylated T cell receptor zeta molecules can inhibit T cell activation.

The T cell receptor complex (TCR) zeta chain is constitutively tyrosine phosphorylated specifically at two of the six zeta immunoreceptor tyrosine-based activation motif (ITAM) tyrosine residues in resting peripheral T cells. Further phosphorylation of zeta is induced by both agonist and antagonist ligands of the TCR, with agonists inducing complete phosphorylation of the zeta ITAM tyrosines. After antagonist stimulation, zeta phosphorylation is incomplete and generates discrete forms of partially phosphorylated ITAMs. Here, we mutate specific tyrosines in chimeric human CD8-zeta molecules to reflect phosphorylation in resting T cells as well as phosphorylation induced by agonist and antagonist ligands. We demonstrate that such partially phosphorylated TCR-zeta species can inhibit IL-2 production in T cell hybridomas and proliferation in T cell clones. This reveals a previously unrecognized, inhibitory function of partially phosphorylated ITAMs. These findings support the concept that TCR antagonism can arise through the generation of an inhibitory signal within the TCR complex and that constitutive zeta phosphorylation in resting T cells is an inhibitory signaling environment.

Animals↗
PubMed1999

Differential requirements for CD4 in TCR-ligand interactions.

The coreceptor molecule, CD4, plays an integral part in T cell activation; it is involved in both extracellular Ag recognition and intracellular signaling. We wanted to examine the functional role of CD4 in the recognition of agonist and altered peptide ligands (APLs). We generated two CD4-deficient T cell lines expressing well-characterized TCRs specific for Hb(64-76)/I-Ek. Although the responsiveness of the T cell lines to the agonist peptide was differently affected by the loss of CD4 expression, the recognition of APLs was in both cases dramatically reduced. Nearly full responsiveness to the agonist peptide was achieved by expression of a CD4 variant that did not associate with p56lck; however, the stimulation by APLs was only partially restored. Importantly, the expression of a CD4 variant in which domains interacting with MHC class II molecules have been mutated failed to restore the reactivity to all ligands. CD4-deficient T cells were able to be antagonized by APLs, indicating that CD4 was not required for antagonism. Overall, these findings support the concepts that CD4 is an integral part of the initial formation of the immunological synapse, and that the requirement for different CD4 functions in T cell activation varies depending upon the potency of the ligand.

Animals↗
PubMed1999

Proline residues in CD28 and the Src homology (SH)3 domain of Lck are required for T cell costimulation.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.

Alanine↗
PubMed1999

The immunological synapse: a molecular machine controlling T cell activation.

The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor-ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of the synapse was a determinative event for T cell proliferation.

Animals↗
PubMed1999

A kinetic threshold between negative and positive selection based on the longevity of the T cell receptor-ligand complex.

We have developed a unique in vivo system to determine the relationship between endogenous altered peptide ligands and the development of major histocompatibility complex class II- restricted T cells. Our studies use the 3.L2 T cell receptor (TCR) transgenic mouse, in which T cells are specific for Hb(64-76)/I-Ek and positively selected on I-Ek plus self-peptides. To this endogenous peptide repertoire, we have individually added one of six well-characterized 3.L2 ligands. This transgenic approach expands rather than constrains the repertoire of self-peptides. We find that a broad range of ligands produce negative selection of thymocytes in vivo. When compared with the in vitro TCR-ligand binding kinetics, we find that these negatively selecting ligands all have a half-life of 2 s or greater. Additionally, one of two ligands examined with no detectable binding to the 3.L2 TCR and no activity on mature 3.L2 T cells (Q72) enhances the positive selection of transgenic thymocytes in vivo. Together, these data establish a kinetic threshold between negative and positive selection based on the longevity of TCR-ligand complexes.

Amino Acid Sequence↗