PubMed Health⌕ Search

Biomedical subjects

R T Kubo

Publications and source records attributed to R T Kubo.

At least 37 records · Page 2Linked to original sources
PubMed1994

Developmental regulation of the TCR zeta-chain. Differential expression and tyrosine phosphorylation of the TCR zeta-chain in resting immature and mature T lymphocytes.

The zeta subunit of the TCR complex targets receptor surface expression, is phosphorylated on tyrosine residues upon T cell activation, and is implicated in signal transduction after TCR ligation. Here we show that, although intrathymic expression of the murine TCR-associated zeta-chain relative to the other chains of the Ag receptor complex remains unchanged during early thymocyte development, there is a doubling of TCR-associated zeta-chain surface expression upon thymocyte maturation. The ratio of tyrosine-phosphorylated relative to nonphosphorylated TCR-associated zeta-chain also changes with thymocyte development. This ratio was quantified after the purification and detergent extraction of receptor complexes from freshly isolated immature or mature thymocytes. Immunoprecipitation of the zeta-chain released from the complex allowed for the isolation of the tyrosine-phosphorylated and nonphosphorylated forms of TCR-associated zeta-chain. Intracellular free zeta-chain was characterized by immunoprecipitation after clearing the cell lysate of intact TCR complexes. Densitometric analysis of immunoblots indicated that surface phosphorylated zeta-chain is more abundant in immature relative to mature T cell populations, whereas the inverse is true of intracellular phosphorylated zeta-chain. Surface phosphorylated zeta-chain also migrated at a higher m.w. than its cytoplasmic counterpart, suggesting that it is more highly modified on some or all of its available tyrosines. These findings demonstrate that the stoichiometry and post-translational modification of the TCR complex are regulated, in vivo, and may determine the functional maturation of T cell signaling, selection, and activation.

Animals↗
PubMed1994

Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins.

We have measured the binding affinity for five HLA-A alleles: HLA-A1 (A*0101), A2.1 (A*0201), A3 (A*0301), A11 (A*1101), and A24 (A*2401); of a set of all possible nonamer peptides (n = 240) of human papillomavirus type 16 E6 and E7 proteins. High affinity binding peptides were identified for each of the alleles, thus allowing us to select several candidates for CTL-based vaccines. Moreover, this unbiased set of peptides allowed an evaluation of the predictive value of HLA motifs derived either from the analysis of sequencing of pools of naturally processed peptides or from the binding analysis of polyalanine nonameric peptides that differed in the amino acids (aa) present at the anchor positions. Whereas pool sequencing-derived motifs were present in only 27% of high affinity binders, the more expanded motif, based on analysis of different aa substitutions at the anchor positions, was present in 73% of high affinity binders. Furthermore, it was found that the presence of anchor residues in a peptide was in itself not sufficient to determine binding to MHC class I molecules, because the majority of motif-containing peptides failed to bind to the relevant MHC. Finally, specific HLA motifs were used to predict peptide binders of 8, 10, and 11 aa in length. Several high affinity binding peptides were identified for each of the various peptide lengths, indicating a significant size heterogeneity in peptides capable of high affinity binding to HLA-A molecules.

Alleles↗
PubMed1994

Definition of specific peptide motifs for four major HLA-A alleles.

Allele-specific motifs for the human MHC class I molecules, HLA-A1, A3, A11, and A24 were characterized by three complementary approaches. First, amino acid sequence analysis of acid eluted peptide pools from affinity purified class I molecules defined putative motifs 9 or 10 amino acids in length and bearing critical anchor residues at position 2 and at the COOH-terminal. These motifs were distinct, with the exception of the HLA-A3 and A11 motifs that were very similar to each other. Second, the correctness of these putative motifs was verified by analyzing the binding capacity of polyalanine peptide analogues to purified HLA-A molecules. Several alternative anchor residues that were not obvious from the pooled peptide sequencing analysis were identified. Third, sequences of individual peptides eluted from HLA-A1, A11, and A24 were determined by tandem mass spectrometry. Nonamers were the predominant species, although peptides of 8, 10, 11, and 12 amino acids in length were also identified. These peptides displayed anchor residues predicted by the specific motifs at position 2 and at the COOH-terminal, regardless of peptide length. Synthetic versions of the naturally processed peptides were shown to bind to the appropriate HLA-A alleles with IC50 values in the 0.3- to 200-nM range. A rational approach to search Ags with known amino acid sequences for epitopes restricted by some of the most common HLA-A types and of potential clinical importance is now feasible.

Alleles↗
PubMed1994

Peptide binding to the most frequent HLA-A class I alleles measured by quantitative molecular binding assays.

Quantitative assays to measure the binding of defined synthetic antigenic peptides and purified MHC class I molecules are described for several common human HLA-A alleles (A1, A2.1, A3, A11 and A24). Under appropriate conditions, the binding of radiolabeled peptides to purified MHC class I molecules is very effective, highly specific, and appears to be dependent on the specific sequence motif of the peptide as defined by critical anchor residue positions. Establishment and optimization of the assay reveals that a relatively high fraction of the MHC class I molecules isolated from EBV transformed B cell line sources is capable of binding exogenously added peptide. Scatchard analysis for all alleles yields 5-10% occupancy values. There is a stringent peptide size requirement that is reflected by the direct influence of peptide length on the binding affinity. The peptide-MHC class I interactions demonstrate remarkable similarity to peptide-MHC class II interactions, both in overall affinity and kinetic behavior. The immunological relevance of the peptide-MHC class I binding assay is also demonstrated by measuring the affinity of a panel of previously described HLA restricted peptides for their HLA restriction element. In 91% (10/11) of the cases, the peptides bound with affinities of 50 nM or less, and in the remaining 9% (1/11) of the cases, in the 50 to 500 nM range. Thus, these data provide the first quantitative estimate of what level of HLA-A binding affinity is associated with a diverse panel of immunodominant CTL epitopes in man.

Amino Acid Sequence↗
PubMed1994

Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides.

Pan DR-binding peptides engineered by introducing anchor residues for different DR motifs within a polyalanine backbone bound 10 of 10 DR molecules tested, with affinities, in most cases, in the nanomolar range. Because of the small methyl group exposed for T cell recognition, these peptides were poor immunogens but effective blockers of DR-restricted antigen presentation. Introduction of bulky and charged residues at positions accessible for T cell recognition yielded extremely powerful Pan DR epitope peptides (PADRE). These peptides elicited powerful responses in vitro from human peripheral blood mononuclear cells (PBMC). Because these cells also cross-react on certain mouse class II alleles, we could also demonstrate that PADRE peptides are active in vivo. In one example of their capacity to elicit T help, they were approximately 1000 times more powerful than natural T cell epitopes. We propose that PADRE peptides may be useful in the development of subunit vaccines.

Alleles↗
PubMed1994

Class I MHC-peptide interaction: structural and functional aspects.

The structural requirements for the interaction between antigens and class I molecules was investigated through the use of a quantitative assay to measure peptide binding to different MHC class I alleles. We determined the permissiveness of the main anchors reported by Rammensee and his group for peptide binding and defined an extended motif for peptides binding to the HLA-A2.1 allele, including the role of non-anchor positions. It was found that the main anchors were necessary, but not sufficient, for good binding. Certain non-anchor positions contributed significantly to overall binding and were referred to a secondary anchors. This finding allowed a better prediction of high affinity binding peptides selected from libraries of different viral and tumor proteins. Furthermore, our data allowed correlation of the structural requirements for binding of peptides with crystallographic data of the MHC molecule. In order to characterize allele-specific motifs for a larger number of alleles, the HLA-A alleles A1, A3, A11, and A24, which represent some of the most common alleles found in different ethnic populations, were chosen. Here, most motifs were found to be highly exclusive; however, HLA-A3 and A11 shared a common motif. The defined motifs were validated further by using naturally processed peptides. Those peptides were also synthesized and tested for binding to the appropriate HLA alleles, giving a binding affinity from 0.3 to 200 nM for sequences of naturally processed peptides. Finally, a set of all possible 9-mer peptides from HPV 16 proteins were synthesized and tested for binding to the five class I alleles. For each allele, high affinity binders were identified, thus allowing for selection of possible peptide candidates for a CTL based vaccine.

Alleles↗
PubMed1994

Inhibition of allograft rejection by anti-T-cell receptor-alpha beta monoclonal antibodies preserving resistance to bacterial infection.

Anti-CD3 monoclonal antibody (mAb) has been administered in clinical organ transplantation to reverse acute allograft rejection; however, severe immunodeficiency can result from such mAb treatment and cause an increased incidence of opportunistic infections. Therefore, new model systems are required in order to establish better methods for suppressing allograft rejection while preserving resistance to opportunistic infections. In this study, we compared the effects of the in vivo administration of anti-T-cell receptor-alpha beta (TcR alpha beta) mAb, H57-597, with those of anti-CD3 mAb, 145-2C11. Much to our surprise, the in vivo administration of anti-TcR alpha beta mAb prior to skin grafting led to a longer allograft survival than that of anti-CD3 mAb at any of the comparable dosages examined. In the lymphoid organs of mice treated with anti-TcR alpha beta mAb, TcR alpha beta-bearing cells were almost completely depleted, while TcR gamma delta-bearing cells remained at a relatively increased level on day 14 after anti-TcR alpha beta mAb treatment. The in vitro stimulation by anti-TcR gamma delta mAb clearly showed that such TcR gamma delta-bearing cells were functionally intact. Furthermore, the mice treated with anti-TcR alpha beta mAb, but not anti-CD3 mAb, were observed to be resistant to infection with Listeria monocytogenes. Finally, treatment with H57-597, but not with 145-2C11, led to a marked prolongation of skin allograft survival in the thymectomized mice. These results strongly suggest that anti-TcR alpha beta mAb, which partially preserved anti-bacterial resistance, may be more effective in preventing graft rejection than anti-CD3 mAb in the periphery, and indicate that anti-TcR alpha beta mAb may thus be potentially applicable for human transplantation. In addition, these results also indicate that the TcR gamma delta-bearing cells alone, at least in the absence of TcR alpha beta-bearing cells, do not contribute to allograft rejection in vivo.

Animals↗
PubMed1993

Expression and function of mixed isotype MHC class II molecules in normal mice.

Mixed isotype MHC class II molecules (E alpha dA beta d) occur at extremely low levels on the surface of normal mouse B cells and macrophages, as determined by surface staining with an E alpha dA beta d-specific hamster mAb, H71-258.41. The surface levels of mixed isotype on the B cell lymphoma line A20 are approximately 1 to 2% that of surface I-A, whereas the levels of these molecules on normal mouse B cells were estimated to be at least two to four times less than those on A20. Nevertheless, other investigators have recently reported that immunization of normal H-2d mice with the sperm whale myoglobin peptide 110-121 (SWM(110-121)) elicits T cells, predominantly, V beta 8.2+, that recognize the peptide only in context of E alpha A beta. We have characterized a large number of SWM(110-121)-specific T cell hybridomas from several strains of H-2d haplotype mice. All of the V beta 8.2+ 110-121-specific hybridomas were found to be restricted by E alpha dA beta d, whereas, of the V beta 8.2- 110-121-specific group, approximately half recognized the peptide through E alpha dA beta d whereas the remainder were restricted by either I-Ad or I-Ed. mAb inhibition experiments revealed that 14-4-4S (E alpha-specific) could block presentation by mixed isotype completely, while MK-D6 (A beta d-specific) and H71-258.41 (E alpha dA beta d-specific) only inhibited presentation when the concentration of peptide was limiting. Although A20 expresses very low levels of mixed isotype, 10 to 100 nmol of the peptide produced a detectable response, illustrating the remarkable efficiency in presenting this peptide through E alpha dA beta d. The ability of normal mouse APC to use this restriction element despite its extremely low expression has important implications for the activation of T cells by low levels of peptide-MHC complexes.

Animals↗
PubMed1993

Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules.

The functional determinants of histocompatibility leukocyte antigen (HLA)-A2.1-peptide interactions have been detailed by the use of quantitative molecular binding assays and a chemically synthesized library of naturally occurring epitopes. The importance of hydrophobic anchor residues in position 2 and the C-terminus was confirmed. These anchors are necessary, but not sufficient, for high affinity binding, as the predictions based solely on these anchors are only about 30% accurate. Prominent roles for several other positions (1, 3, and 7) were also demonstrated. The location of these residues within the peptides matches secondary A2.1 pockets previously demonstrated by X-ray crystallography. From a functional standpoint, similar dominant negative effects on binding were observed for charged residues in both nonamers and decamers, while positive effects differed between nonamers and decamers. An extended motif taking into account secondary anchors increased the predictability of A2.1-binding epitopes to a level of 70%, underscoring the practical usefulness of extended motifs.

Acid Phosphatase↗
PubMed1992

Invariant chain peptides in most HLA-DR molecules of an antigen-processing mutant.

Class II major histocompatibility complexes bind peptides in an endosome-like compartment. When the class II null cell line 721.174 was transfected with class II DR3 genes, DR molecules were produced in normal amounts. However, the DR molecules were abnormally conformed and unstable because deletion of an antigen-processing gene had impaired intracellular formation of most class II-peptide complexes. Yet, 70 percent of the DR molecules still bore peptides, 80 percent of which were 21- to 24-amino acid fragments of the class II-associated invariant chain. These peptides were rare on DR3 from control cells. Thus, a defect in the main antigen-processing pathway revealed a process in which DR molecules bind long peptides derived from proteins present in the same compartment.

Amino Acid Sequence↗
PubMed1992

Translocation of spectrin and protein kinase C to a cytoplasmic aggregate upon lymphocyte activation.

We have previously reported that mammalian tissue lymphocytes exhibit significant heterogeneity with respect to the subcellular distribution of spectrin and that this phenomenon may result from a dynamic behavior of spectrin in response to activation signals. Here, we further characterize the involvement of spectrin in lymphocyte activation by examining its relationship with protein kinase C (PKC). PKC isoenzymes are a family of cytosolic kinases that translocate from the soluble to particulate fraction upon cell stimulation. It is reported here that activation of lymph node T cells through the antigen-specific receptor, or direct activation of PKC by phorbol esters, results in a striking increase in cells expressing a cytoplasmic aggregate of spectrin. Additionally, a concurrent increase in cells expressing aggregates of the beta II isozyme of PKC is observed. Immunofluorescence staining revealed that spectrin and PKC beta II are colocalized in untreated lymphocytes and that these two proteins are coincidently translocated to the same focal aggregate within the cytoplasm following stimulation. This redistribution of spectrin and PKC beta is blocked by pretreatment with calphostin C, a specific inhibitor of PKC. Solubility studies showed that there is an increase of both proteins in the detergent-insoluble fraction of lymphocytes upon activation, and immunoprecipitation studies indicated that the soluble form of these molecules may be associated directly or indirectly as part of a complex of proteins. These data indicate that the positioning of the spectrin-based cytoskeleton is sensitive to activation signals and may play a role in the function or positioning of PKC beta II.

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

Glycosylphosphatidyl inositol-linked membrane protein expression by intestinal intraepithelial lymphocytes.

Murine intestinal intraepithelial lymphocytes (ilEL) represent a heterogeneous population of cells which contains both TCR alpha beta+ and TCR gamma delta+ cells. In contrast to the TCR alpha beta+ cell population in peripheral lymphoid organs, the TCR alpha beta+ ilEL contain high numbers of Thy-1- cells. This negativity of ilEL for Thy-1, a glycosylphosphatidyl inositol (GPI)-linked membrane protein, is not the result of conformational changes of the Thy-1 molecule. The absence of Thy-1 on TCR gamma delta+ and a subset of TCR alpha beta+ ilEL is furthermore not caused by a general defect in the processing of GPI-linked proteins, since Thy-1- ilEL mainly express another GPI-linked protein, Qa-2. However, TCR alpha beta+ and TCR gamma delta+ ilEL are also virtually negative for two other members of this family of activation-associated GPI-linked proteins, Ly-6A and Ly-6C. This unusual pattern of GPI-linked protein expression on TCR+ ilEL may be related to differences in cellular activation and signal transduction pathways between TCR+ ilEL and regular peripheral T cells.

Animals↗
PubMed1992

Characterization of defective I-A surface expression in a mixed isotype expressing murine B cell lymphoma: continued expression of E alpha d A beta d despite competition from restored A alpha d A beta d pairs.

The BALB/c-derived mouse B cell lymphoma line, 2PK3, expresses mixed isotype E alpha dA beta d and classical I-E class II molecules on its surface, but normal surface I-A expression is not detectable. Northern blot analysis showed comparable amounts of A alpha mRNA in 2PK3 as compared to another Iad expressing B cell lymphoma, A20, which predominantly expresses I-A and I-E. Sequence analysis of 2PK3 A alpha cDNA revealed a single nucleotide difference in the signal sequence that would result in a proline for leucine substitution at position - 12. In vitro translation of 2PK3 A alpha mRNA gave results suggesting that the signal peptide mutation prevented translocation of the A alpha protein across the rough endoplasmic reticulum which would provide an explanation for the lack of I-A expression in 2PK3. I-A expression was restored by transfecting a functional A alpha d gene into 2PK3. Although I-A was expressed at high levels in some transfectants, in all cases significant levels of mixed isotype were still detected. Functional studies performed using antigen-specific I-A(d)-restricted and E alpha d-A beta d-specific T cell hybridomas confirmed the levels of expression of I-A(d) and E alpha dA beta d respectively on the transfectants and showed that these molecules were functional. An interesting observation from this study is the continued expression of significant levels of E alpha dA beta d in spite of competition from restored expression of I-A(d).

Amino Acid Sequence↗
PubMed1991

Surface expression of a T cell receptor beta (TCR-beta) chain in the absence of TCR-alpha, -delta, and -gamma proteins.

The antigen receptor expressed by mature T cells has been described as a disulfide-linked alpha/beta or gamma/delta heterodimer noncovalently associated with CD3, a complex of transmembrane proteins that communicates signals from the T cell receptor (TCR) to the cell interior. Studies suggest that all component chains must assemble intracellularly before surface expression can be achieved. We described, however, a CD4+/CD8+ transformed murine thymocyte, KKF, that expresses surface TCR-beta chains in the absence of gamma, delta, and alpha proteins; these beta chains are only weakly associated with CD3-epsilon and CD3-zeta. Furthermore, KKF responds differently to stimulation through TCR-beta and CD3-epsilon, a functional dissociation that has been ascribed to a CD4+/CD8+ subpopulation of normal thymocytes. KKF's unique TCR structure may offer an explanation for the functional anomalies observed.

Animals↗
PubMed1991

In vivo administration of monoclonal antibodies to the CD3 T cell receptor complex induces cell death (apoptosis) in immature thymocytes.

Some thymocytes, upon activation via the TCR complex in vitro, undergo apoptotic cell death. In this report, we examine the cell death induced in the thymus after administration of anti-CD3 or anti-TCR antibodies. We found that shortly after antibody injection, cortical thymocytes undergo apoptosis as characterized by morphologic changes and DNA fragmentation. Anti-CD3 administration led to depletion of nearly all CD4+CD8+ thymocytes, and approximately 50% of CD4+CD8- thymocytes. This depletion predominantly affected cells bearing low levels of CD3, although some depletion also occurred among cells expressing intermediate and high levels. Administration of an anti-TCR antibody also induced apoptosis, but affected significantly fewer thymocytes than anti-CD3. This effect was probably not due to different binding affinities for the two antibodies, because both antibodies show similar dose response effects in an in vitro model of activation-induced apoptosis. This work demonstrates that findings on activation-induced apoptosis in vitro can be extended to the in vivo situation, and further, that the activation of cortical thymocytes, in situ, results in apoptosis and removal of the activated cells. The possible relationships between this activation-induced cell death in immature thymocytes and the process of negative selection of autoreactive T cells is discussed.

Animals↗
PubMed1991

Analysis of the interaction site for the self superantigen Mls-1a on T cell receptor V beta.

Superantigen bound to major histocompatibility complex (MHC) products have been shown to stimulate T cells in a V beta-specific manner. Mouse T cells bearing V beta 8.1 usually respond to the self superantigen, Mls-1a, whereas T cells bearing V beta 8.2a do not. Previously, using site-directed mutational analysis, we identified the residues of natural variants of T cell receptor (TCR) V beta 8.2 that conferred Mls-1a reactivity. These residues are predicted to lie on a beta-pleated sheet of the TCR V beta element, well away from the expected binding site for antigen and MHC proteins. This study was undertaken to determine the effect of glycosylation on this beta-pleated sheet on Mls-1a reactivity and to map the extent of the interaction site on V beta 8.2 for Mls-1a. to Mls-1a, as well as to peptides derived from the conventional protein antigen, chicken ovalbumin. Here we demonstrate that first, N-linked carbohydrate on the lateral surface of V beta blocks the interaction of the TCR V beta with the self superantigen, Mls-1a, but has no effect on the TCR interaction with peptide antigen and MHC, second, that the interaction site for Mls-1a extends over the surface of the solvent-exposed beta-pleated sheet on the side of the TCR, and third, that mutations which affect both superantigen and peptide antigen reactivity lie at the beginning of the first complementarity determining region of V beta, consistent with models of the trimolecular complex of TCR-peptide-MHC.

Animals↗