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F R Carbone

Publications and source records attributed to F R Carbone.

At least 73 records · Page 4Linked to original sources
PubMed1993

Identification of conserved T cell receptor CDR3 residues contacting known exposed peptide side chains from a major histocompatibility complex class I-bound determinant.

We have analyzed the T cell receptor (TCR) repertoire found in the major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response to the protein ovalbumin (OVA). Despite skewing towards the expression of V beta 5.2+TCR by OVA-specific CTL from C57BL/6 mice, we found a relatively high degree of diversity in V(D)J usage in both TCR alpha- and beta-chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR alpha- and beta-chain VJ or VDJ junctions. These junctions form the third complementarity-determining regions (CDR3) of the TCR polypeptides involved in the direct interaction with the class I-bound peptide. Crystallographic analyses of Kb-peptide complexes predict that the major determinant from OVA, peptide OVA257-264 (SIINFEKL), contains two exposed charged side chains which can contact the TCR. These are the negatively charged glutamic acid at determinant position 6 (P6) and the positively charged lysine at P7. To examine whether the TCR alpha-chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR beta-chain derived from the V beta 5.2+ clone B3. OVA-specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR alpha-chains that also contained negatively charged junctional residues despite some further variation in V alpha and J alpha sequences. Stimulation of naive TCR beta-chain transgenic T cells with a P7 substitution peptide analogue induced a T cell response that was no longer cross-reactive with the wild-type OVA257-264 determinant, suggesting that the TCR alpha-chain from the T cell clone B3 can determine the specificity for this residue. Consequently, these results reveal the existence of conserved residues in the CDR3 of TCR alpha- and beta-chains specific for OVA257-264 and identify their possible orientation over the peptide-class I complex.

Amino Acid Sequence↗
PubMed1993

Salmonella typhimurium delta aroA delta aroD mutants expressing a foreign recombinant protein induce specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes in mice.

Recombinant Salmonella typhimurium aroA aroD mutants which expressed ovalbumin were constructed. The two expression constructs used were based on either pUC18 or pBR322. The pBR322-based construct was more stable in vitro and in vivo than the pUC-based construct. Salmonellae containing the stable pBR322-based plasmid induced major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL), in contrast to salmonellae containing the pUC18-based expression construct. The priming of MHC class I-restricted CTL was increased by multiple immunizations. The study described in this report suggest that S. typhimurium delta aro mutants have the capacity to induce MHC class I-restricted CTL against carried antigens and that MHC class I-restricted CTL responses require stable in vivo expression of the target antigen. Further, the results indicate that the Salmonella typhi delta aro mutants currently undergoing evaluation in studies with humans may be good carriers of viral antigens with CTL determinants.

Animals↗
PubMed1992

Both intrathymic and peripheral selection modulate the differential expression of V beta 5 among CD4+ and CD8+ T cells.

Murine T cells expressing V beta 5 are characterized by (a) intrathymic deletion in the presence of I-E and products of endogenous mouse mammary tumor viruses, and (b) a greater representation in CD8+ relative to CD4+ peripheral T cells, thought to be due to more efficient intrathymic positive selection on class I rather than class II major histocompatibility complex antigens. We have engineered mice that are transgenic for a rearranged gene encoding a V beta 5+ beta chain of the T cell receptor for antigen. Deletion is not predicted in I-E- V beta 5+ transgenic mice, and until the age of 2 wk, the CD4/CD8 ratio of peripheral T cells is > 3:1 and indistinguishable between transgenic and nontransgenic mice. Transgenic mice then show a rapid, age-dependent decline in the ratio of CD4+ to CD8+ T cells in the lymphoid periphery, reaching a low of 1:10 by 7 mo of age. Furthermore, the percent of peripheral CD4+ cells that express the transgene drops with age, reaching a low of about 60% at 7 mo, while the percent of CD8+ cells that express V beta 5 remains greater than 95% at all ages. The lymphoid periphery is implicated in this selection against CD4+ V beta 5+ T cells as it occurs more rapidly in thymectomized transgenic mice, and can be delayed in mice whose peripheral T cells are replaced by recent thymic emigrants after depletion by in vivo treatment with anti-Thy-1 antibodies. These results indicate that the relative expression of V beta 5 in T cell subsets can be influenced not only intrathymically in I-E+ V beta 5+ transgenic mice, but also by events in the periphery, in the absence of I-E expression.

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PubMed1992

Murine AIDS superantigen reactivity of the T cells bearing V beta 5 T cell antigen receptor.

A B cell line, B6-1710, that expresses the defective virus known to induce murine AIDS stimulates a large fraction of nonprimed splenic T cells. Analysis of the T cell population responding to the B6-1710 for TCR V beta-chain usage revealed that, in addition to the previously reported V beta 5-chain-positive T cells, T cells bearing V beta 11 and V beta 12 are also specifically enriched. We have established V beta 5+ T cell lines, clones, and hybridomas expressing identical TCR with different CD4/CD8 phenotypes and demonstrated that T cell reactivity to B6-1710 is, although not absolute, dependent on the presence of CD4 molecules. Further analysis of T cell hybridomas with known J beta-chain usage revealed that D beta- and J beta-chain usage do not play crucial roles in T cell reactivity to B6-1710 B cells. However, T cell hybridomas derived from TCR-V beta gene transgenic mice were found to be heterogeneous for their reactivity to B6-1710, suggesting that the V alpha-chains associating with the transgenic V beta-chain determine T cell responsiveness to B6-1710. These data clearly demonstrate that T cell reactivity to a murine AIDS virus expressing B cell line resembles that previously reported for Mls-like superantigens.

Animals↗
PubMed1992

T cell receptor alpha-chain pairing determines the specificity of residue 262 within the Kb-restricted, ovalbumin257-264 determinant.

Ovalbumin-specific, Kb-restricted T cells recognize the minimal fully active synthetic peptide ovalbumin (OVA)257-264. This sequence coincides with the eight residue, allele-specific peptide binding motif previously predicted from direct sequencing of naturally occurring Kb-associated peptides (Falk, K., Rotzscke, O., Stevanovic, S., Jung, G., and Rammensee, H.-G., Nature 351:290, 1990). T cell recognition of a panel of analogs with single residue substitutions between the two putative Kb anchor residues at OVA261 and OVA264 suggested that at least one residue, Glu at position 262, is involved in TCR interaction. OVA-specific cytotoxic T lymphocytes (CTL) derived from TCR beta-chain transgenic mice, where the beta-chain originates from an OVA-specific, Kb-restricted CTL B3, showed that differences in TCR alpha-chain pairing determined the specificity for OVA residue 262. These data support the notion that residue 262 of the OVA T cell determinant, corresponding to position 6 within the Kb-binding motif, represents a contact site for TCR. This residue interacts directly with the TCR alpha-chain or with a site on the TCR beta-chain whose conformation is affected by TCR alpha-chain pairing.

Amino Acid Sequence↗
PubMed1991

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.

Animals↗
PubMed1991

Peptide presentation by class-I major histocompatibility complex molecules.

MHC class-I molecules express distinct peptide-binding pockets within their antigen-binding groove. These are critically involved in the binding of antigenic peptides. The amino acid composition of a pocket dictates the structure of a peptide which can be bound in it. This is evident as a consensus amino acid motif which has to exist within a peptide in order for it to bind to a particular MHC allele. Perturbation of a MHC pocket by amino acid substitution can result in the abolition of peptide binding. Less drastic mutations of the peptide-binding groove, particularly the ones away from the critical pocket, can subtly alter the conformation of bound peptide. Both types of substitution exert an influence on the TCR recognition of antigenic peptide. Peptides are also critically involved in the positive selection of the class-I-restricted TCR repertoire in the thymus. These self peptides act by mimicking their foreign antigens. This mimicking involves the binding of self peptides and foreign antigenic peptides to the same pockets of the MHC class-I-antigen binding groove. Consequently, MHC class-I polymorphism in the antigen binding groove controls the intrathymic positive selection and peripheral antigen presentation by the same mechanisms. The majority of positively selecting self peptides could well originate from the extracellular processing of circulating self proteins. Using the diverse, extracellularly generated self peptides and the different determinant density requirements for positive versus negative selection, the immune system can ensure the repertoire diversity, avoiding both the massive clonal deletion of the selected repertoire and the autoreactivity of its T cells.

Amino Acid Sequence↗
PubMed1991

Conformational constraints involved in MHC class I restricted antigen presentation.

The realization that class I-restricted antigen presentation can be mimicked using synthetic peptides and the description of the class I three-dimensional structure has provided a basis with which to study the conformational constraints associated with cytotoxic T cell (CTL) recognition. Recent experiments have suggested that antigenic fragments can bind to class I molecules in extended conformations. This binding is dependent on the presence of predominant motif arrangements in the peptides which are likely to correspond to specific subsites within the class I binding cleft. Changes within this cleft can affect T cell receptor recognition by inhibiting peptide binding or by altering the conformation of the presented peptide. These changes can result in dramatic modifications in peripheral T cell recognition and in the thymic development of the T cells themselves. Consequently, class I molecules apparently control the T cell response via their effect on the binding and conformation of the presented peptide determinants.

Amino Acid Sequence↗
PubMed1990

Class I-restricted processing and presentation of exogenous cell-associated antigen in vivo.

MHC class I-restricted T lymphocyte responses are usually directed to cellular antigenic components resulting from endogenous gene expression. Exogenous, non-replicating antigens, such as soluble proteins, usually fail to enter the class I pathway of antigen processing and presentation. Consistent with this notion, we have recently shown that soluble, exogenous proteins can be efficiently processed for class I presentation in vitro only if they are introduced directly into the target cell cytoplasm. In this report we extend this work to the in vivo situation by introducing soluble protein into the cytoplasm of mouse splenocytes via the osmotic lysis of pinosomes and then using these cells for in vivo immunization. Our results show that cytoplasmic loading of OVA and beta-GAL into H-2b and H-2d splenocytes respectively, resulted in effective in vivo immunogens for class I-restricted CTL. To our surprise, control spleen cell preparations simply incubated with the exogenous, native protein for 10 min at 37 degrees C in isotonic medium and then washed could also induce a comparable class I-restricted CTL response following intravenous injection. Experiments using (H-2b X H-2d)F1 mice showed that protein pulsed splenocytes from one parental strain could effectively "cross prime" T cells restricted to the MHC of the other parental strain. In all cases, target cell recognition by the effector CTL generated by immunization with spleen cell-associated antigen required the antigen to be present in the cell cytoplasm. Thus the CTL do not recognize target cells exposed to soluble, exogenous antigen. These results, reminiscent of analogous experiments with cross priming by minor histocompatibility antigens, argue that class I-restricted processing and presentation of exogenous antigen can occur in vivo following immunization with cell-associated antigen.

Animals↗
PubMed1990

The effect of mutations in the MHC class I peptide binding groove on the cytotoxic T lymphocyte recognition of the Kb-restricted ovalbumin determinant.

The H-2Kb-restricted cytotoxic T lymphocyte (CTL) response directed against ovalbumin (OVA) is specific for a region contained within the sequence OVA253-276. In this study we have characterized this response by examining the class I-restricted presentation of OVA peptides by the naturally occurring Kb mutant (Kbm) glycoproteins Kbm1, Kbm3, Kbm5, Kbm8, Kbm10, Kbm11 and Kbm23. To facilitate this study we derived a series of somatic cell hybrid targets expressing the various Kbm class I molecules. Experiments using bulk OVA-specific CTL from C57BL/6 mice demonstrated that all the Kbm molecules except for Kbm1 and Kbm8 could present OVA peptides for effective T cell recognition. Clonal analysis revealed a more complex and relatively diverse pattern of CTL recognition of the Kbm/peptide combinations. This diversity is unlikely to result from the existence of multiple, independent Kb-restricted T cell determinants within OVA, since all CTL tested were specific for a single region between residues 259 and 273. Examination of the fine specificity of Kbm presentation identified individual changes at residues 77, 80 and 116 which affected T cell recognition. The results imply that these changes do not inhibit peptide binding since some clones could recognize peptide presented by a particular Kbm molecule, while other clones could not. All three residues reside within the peptide-binding cleft of the class I protein and are not expected to directly contact the T cell receptor. Although we did not formally demonstrate that OVA binding by Kbm vs. Kb is quantitatively identical, our results are best explained by postulating that the changes at residues 77, 80 and 116 indirectly affect T cell recognition by altering peptide conformation. Taken together our results suggest that changes within the class I binding site can profoundly modify peptide presentation without significantly inhibiting peptide-class I association.

Animals↗
PubMed1989

Peptide-dependent recognition of H-2Kb by alloreactive cytotoxic T lymphocytes.

Antigen-specific T lymphocytes appear to recognize foreign antigens in the form of peptide fragments presented within the antigen-binding groove of class I or class II molecules encoded by the major histocompatibility complex (MHC). Alloreactive T cells also show specificity for MHC molecules, and various reports suggest that residues of the MHC molecules constitute at least part of the ligand to which alloreactive T-cell receptors bind. The X-ray crystal structure of the human MHC class I molecule, HLA-A2, has provided evidence to strengthen the argument that MHC-bound self-peptide might also contribute to such recognition. We now provide direct evidence for this, showing that at least some alloreactive cytotoxic T lymphocyte clones recognize peptide fragments derived from cytoplasmic proteins. We reasoned that if self-peptides were involved in allorecognition, then the sequence of some of these peptides could vary between species, resulting in species-restricted distribution of the relevant ligand(s). Several alloreactive cytotoxic T lymphocyte clones specific for H-2Kb, expressed by the murine cell line EL4, did not lyse a human-cell transfectant expressing the H-2Kb molecule (Jurkat-Kb cells). However, these clones were able to lyse Jurkat-Kb cells sensitized by preincubation with an EL4 cytoplasmic extract cleaved by cyanogen bromide. The sensitizing activity from this extract was destroyed by protease and appeared to be due to a peptide consisting of 10 to 15 amino acids.

Animals↗
PubMed1989

Induction of ovalbumin-specific cytotoxic T cells by in vivo peptide immunization.

CTL recognize peptide forms of processed, foreign antigens in association with class I molecules encoded by the MHC and are usually directed against endogenously synthesized "cellular antigens," such as those expressed by virus-infected cells. In vitro studies have shown that small exogenous peptides can directly associate with class I molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. We have recently generated OVA-specific, H-2Kb-restricted CTL by immunizing C57BL/6 mice with a syngeneic tumor line transfected with the OVA cDNA. The CTL recognize the OVA transfectant E.G7-OVA and the synthetic peptide OVA258-276, but fail to recognize the native protein. We reasoned that given the potential for direct peptide/class I association observed in vitro, OVA258-276 may induce CTL after in vivo priming. However, we found that this is not the case. OVA258-276 and peptides of increasing lengths up to OVA242-276 and OVA242-285, which are all able to form the target complex in vitro, are inefficient at priming E.G7-OVA-specific CTL responses after intravenous injection. This is also true for both native and denatured OVA. In contrast to these results the synthetic peptide OVA229-276 corresponding to a peptide in a partial tryptic digestion of OVA can efficiently prime C57BL/6 mice in vivo after intravenous injection. This peptide elicits CTL that appear identical to those derived from animals immunized with syngeneic cells producing OVA endogenously. These results are discussed in terms of separate class I and class II antigen presentation pathways and the ability of only certain, exogenous antigens to enter the cytoplasmic, class I pathway.

Animals↗
PubMed1989

Class I-restricted presentation occurs without internalization or processing of exogenous antigenic peptides.

Previous studies have shown that glutaraldehyde-fixed cells can present fragmented, but not native, Ag to class II-restricted T cells. This presumably occurs via direct binding of peptides to class II molecules at the cell surface. More recently, it has been shown that viable target cells can present peptides and endogenous, but not exogenous, protein Ag in association with class I MHC molecules to CTL. We have derived CTL specific for a chicken OVA peptide (OVA258-276) recognized in association with H-2Kb. These CTL recognize target cells that endogenously synthesize OVA and cells "loaded" with native OVA but fail to recognize target cells in the presence of exogenous native OVA. Thus, OVA must be intracellularly located to be processed and presented for CTL recognition. It remains unclear, however, whether exogenous peptides require internalization and further processing by target cells or are able to associate directly with class I molecules at the cell surface for CTL recognition. We provide evidence that glutaraldehyde-fixed cells can present synthetic peptides to H-2Kb- and H-2Db-restricted CTL and that such presentation does not require internalization or processing. The peptides used range in size from 16 to 48 amino acids in length. In contrast, glutaraldehyde-fixed cells are incapable of presenting Ag to CTL specific for influenza nucleoprotein and OVA if the cells are fixed within 1 h of viral influenza infection or loading with OVA. Thus, CTL recognition of antigenic peptides appears to occur via direct binding of peptides to class I molecules at the cell surface and does not require any intracellular processing events.

Animals↗
PubMed1988

Introduction of soluble protein into the class I pathway of antigen processing and presentation.

In order to investigate how peptides associate with class I major histocompatibility complex (MHC) glycoproteins intracellularly, we generated cytotoxic T lymphocytes (CTL) specific for a readily available soluble protein in association with class I. C57BL/6 (H-2b) mice immunized against a syngeneic tumor cell transfected with chicken ovalbumin (OVA) cDNA gave rise to H-2Kb-restricted CTL specific for the OVA258-276 peptide. This synthetic peptide and CNBr fragments of OVA (242-285 and 242-273) were able to target H-2b cells for lysis by the CTL in a 3 hr assay. Cells incubated with native OVA for up to 24 hr did not become sensitized for recognition and lysis. However, when OVA was introduced directly into the cytoplasm of cells by the osmotic lysis of pinosomes, the Kb restricted determinant formed readily.

Animals↗
PubMed1988

The activation of pigeon cytochrome c-specific T cell hybridomas by antigenic peptides is influenced by non-native sequences at the amino terminus of the determinant.

We recently demonstrated that the sequence 95-104 contains all the residues necessary for direct recognition of the I-Ek restricted pigeon cytochrome c determinant but that residues located in sequences to the amino-terminal side of residue 95 improve the ability of peptides containing the sequence 95-104 to stimulate Ag-specific T cell clones. In this study we use synthetic peptides with amino-terminal leader sequences containing residues that differ with respect to their conformational stabilizing effects, charge, and hydrophilicity to examine the mechanism by which they modulate T cell recognition. Our findings indicate that the role of these residues in T cell stimulation is not related to their ability to stabilize alpha-helical secondary structure, nor do they appear to be processed differently. The leader sequences do not differentially influence the ability of the peptides to be presented by APC displaying Ia molecules of related haplotype, i.e., E alpha kE beta k, E alpha kE beta b, and E alpha kE beta s, to T cells which recognize the pigeon cytochrome c determinant on such presenting cells. Because antigenic potency correlates with the inclusion of hydrophobic residues and positively charged residues in the leader sequences, we discuss our findings with reference to the possibility that they non-specifically enhance the interaction of the antigenic peptides with the APC membrane.

Amino Acid Sequence↗
PubMed1988

Induction of cytotoxic T lymphocytes by primary in vitro stimulation with peptides.

Antigen-specific cytotoxic T cells can be generated by primary in vitro stimulation of spleen cells from C57BL/6 mice with appropriate peptide fragments. This response can be elicited without prior in vivo immunization. Chicken OVA fragmented with either cyanogen bromide (CN OVA) or trypsin (T OVA) was used as a source of mixed peptides. A synthetic peptide, NP365-380, representing the sequence 365-380 from influenza virus A/PR/8 nucleoprotein, was also used, since this contains the main determinants recognized by CTL generated from H-2b mice infected with A/PR/8 virus. The primary in vitro cytotoxic T cell response was peptide specific, since targets were lysed only in the presence of appropriate peptide antigens. Native OVA could not elicit primary effectors in vitro nor could it sensitize targets for lysis by OVA digest-specific CTL. A synthetic peptide corresponding to residues 111-122 within the OVA sequence could sensitize targets for lysis by effectors induced against T OVA. Effectors generated by in vitro stimulation were CD8+, CD4-, and H-2Db-restricted for NP365-380 and T OVA recognition. CN OVA-specific effectors were also CD8+, CD4-, but surprisingly, were able to lyse a range of H-2-different targets in an antigen-specific manner. These effectors failed to lyse a tumor line that does not express class I MHC molecules. This broad MHC restriction pattern was also apparent at the clonal level. In all cases, the antipeptide CTL generated by primary in vitro stimulation were inefficient in lysing target cells expressing endogenous forms of antigens, such as influenza virus-infected cells or cells transfected with the OVA cDNA. However, cytotoxic T cell lines generated in vitro against the NP365-380 peptide did contain a minor population of virus-reactive cells that could be selectively expanded by stimulation with A/PR/8-infected spleen cells. These results are discussed in terms of class I-restricted T cell stimulation in the absence of antigen processing by high surface densities of peptide/MHC complexes.

Animals↗
PubMed1988

Processing of a minimal antigenic peptide alters its interaction with MHC molecules.

Before their recognition by T lymphocytes, protein antigens generally require processing by antigen-presenting cells. In a poorly understood series of events, the protein antigen is internalized, transformed and re-expressed on the surface of the antigen-presenting cell in association with gene products of the major histocompatibility complex (MHC). Small peptides derived from the native protein can be recognized in the absence of antigen processing, suggesting that processing involves proteolytic degradation. These peptides are thought to mimic the naturally produced peptide fragment. We describe here a synthetic peptide antigen of this type which does not require processing but which is nevertheless further processed by splenic antigen-presenting cells. Interestingly, this processing event specifically alters the interaction of the peptide with the class II MHC (Ia) molecule, markedly affecting both its potency as an antigen in vitro and its immunogenicity in vivo (IR gene control).

Animals↗