PubMed Health⌕ Search

Biomedical subjects

F R Carbone

Publications and source records attributed to F R Carbone.

At least 37 records · Page 2Linked to original sources
PubMed1999

CD30 prevents T-cell responses to non-lymphoid tissues.

Self antigens can induce T-cell tolerance via a mechanism termed cross-tolerance. This involves the transfer of peripheral tissue antigens to professional APC for presentation in the draining lymph nodes. In this site, CD8+ T cells are activated, proliferate, and are slowly deleted by a CD95-dependent mechanism. Prior to their deletion, some activated cells leave the lymph nodes and encounter antigens on peripheral parenchymal tissues. Without functional CD30, these cells proliferate extensively and cause substantial tissue damage. Thus, CD30 limits autoreactivity, acting as a 'brake' on T-cell proliferation after recognition of autoantigens on parenchymal tissues.

Animals↗
PubMed1999

The cytotoxic T-cell response to herpes simplex virus type 1 infection of C57BL/6 mice is almost entirely directed against a single immunodominant determinant.

Many virus infections give rise to surprisingly limited T-cell responses directed to very few immunodominant determinants. We have been examining the cytotoxic T-lymphocyte (CTL) response to herpes simplex virus type 1 (HSV-1) infection. Previous studies have identified the glycoprotein B-derived peptide from residues 498 to 505 (gB(498-505)) as one of at least three determinants recognized by HSV-1-specific CTLs isolated from C57BL/6 mice. We had previously found that in vitro-derived CTLs directed to gB(498-505) show a characteristic pattern of T-cell receptor (TCR) usage, with 60% of gB(498-505)-specific CD8(+) T cells expressing BV10(+) TCR beta chains and a further 20% expressing BV8S1. In this report, we confirm that this TCR V-region bias is also reflected in the ex vivo response to HSV-1 infection. A high proportion of activated CD8(+) draining lymph node cells were found to express these dominant V regions, suggesting that a substantial number of in vivo responding T cells were directed to this one viral determinant. The use of an HSV-1 deletion mutant lacking the gB(498-505) determinant in combination with accurate intracellular gamma interferon staining allowed us to quantify the extent of gB-specific T-cell dominance. Together, these results suggested that between 70 and 90% of all CD8(+) HSV-1-specific T cells target gB(498-505). While deletion of this determinant resulted in an attenuated CD8(+) T-cell response, it also permitted the emergence of one or more previously unidentified cryptic specificities. Overall, HSV-1 infection of C57BL/6 mice results in an extremely focused pattern of CD8(+) T-cell selection in terms of target specificity and TCR expression.

Animals↗
PubMed1998

B cells directly tolerize CD8(+) T cells.

This report investigates the response of CD8(+) T cells to antigens presented by B cells. When C57BL/6 mice were injected with syngeneic B cells coated with the Kb-restricted ovalbumin (OVA) determinant OVA257-264, OVA-specific cytotoxic T lymphocyte (CTL) tolerance was observed. To investigate the mechanism of tolerance induction, in vitro-activated CD8(+) T cells from the Kb-restricted, OVA-specific T cell receptor transgenic line OT-I (OT-I cells) were cultured for 15 h with antigen-bearing B cells, and their survival was determined. Antigen recognition led to the killing of the B cells and, surprisingly, to the death of a large proportion of the OT-I CTLs. T cell death involved Fas (CD95), since OT-I cells deficient in CD95 molecules showed preferential survival after recognition of antigen on B cells. To investigate the tolerance mechanism in vivo, naive OT-I T cells were adoptively transferred into normal mice, and these mice were coinjected with antigen-bearing B cells. In this case, OT-I cells proliferated transiently and were then lost from the secondary lymphoid compartment. These data provide the first demonstration that B cells can directly tolerize CD8(+) T cells, and suggest that this occurs via CD95-mediated, activation-induced deletion.

Animals↗
PubMed1998

Major histocompatibility complex class I-restricted cross-presentation is biased towards high dose antigens and those released during cellular destruction.

Naive T cells recirculate mainly within the secondary lymphoid compartment, but once activated they can enter peripheral tissues and perform effector functions. To activate naive T cells, foreign antigens must traffic from the site of infection to the draining lymph nodes, where they can be presented by professional antigen presenting cells. For major histocompatibility complex class I-restricted presentation to CD8+ T cells, this can occur via the cross-presentation pathway. Here, we investigated the conditions allowing antigen access to this pathway. We show that the level of antigen expressed by peripheral tissues must be relatively high to facilitate cross-presentation to naive CD8+ T cells. Below this level, peripheral antigens did not stimulate by cross-presentation and were ignored by naive CD8+ T cells, although they could sensitize tissue cells for destruction by activated cytotoxic T lymphocytes (CTLs). Interestingly, CTL-mediated tissue destruction facilitated cross-presentation of low dose antigens for activation of naive CD8+ T cells. This represents the first in vivo evidence that cellular destruction can enhance access of exogenous antigens to the cross-presentation pathway. These data indicate that the cross-presentation pathway focuses on high dose antigens and those released during tissue destruction.

Animals↗
PubMed1998

The peripheral deletion of autoreactive CD8+ T cells induced by cross-presentation of self-antigens involves signaling through CD95 (Fas, Apo-1).

Recently, we demonstrated that major histocompatibility complex class I-restricted cross-presentation of exogenous self-antigens can induce peripheral T cell tolerance by deletion of autoreactive CD8+ T cells. In these studies, naive ovalbumin (OVA)-specific CD8+ T cells from the transgenic line OT-I were injected into transgenic mice expressing membrane-bound OVA (mOVA) under the control of the rat insulin promoter (RIP) in pancreatic islets, kidney proximal tubules, and the thymus. Cross-presentation of tissue-derived OVA in the renal and pancreatic lymph nodes resulted in activation, proliferation, and then the deletion of OT-I cells. In this report, we investigated the molecular mechanisms underlying this form of T cell deletion. OT-I mice were crossed to tumor necrosis factor receptor 2 (TNFR2) knockout mice and to CD95 (Fas, Apo-1) deficient mutant lpr mice. Wild-type and TNFR2-deficient OT-I cells were activated and then deleted when transferred into RIP-mOVA mice, whereas CD95-deficient OT-I cells were not susceptible to deletion by cross-presentation. Furthermore, cross-presentation led to upregulation of the CD95 molecule on the surface of wild-type OT-I cells in vivo, consistent with the idea that this is linked to rendering autoreactive T cells susceptible to CD95-mediated signaling. This study represents the first evidence that CD95 is involved in the deletion of autoreactive CD8+ T cells in the whole animal.

Animals↗
PubMed1998

Help for cytotoxic-T-cell responses is mediated by CD40 signalling.

Cytotoxic T lymphocytes (CTLs) which carry the CD8 antigen recognize antigens that are presented on target cells by the class I major histocompatibility complex. CTLs are responsible for the killing of antigen-bearing target cells, such as virus-infected cells. Although CTL effectors can act alone when killing target cells, their differentiation from naive CD8-positive T cells is often dependent on 'help' from CD4-positive helper T (TH) cells. Furthermore, for effective CTL priming, this help must be provided in a cognate manner, such that both the TH cell and the CTL recognize antigen on the same antigen-presenting cell. One explanation for this requirement is that TH cells are needed to convert the antigen-presenting cell into a cell that is fully competent to prime CTL. Here we show that signalling through CD40 on the antigen-presenting cells can replace the requirement for TH cells, indicating that T-cell 'help', at least for generation of CTLs by cross-priming, is mediated by signalling through CD40 on the antigen-presenting cell.

Animals↗
PubMed1998

Cross-presentation of self antigens to CD8+ T cells: the balance between tolerance and autoimmunity.

Upon encounter with foreign antigen, tissue-associated antigen-presenting cells (APCs) migrate to draining lymph nodes to prime specific T cells. Using the transgenic RIP-mOVA model, we recently demonstrated that self antigens derived from peripheral tissues are constitutively transported to draining lymph nodes, and can be presented in association with MHC class I molecules by a bone marrow-derived APC population. This form of class I-restricted presentation of exogenous antigen has been referred to as cross-presentation and can induce activation and proliferation of antigen-specific CD8+ T cells. In the absence of CD4+ T cell help, activation of CD8+ T cells is inefficient, and cross-presentation leads to peripheral deletion of autoreactive CD8+ T cells, acting as a mechanism to maintain self-tolerance. If CD4+ T cell help is available, CD8+ T cell responses to self antigens can be rendered immunogenic, leading to autoreactive responses. Whether autoimmunity results from such responses also depends on the tissue location of the antigen. In RIP-mOVA mice, which express the model antigen mOVA (a membrane-bound form of ovalbumin) in the pancreatic beta cells and kidney proximal tubules, OVA-specific CD8+ T cells, activated by cross-presentation, infiltrated the pancreas and caused B cell destruction. Interestingly, however, these cells did not infiltrate the kidney, suggesting that proximal tubular cells are to some extent protected from immune destruction. Analysis of the role of antigen concentration indicates that high doses were required for efficient cross-presentation, suggesting that this pathway is directed towards immune responses to high-dose antigens, such as may be present during viral infection.

Animals↗
PubMed1998

A dominant V beta bias in the CTL response after HSV-1 infection is determined by peptide residues predicted to also interact with the TCR beta-chain CDR3.

Many T cell responses are dominated by restricted TCR expression and can range from repeated usage of particular TCR Vbeta- and/or Valpha-elements, to the preferential usage of both V- and J-elements, often in conjunction with conserved V-D-J or V-J junctional sequences. Cytotoxic T lymphocytes specific for a Kb-restricted determinant from the herpes simplex virus glycoprotein B (gB) preferentially express a dominant TCRBV10 beta-chain with sequence conservation of a tryptophan-glycine located in the V-D junction. Here we have examined whether immunisation of C57BL/6 mice with the gB-peptide can mimic the CTL response seen after HSV-1 infection. Immunisation with the gB-peptide resulted in the generation of gB-specific CTL that showed a similar TCRBV10 bias to that observed after HSV-1 infection. When the gB-determinant was expressed as a part of a fusion protein, immunised mice again exhibited the TCRBV10 bias with the junctional sequence conservation in the responding CTL. C57BL/6 mice were then immunised with variants of the gB-peptide that contained amino acid substitutions at positions previously predicted to contact the TCR beta-chain CDR3. Analysis of the TCRBV usage of variant specific CTL lines showed that substitutions at the TCR-contact positions 4, 6 and 7 of the gB-peptide resulted in a loss of the TCRBV10 bias. These results suggest that the TCRBV10 bias seen in gB-specific CTL after HSV-1 infection is due to antigenic selection by the minimal peptide and is determined by residues proposed to contact the TCR beta-chain CDR3.

Amino Acid Sequence↗
PubMed1998

Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements.

We describe the generation of ovalbumin (OVA)-specific, MHC class II-restricted alpha beta T cell receptor (TCR) transgenic mice. Initial attempts at generating these transgenic mice utilized heterologous regulatory elements to drive the expression of cDNA genes encoding the separate alpha- and beta-chains of the TCR. Unexpectedly, T cells bearing the transgenic alpha beta TCR failed to emerge from the thymus in these mice, although the transgenes did modify endogenous TCR expression. However, subsequent modification of the approach which enabled expression of the TCR beta-chain under the control of its natural regulatory elements generated mice whose peripheral T cells expressed the transgenic TCR and were capable of antigen-dependent proliferation. These results show that successful generation of MHC class II-restricted, OVA-specific alpha beta TCR transgenic mice was dependent upon combining cDNA- and genomic DNA-based constructs for expression of the respective alpha- and beta-chains of the TCR.

Animals↗
PubMed1998

Loss of antiviral cytotoxic T-lymphocyte activity during high-level antigen stimulation.

High levels of antigenic stimulation can result in deactivation of CD8+ T cells through a variety of mechanisms, including insufficient T-cell help. In the present study, an adoptive transfer system was established in which ovalbumin (OVA)-specific CD8+ T cells were transferred to irradiated mice infected with a recombinant vaccinia virus encoding OVA (VV-OVA). Prolonged activation of OVA-specific CD8+ T cells resulted in a proliferative block in these cells, although cytotoxic function was maintained. Unlike naive and recently activated OVA-specific T cells, these nonproliferative cytotoxic CD8+ T cells did not have antiviral activity following further transfer to mice infected with VV-OVA. Provision of interleukin-2 (IL-2) at the site of virus infection using a recombinant virus encoding antigen and IL-2, as well as the addition of helper T cells, had no effect on the generation of these dysfunctional T cells. Thus, there was no evidence that lack of T-cell help was responsible for CD8+ T-cell deactivation in this model.

Animals↗
PubMed1997

CD4+ T cell help impairs CD8+ T cell deletion induced by cross-presentation of self-antigens and favors autoimmunity.

Self-antigens expressed in extrathymic tissues such as the pancreas can be transported to draining lymph nodes and presented in a class I-restricted manner by bone marrow-derived antigen-presenting cells. Such cross-presentation of self-antigens leads to CD8+ T cell tolerance induction via deletion. In this report, we investigate the influence of CD4+ T cell help on this process. Small numbers of autoreactive OVA-specific CD8+ T cells were unable to cause diabetes when adoptively transferred into mice expressing ovalbumin in the pancreatic beta cells. Coinjection of OVA-specific CD4+ helper T cells, however, led to diabetes in a large proportion of mice (68%), suggesting that provision of help favored induction of autoimmunity. Analysis of the fate of CD8+ T cells indicated that CD4(+) T cell help impaired their deletion. These data indicate that control of such help is critical for the maintenance of CD8+ T cell tolerance induced by cross-presentation.

Adoptive Transfer↗
PubMed1997

Functional mapping of the orientation for TCR recognition of an H2-Kb-restricted ovalbumin peptide suggests that the beta-chain subunit can dominate the determination of peptide side chain specificity.

T cells recognize a complex of antigenic peptide bound to the class I or class II products of the MHC. Crystallographic analysis of the interaction between MHC class I-bound peptide fragments and specific TCR have recently been described and highlight the importance of the CDR3 in determining peptide specificity. The results presented here show functional data for TCR recognition of the H2-Kb class-I restricted determinant derived from OVA (SIINFEKL) that are consistent with the TCR orientation defined by these crystal structures. In addition, we also found that the beta-chain CDR3 dominates side chain specificity for the most exposed regions within this peptide. The data also suggest that this orientation and pattern of beta-chain dominance may extend to the recognition of a second H2-Kb-restricted peptide from the herpes simplex virus type 1 glycoprotein B (SSIEFARL), which shares a common alpha-chain contact with the OVA peptide. These results are discussed in terms of a common orientation for TCR-ligand interaction and the greater potential for TCR beta-chain CDR3 diversity in determining peptide side chain specificity.

Amino Acid Sequence↗
PubMed1997

Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.

In this report, we show that cross-presentation of self-antigens can lead to the peripheral deletion of autoreactive CD8(+) T cells. We had previously shown that transfer of ovalbumin (OVA)-specific CD8(+) T cells (OT-I cells) into rat insulin promoter-membrane-bound form of OVA transgenic mice, which express the model autoantigen OVA in the proximal tubular cells of the kidneys, the beta cells of the pancreas, the thymus, and the testis of male mice, led to the activation of OT-I cells in the draining lymph nodes. This was due to class I-restricted cross-presentation of exogenous OVA on a bone marrow-derived antigen presenting cell (APC) population. Here, we show that adoptively transferred or thymically derived OT-I cells activated by cross-presentation are deleted from the peripheral pool of recirculating lymphocytes. Such deletion only required antigen recognition on a bone marrow-derived population, suggesting that cells of the professional APC class may be tolerogenic under these circumstances. Our results provide a mechanism by which the immune system can induce CD8(+) T cell tolerance to autoantigens that are expressed outside the recirculation pathway of naive T cells.

Adoptive Transfer↗
PubMed1997

Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help.

Class I-restricted presentation is usually associated with cytoplasmic degradation of cellular proteins and is often considered inaccessible to exogenous antigens. Nonetheless, certain exogenous elements can gain entry into this so-called endogenous pathway by a mechanism termed cross-presentation. This is known to be effective for class I-restricted cytotoxic T lymphocyte (CTL) cross-priming directed against a variety of exogenous tumor, viral, and minor transplantation antigens. The related effect of cross-tolerance can also effectively eliminate responses to selected self components. In both cases, this presentation appears to require the active involvement of a bone marrow-derived antigen presenting cell (APC). Here, we show that CTL induction by cross-priming with cell-associated ovalbumin requires the active involvement of CD4+ helper T cells. Importantly, this CD4+ population is only effective when both the helper and CTL determinants are recognized on the same APC. Moreover, we would argue that the cognitive nature of this event suggests that the CD4+ T cell actively modifies the APC, converting it into an effective stimulator for the successful priming of the CTL precursor.

Animals↗
PubMed1997

Down-modulation of CD8 beta-chain in response to an altered peptide ligand enables developing thymocytes to escape negative selection.

Mice expressing a Kb-restricted transgenic T cell receptor (TCR) and a naturally occurring MHC class I variant molecule, Kbm8, were used to study thymic selection. The transgenic TCR was specific for the major peptide determinant from ovalbumin (OVA(257-264)), while Kbm8 has a mutation that alters the position 2 binding pocket of the Kb molecule, abolishing antigenic peptide presentation and positive selection of transgenic T cells. Peptide presentation was restored by identifying a position 2 analog peptide with Kbm8-binding capacity. In combination with Kbm8, the E2 peptide variant was capable of deleting immature double-positive thymocytes in suspension culture. Similarly, addition of exogenous E2 peptide to fetal thymic organ culture resulted in efficient deletion of double-positive thymocytes. However, there remained a population of CD8 single-positive T cells that exhibited impaired responsiveness to the antigenic peptide and lacked expression of the CD8 beta-chain. These results suggest a mechanism whereby developing thymocytes bearing an alphabetaTCR can modify their expression of the CD8 coreceptor to escape thymic deletion and achieve self-tolerance.

Animals↗
PubMed1997

Antigen-specific CD8+ T cell subset distribution in lymph nodes draining the site of herpes simplex virus infection.

Inoculation with replicating virus leads to an increase in T cell numbers within lymph nodes that drain the site of infection. This increase has been associated with a nonspecific proliferation of bystander cells, with only a minority thought to be directed to the infectious agent. Such an assumption is largely based on precursor cytotoxic T lymphocyte (CTL) estimations using limiting dilution analysis. Recently, studies using more advanced molecular approaches have suggested that such functionally derived precursor frequencies considerably underestimate the proportion of T cells specific for the antigen under investigation. We have defined T cell receptor sequences characteristic of CTL populations directed to a dominant determinant of the herpes simplex virus (HSV) glycoprotein B (gB). In this investigation, we used this receptor signature as a probe to directly monitor changes occurring within lymph nodes draining the sites of active infection with HSV. We found that although lymph node CD8+ T cell numbers increase as a consequence of HSV infection, the majority of these cells are small resting cells that are not enriched for gB-specific receptors. In contrast, a significant proportion of activated T cells are highly enriched for CTL bearing gB-specific receptors. Our results are therefore consistent with a nonspecific migration of CTL precursors into the lymph nodes draining the site of infection, followed by the activation and proliferation of the antigen-specific subset that normally makes up a small proportion of the naive T cell repertoire.

Amino Acid Sequence↗