Biomedical subjects

E J Collins

Publications and source records attributed to E J Collins.

At least 19 recordsLinked to original sources

PubMed2001

T cell activity correlates with oligomeric peptide-major histocompatibility complex binding on T cell surface.

Recognition of virally infected cells by CD8+ T cells requires differentiation between self and nonself peptide-class I major histocompatibility complexes (pMHC). Recognition of foreign pMHC by host T cells is a major factor in the rejection of transplanted organs from the same species (allotransplant) or different species (xenotransplant). AHIII12.2 is a murine T cell clone that recognizes the xenogeneic (human) class I MHC HLA-A2.1 molecule (A2) and the syngeneic murine class I MHC H-2 D(b) molecule (D(b)). Recognition of both A2 and D(b) are peptide-dependent, and the sequences of the peptides recognized have been determined. Alterations in the antigenic peptides bound to A2 cause large changes in AHIII12.2 T cell responsiveness. Crystal structures of three representative peptides (agonist, null, and antagonist) bound to A2 partially explain the changes in AHIII12.2 responsiveness. Using class I pMHC octamers, a strong correlation is seen between T cell activity and the affinity of pMHC complexes for the T cell receptor. However, contrary to previous studies, we see similar half-lives for the pMHC multimers bound to the AHIII12.2 cell surface.

Amino Acid Sequence↗

PubMed2001

Antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers.

Soluble MHC/peptide tetramers that can directly bind the TCR allow the direct visualization and quantitation of Ag-specific T cells in vitro and in vivo. We used HY-D(b) tetramers to assess the numbers of HY-reactive CD8+ T cells in HYTCR-transgenic mice and in naive, wild-type C57BL/6 (B6) mice. As expected, tetramer staining showed the majority of T cells were male-specific CD8+ T cells in female HY-TCR mice. Staining of B6 mice showed a small population of male-specific CD8+ T cells in female mice. The effect of administration of soluble MHC class I tetramers on CD8+ T cell activation in vivo was unknown. Injection of HY-D(b) tetramer in vivo effectively primed female mice for a more rapid proliferative response to both HY peptide and male splenocytes. Furthermore, wild-type B6 female mice injected with a single dose of HY-D(b) tetramer rejected B6 male skin grafts more rapidly than female littermates treated with irrelevant tetramer. In contrast, multiple doses of HY-D(b) tetramer did not further decrease graft survival. Rather, female B6 mice injected with multiple doses of HY-D(b) tetramer rejected male skin grafts more slowly than mice primed with a single injection of tetramer or irradiated male spleen cells, suggesting clonal exhaustion or anergy. Our data highlight the ability of soluble MHC tetramers to identify scarce alloreactive T cell populations and the use of such tetramers to directly modulate an Ag-specific T cell response in vivo.

Animals↗

PubMed2001

Class I major histocompatibility complex anchor substitutions alter the conformation of T cell receptor contacts.

An immunogenic peptide (GP2) derived from HER-2/neu binds to HLA-A2.1 very poorly. Some altered-peptide ligands (APL) of GP2 have increased binding affinity and generate improved cytotoxic T lymphocyte recognition of GP2-presenting tumor cells, but most do not. Increases in binding affinity of single-substitution APL are not additive in double-substitution APL. A common first assumption about peptide binding to class I major histocompatibility complex is that each residue binds independently. In addition, immunologists interested in immunotherapy frequently assume that anchor substitutions do not affect T cell receptor contact residues. However, the crystal structures of two GP2 APL show that the central residues change position depending on the identity of the anchor residue(s). Thus, it is clear that subtle changes in the identity of anchor residues may have significant effects on the positions of the T cell receptor contact residues.

Amino Acid Sequence↗

PubMed2001

Gorillas with spondyloarthropathies express an MHC class I molecule with only limited sequence similarity to HLA-B27 that binds peptides with arginine at P2.

The human MHC class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloarthropathies (SpAs). HLA-B27-transgenic rodents develop SpAs, implicating HLA-B27 in the etiology of these disorders. Several nonhuman primates, including gorillas, develop signs of SpAs indistinguishable from clinical signs of humans with SpAs. To determine whether SpAs in gorillas have a similar HLA-B27-related etiology, we analyzed the MHC class I molecules expressed in four affected gorillas. Gogo-B01, isolated from three of the animals, has only limited similarity to HLA-B27 at the end of the alpha1 domain. It differs by several residues in the B pocket, including differences at positions 45 and 67. However, the molecular model of Gogo-B*0101 is consistent with a requirement for positively charged residues at the second amino acid of peptides bound by the MHC class I molecule. Indeed, the peptide binding motif and sequence of individual ligands eluted from Gogo-B*0101 demonstrate that, like HLA-B27, this gorilla MHC class I molecule binds peptides with arginine at the second amino acid position of peptides bound by the MHC class I molecule. Furthermore, live cell binding assays show that Gogo-B*0101 can bind HLA-B27 ligands. Therefore, although most gorillas that develop SpAs express an MHC class I molecule with striking differences to HLA-B27, this molecule binds peptides similar to those bound by HLA-B27.

Amino Acid Motifs↗

PubMed2001

CD8(+) T cells express a T-helper 1--like phenotype after burn injury.

BACKGROUND: Previous studies suggest that CD8(+) T cells are immunosuppressive after burn injury, but recent reports indicate that CD8(+) T cells have several functions similar to CD4(+) T cells, including the secretion of cytokines. This study uses HY male antigen in transgenic HY female mice to determine the antigen-specific response of activated CD8(+) T cells after burn injury. METHODS: HY TCR transgenic female mice underwent burn or sham injury. Seventy-two hours after the burn, splenocytes were stimulated with 20 micromol/L HY peptide for 16, 48, and 64 hours; cellular proliferation, intracellular interferon-gamma and interleukin-2, and apoptosis were measured. RESULTS: Burn injury significantly impaired proliferation to HY antigen (P < or =.05). Activated CD8(+) T cells from burned mice showed increased intracellular interferon-gamma and interleukin-2 16 hours after stimulation compared with sham (P < or =.05) and at no time was less than control mice. The percent of CD8(+) T cells decreased with the time of stimulation but was not due to apoptosis by Annexin V staining. CONCLUSIONS: Activated CD8(+) T cells express a T(h1)-like phenotype after burn injury. This provides evidence that CD8(+) T cells are not simply suppressive and that is consistent with data that CD4(+) T cells are primed for a T(h1) response after burn injury.

Animals↗

PubMed2001

CYP1A2 activity as measured by a caffeine test predicts clozapine and active metabolite steady-state concentrationin patients with schizophrenia.

Clozapine is an atypical antipsychotic drug and displays efficacy in 30% to 60% of patients with schizophrenia who do not respond to traditional antipsychotics. A clozapine concentration greater than 1,150 nmol/L increases the probability of antipsychotic efficacy. However, plasma clozapine concentration can vary more than 45-fold during long-term treatment. The aim of this study was to assess the contribution of CYP1A2 to variability in steady-state concentration of clozapine and its active metabolite norclozapine. Patients with schizophrenia or schizoaffective disorder were prospectively monitored during clozapine treatment (N = 18). The in vivo CYP1A2 activity was measured using the caffeine metabolic ratio (CMR) in overnight urine. Trough plasma samples were drawn after at least 5 days of treatment with a constant regimen of clozapine. A significant negative association was found between the CMR and the dose-corrected clozapine (r(s) = -0.87,p < 0.01) and norclozapine (r(s) = -0.76,p < 0.01) concentrations. Nonsmokers displayed a higher clozapine (3.2-fold) and norclozapine (2.3-fold) concentration than smokers (p < 0.05). Furthermore, there was marked person-to-person variation in CYP1A2 activity during multiple-dose clozapine treatment (coefficient of variation = 60%). Age, weight, serum creatinine, and grapefruit juice consumption did not significantly contribute to variability in clozapine and norclozapine concentration (p > 0.05). In conclusion, CYP1A2 is one of the important contributors to disposition of clozapine during multiple-dose treatment. Although further in vitro experiments are necessary, the precise metabolic pathways catalyzed by CYP1A2 seem to be subsequent to the formation of norclozapine, hitherto less recognized quantitatively important alternate disposition routes, or both. From a clinical perspective, an environmentally induced or constitutively high CYP1A2 expression can lead to a decrease in steady-state concentration of clozapine as well as its active metabolite norclozapine. Thus, interindividual variability in CYP1A2 activity may potentially explain treatment resistance to clozapine in some patients. CYP1A2 phenotyping with a simple caffeine test may contribute to individualization of clozapine dosage and differentiate between treat ment noncompliance and high CYP1A2 activity.

Adult↗

PubMed2001

Treatment-resistance to clozapine in association with ultrarapid CYP1A2 activity and the C-->A polymorphism in intron 1 of the CYP1A2 gene: effect of grapefruit juice and low-dose fluvoxamine.

Antipsychotic response to clozapine varies markedly among patients with schizophrenia. The disposition of clozapine is dependent, in part, on the cytochrome P-450 (CYP) 1A2 enzyme in vivo. In theory, a very high CYP1A2 activity may lead to subtherapeutic concentrations and treatment resistance to clozapine. This prospective case study evaluates the clinical significance of ultrarapid CYP1A2 activity and a recently discovered single nucleotide (C --> A) polymorphism in intron 1 of the CYP1A2 gene (CYP1A2*F) for treatment resistance to clozapine. In addition, we describe the effect of grapefruit juice or low-dose fluvoxamine (25-50 mg/d) coadministration on clozapine and active metabolite norclozapine steady-state plasma concentration and antipsychotic response.

Adult↗

PubMed2001

Enhancing cytotoxic T cell responses with altered-peptide ligands.

Interest in class I MHC-mediated immunotherapy is growing rapidly. In order to fight a virus or cancer effectively, a successful immunotherapeutic must activate a large number of specific CD8+ T cells and also generate immunological memory. Attempts to generate immune responses towards tumor- or virus-derived peptides have frequently been frustrated by the nature of the peptide antigen itself. Either the peptide does not bind well to its cognate MHC, or the T cells directed towards it have been functionally inactivated in vivo. Altered-peptide ligands (APL) are an effective way to circumvent these problems. However, generating enhanced binding of altered peptides to class I MHC while still maintaining recognition of the wild-type peptide is not straightforward. Many groups design enhanced binding peptides by substituting the observed anchor residues with those that are most preferred by the class I MHC molecule. For many antigenic peptides, this approach does not work. Furthermore, if a higher affinity peptide is designed, the substitutions may result in reduced recognition by CD8+ T cells. Therefore, the design of APL requires careful testing of each candidate therapeutic in terms of affinity for class I MHC and immunological reactivity. Lastly, immunotherapy using class I MHC must also take into account the large genetic heterogeneity in the population. A therapeutic that is only effective for 5-10% of the population is not as attractive as one that works for over 90% of the population. The use of MHC supertypes (groups of class I MHC allotypes that share similar peptide-binding characteristics) shows great promise in overcoming this problem.

Amino Acid Sequence↗

PubMed2000

Tumor-specific CD4+ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity.

A number of tumor studies have indicated a link between CD4 help and the magnitude and persistence of CTL activity; however, the mechanisms underlying this have been largely unclear. To evaluate and determine the mechanisms by which CD4(+) T cells synergize with CD8(+) T cells to prevent tumor growth, we used the novel technique of monitoring in vivo CTL by labeling target cells with CFSE. This approach was supported by the direct visualization of CTL using peptide-MHC tetramers to follow tumor-specific T cells. The data presented demonstrate that while cotransfer of Ag-specific CD4(+) T cells was not required for the generation of CTLs, because adoptive transfer of CD8(+) T cells alone was sufficient, CD4(+) T cells were required for the maintenance of CD8(+) T cell numbers. Our data suggest that there is a correlation among the number of CD8(+) T cells, in vivo CTL function, and IFN-gamma production, with no evidence of a partial or nonresponsive phenotype among tetramer-positive cells. We also show that CD4(+) T cells are required for CD8(+) T cell infiltration of the tumor.

Adoptive Transfer↗

PubMed2000

Class I MHC is stabilized against thermal denaturation by physiological concentrations of NaCl.

Class I MHC molecules are ternary complexes composed of an allotype specific heavy chain, a noncovalently associated protein beta(2)-microglobulin (beta(2)m), and a peptide. The complexes are assembled in the endoplasmic reticulum by a complex series of chaperones and peptide-loading mechanisms. In the absence of beta(2)m or peptide, very little class I heavy chain is transported to the surface of the cell. Complexes that do not contain all three parts of the protein are not made productively in vivo and not at all in vitro. The ability of the complex to withstand thermal denaturation in vitro has been shown to be related to the binding affinity of the peptide. Paradoxically, some low-affinity peptide complexes denature at or below human basal body temperatures in vitro but are effective biological agents in vivo. Here we show that these complexes are stabilized against thermal denaturation by physiological cosolvents and maximally stabilized by 150 mM NaCl. While the degree of stabilization by 150 mM NaCl is greatest for low-affinity peptide/MHC complexes, the mechanism of stabilization is independent of peptide sequence. This effect is hypothesized to occur by multiple mechanisms including increasing the affinity of beta(2)m for the complex and charge screening.

Amino Acid Sequence↗

PubMed2000

Correlated switch binding and signaling in bacterial chemotaxis.

In Escherichia coli, swimming behavior is mediated by the phosphorylation state of the response regulator CheY. In its active, phosphorylated form, CheY exhibits enhanced binding to a switch component, FliM, at the flagellar motor, which induces a change from counterclockwise to clockwise flagellar rotation. When Ile(95) of CheY is replaced by a valine, increased clockwise rotation correlates with enhanced binding to FliM. A possible explanation for the hyperactivity of this mutant is that residue 95 affects the conformation of nearby residues that potentially interact with FliM. In order to assess this possibility directly, the crystal structure of CheY95IV was determined. We found that CheY95IV is structurally almost indistinguishable from wild-type CheY. Several other mutants with substitutions at position 95 were characterized to establish the structural requirements for switch binding and clockwise signaling at this position and to investigate a general relationship between the two properties. The various rotational phenotypes of these mutants can be explained solely by the amount of phosphorylated CheY bound to the switch, which was inferred from the phosphorylation properties of the mutant CheY proteins and their binding affinities to FliM. Combined genetic, biochemical, and crystallographic results suggest that residue 95 itself is critical in mediating the surface complementarity between CheY and FliM.

Bacterial Proteins↗

PubMed2000

T cell activity after dendritic cell vaccination is dependent on both the type of antigen and the mode of delivery.

Previous work in both human and animal models has shown that CTL responses can be generated against proteins derived from tumors using either peptide-pulsed dendritic cells (DCs) or nucleic acids from the tumor transfected into autologous DCs. Despite the efficacy of this approach for vaccine therapy, many questions remain regarding whether the route of administration, the frequency of administration, or the type of Ag is critical to generating T cell responses to these Ags. We have investigated methods to enhance CTL responses to a peptide derived from the human proto-oncogene HER-2/neu using mice containing a chimeric HLA A2 and H2Kb allele. Changes in amino acids in the anchor positions of the peptide enhanced the binding of the peptide to HLA-A2 in vitro, but did not enhance the immunogenicity of the peptide in vivo. In contrast, when autologous DCs presented peptides, significant CTL activity was induced with the altered, but not the wild-type, peptide. We found that the route of administration affected the anatomic site and the time to onset of CTL activity, but did not impact on the magnitude of the response. To our surprise, we observed that weekly administration of peptide-pulsed DCs led to diminishing CTL activity after 6 wk of treatment. This was not found in animals injected with DCs every 3 wk for six treatments or in animals initially given DCs weekly and then injected weekly with peptide-pulsed C1R-A2 transfectants.

Adoptive Transfer↗

PubMed2000

Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells.

Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8+ T cells can be activated via the signal generated through the TCR-alphabeta in the absence of any potential costimulatory molecules. Using T cells from two different TCR transgenic mice in vitro, we find that TCR engagement by peptide/MHC tetramers is sufficient for the activation of naive CD8+ T cells. Furthermore, these T cells proliferate, produce cytokines, and differentiate into cytolytic effectors. Under the conditions where anti-CD28 is able to enhance proliferation of normal B6 CD4+, CD8+, and TCR transgenic CD8+ T cells with anti-CD3, we see no effect of anti-CD28 on proliferation induced by tetramers. The results of this experiment argue that given a strong signal delivered through the TCR by an authentic ligand, no costimulation is required.

Animals↗

PubMed2000

Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes.

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.

Animals↗

PubMed1999

The structural basis for the increased immunogenicity of two HIV-reverse transcriptase peptide variant/class I major histocompatibility complexes.

Designing altered peptide ligands to generate specific immunological reactivity when bound to class I major histocompatibility complexes is important for both therapeutic and prophylactic reasons. We have previously shown that two altered peptides, derived from human immunodeficiency virus (HIV)-reverse transcriptase (RT) residues 309-317, are more immunogenic in vitro than the wild-type peptide. One peptide variant, I1Y, was able to stimulate RT-specific cytotoxic T cells from the blood of three HIV-infected individuals better than the wild-type RT peptide. Both I1Y and I1F peptide variants increase the cell surface half-life of the peptide-class I complex approximately 3-fold over that of the RT peptide but have different immunological activities. These peptides are candidates for the design of vaccines for HIV due to their increased immunogenicity. To understand the basis for the increased cell surface stability compared with wild-type peptide and to understand the differences in T cell recognition between I1Y and I1F, we determined the x-ray crystal structures of the two class I MHC-peptide complexes. These structures indicate that the increased cell surface half-life is due to pi-pi stacking interactions between Trp-167 of HLA-A2.1 and the aromatic P1 residues of I1F and I1Y. Comparison of the structures and modeling potential T cell receptor (TCR) interactions suggests that T cell interactions and immunogenicity are different between I1Y and I1F for two reasons. First, subtle changes in the steric and polar properties of the I1Y peptide affect TCR engagement. Second, water-mediated hydrogen bond interactions between the P1-Tyr and the P4-Glu peptide residues increase peptide side chain rigidity of residues critical for TCR engagement.

Circular Dichroism↗

PubMed1999

Poor binding of a HER-2/neu epitope (GP2) to HLA-A2.1 is due to a lack of interactions with the center of the peptide.

Class I major histocompatibility complex (MHC) molecules bind short peptides derived from proteins synthesized within the cell. These complexes of peptide and class I MHC (pMHC) are transported from the endoplasmic reticulum to the cell surface. If a clonotypic T cell receptor expressed on a circulating T cell binds to the pMHC complex, the cell presenting the pMHC is killed. In this manner, some tumor cells expressing aberrant proteins are recognized and removed by the immune system. However, not all tumors are recognized efficiently. One reason hypothesized for poor T cell recognition of tumor-associated peptides is poor binding of those peptides to class I MHC molecules. Many peptides, derived from the proto-oncogene HER-2/neu have been shown to be recognized by cytotoxic T cells derived from HLA-A2(+) patients with breast cancer and other adenocarcinomas. Seven of these peptides were found to bind with intermediate to poor affinity. In particular, GP2 (HER-2/neu residues 654-662) binds very poorly even though it is predicted to bind well based upon the presence of the correct HLA-A2.1 peptide-binding motif. Altering the anchor residues to those most favored by HLA-A2.1 did not significantly improve binding affinity. The crystallographic structure shows that unlike other class I-peptide structures, the center of the peptide does not assume one specific conformation and does not make stabilizing contacts with the peptide-binding cleft.

Amino Acid Sequence↗

PubMed1999

Structural evidence of T cell xeno-reactivity in the absence of molecular mimicry.

The T cell receptor (TCR), from a xeno-reactive murine cytotoxic T lymphocyte clone AHIII12.2, recognizes murine H-2Db complexed with peptide p1027 (FAPGVFPYM), as well as human HLA-A2.1 complexed with peptide p1049 (ALWGFFPVL). A commonly proposed model (the molecular mimicry model) used to explain TCR cross-reactivity suggests that the molecular surfaces of the recognized complexes are similar in shape, charge, or both, in spite of the primary sequence differences. To examine the mechanism of xeno-reactivity of AHIII12.2, we have determined the crystal structures of A2/p1049 and Db/p1027 to 2.5 A and 2.8 A resolution, respectively. The crystal structures show that the TCR footprint regions of the two class I complexes are significantly different in shape and charge. We propose that rather than simple molecular mimicry, unpredictable arrays of common and differential contacts on the two class I complexes are used for their recognition by the same TCR.

Animals↗

PubMed1999

Extensive alanine substitutions increase binding affinity of an influenza nucleoprotein peptide to HLA-Aw68 and do not abrogate peptide-specific CTL recognition.

Class I MHC molecules bind peptides in the endoplasmic reticulum and present them at the cell surface to circulating CD8+ T cells for analysis. We have examined binding of peptides and stabilization of HLA-Aw68 class I molecules using synthetic peptide variants of an influenza virus nucleoprotein peptide, NP91-99 (KTGGPIYKR). We have demonstrated that insertion of increasing numbers of alanines in the center of the peptide (between P and I), to increase a natural bulging out of the peptide-binding cleft, results in a large decrease in thermal stability. Although there is a great decrease in the t 1/2 of the MHC/peptide complex for NP-1A compared with NP91-99, a T cell line, stimulated by NP91-99, recognizes NP-1A efficiently. Peptide variants with three or more alanines do not show saturable binding to HLA-Aw68 and also are not recognized by the T cell line. Thermal studies show that polyalanine peptides with minimal anchors and nearly all TCR contact residues exchanged stabilized HLA-Aw68 to high temperatures. Additionally, some of these polyalanine peptides are recognized by T cell lines generated against NP91-99. Analysis of the peptide sequences show that the stabilization effects are not due to the hydrophobicity of the peptide. These data suggest that the strength of binding of peptides to HLA-Aw68 is not only dictated by the presence of anchor residues but also by the lack of unfavorable contacts between the peptide ligand and class I MHC-binding cleft.

Alanine↗