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P J Fairchild

Publications and source records attributed to P J Fairchild.

At least 19 recordsLinked to original sources
PubMed2001

Regulation of CD40 function by its isoforms generated through alternative splicing.

CD40 is a member of the tumor necrosis factor receptor superfamily. The interaction between CD40 and CD40 ligand (CD154) activates NF-kappa B, Jun N-terminal kinase, and Janus kinase/signal transducers and activators of transcription pathways and promotes B cell growth, differentiation, and survival as well as IL-12 production in macrophages and dendritic cells. We demonstrate here the existence of multiple isoforms of CD40 mRNA generated by alternative splicing and show that their expression is regulated differentially in activated macrophages and dendritic cells. Pre-CD40 RNA is spliced preferentially out to signal-transducible CD40 mRNA in the early stage of activation; half of the CD40 mRNA is replaced by the signal-nontransducible CD40 mRNAs in the later stages (24 h). Using IL-12 p40 gene expression as a reporter for CD40 signaling, we show that three of the alternative isoforms can disable signaling through CD40. The major alternative isoform lacks the membrane-associated endodomain and seems to reduce the amount of the signal-transducible form available on the cell surface. It would seem, therefore, that CD40 expression is controlled by posttranscriptional and posttranslational regulation through alternative splicing. Modulation of isoform expression may provide a mechanism by which cells regulate their susceptibility to CD40L signaling.

Alternative Splicing↗
PubMed2000

Directed differentiation of dendritic cells from mouse embryonic stem cells.

Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.

Animals↗
PubMed2000

Extrathymic signals regulate the onset of T cell repertoire selection.

The thymus is the principal site of T cell development, coordinating positive and negative selection of an immunocompetent repertoire. The ability of fetal thymi from C57BL/6 mice to support these selection events in culture is, however, critically dependent on the timing of their excision: whereas thymi from 16 day embryos generate mature CD4+8(-) and CD4(-)8+ thymocytes, 14 day thymi show a profound blockage in positive selection at the CD4+8+ stage. Here we show that this blockage is not due to intrinsic deficiencies in the ability of thymocytes to transduce positive selection signals, suggesting the defect to lie within the thymic microenvironment. Since vascularization of the thymus occurs at day 15 of gestation, we investigated whether the exclusion of plasma proteins from 14 day thymi was responsible. Accordingly, the addition of serum from 16 day embryos to organ cultures of 14 day thymi rescued mature CD4+8(-) and CD4(-)8+ subsets. Activity was found to reside in a low molecular weight fraction of serum, sensitive to proteolysis, which was present only transiently during ontogeny. Our data suggest that repertoire selection is initiated following "priming" of the thymic microenvironment by plasma proteins, thereby ensuring the onset of positive selection to be delayed until the entry of extrathymic proteins to which self tolerance must be established.

Animals↗
PubMed2000

Dendritic cells and prospects for transplantation tolerance.

The past year has witnessed the resolution of some long-standing enigmas surrounding the immunobiology of dendritic cells, illuminating their opposing roles in peripheral tolerance and allograft rejection. Nevertheless these advances have posed many new questions, the answers to which may subtly influence our approach to the treatment of rejection while bringing ever closer the prospect of donor-specific transplanation tolerance.

Animals↗
PubMed1999

Expression of murine IL-12 is regulated by translational control of the p35 subunit.

IL-12 is a heterodimer of two subunits, p35 and p40, encoded by separate genes that are regulated independently. To investigate the mechanisms underlying the regulation of the p35 gene, we characterized murine p35 expression in the B cell lymphoma line A20 and in bone marrow-derived dendritic cells. Multiple transcription start sites were identified in both cell types, resulting in four p35 mRNA isoforms (types I-IV) that differ in the number and position of upstream ATGs in their 5' untranslated regions. In nonstimulated cells, the predominant forms of p35 message (types II and IV) contained an additional upstream ATG, whose presence was shown to inhibit the downstream translation of the p35 subunit. After LPS stimulation, however, transcription initiated from alternate positions, so that the proportion of transcripts not containing this upstream ATG (types I and III) was significantly increased in the population of p35 mRNA. These type I and type III transcripts readily supported translation of the p35 subunit and its incorporation into bioactive IL-12. Furthermore, p35 mRNA levels were substantially up-regulated after LPS stimulation in both cell types. Thus, our results show that p35 gene expression is highly regulated by both transcriptional and translational mechanisms.

5' Untranslated Regions↗
PubMed1999

Reversal of immunodominance among autoantigenic T-cell epitopes.

Studies spanning several decades have revealed how the complex forces of antigen processing distinguish those epitopes of a protein that dominate the immune response from those that remain cryptic. Since foreign antigens and self-proteins are subjected to the same proteolytic pathways before presentation to the T-cell repertoire, it has long been assumed that they comply equally with the established rules of immunodominance. Nevertheless, the pathological determinants of some autoantigens appear ill-equipped for the dominant role they adopt, displaying features more befitting subdominant or cryptic epitopes, such as low affinity for their MHC restriction element. These findings may be reconciled by suggesting that, far from remaining sequestered during ontogeny, many classical autoantigens participate in the establishment of self-tolerance, the efficiency with which individual epitopes purge the T-cell repertoire being determined by the conventional rules of immunodominance: while those epitopes that are truly dominant induce profound non-responsiveness, those that are poorly presented may leave residual reactivity, manifest in the periphery as responses to epitopes that appear inappropriately dominant. Here we review recent evidence showing the process of self-tolerance to be uniquely responsible for the reversal of immunodominance which promotes such epitopes to an undeserved position of importance within the determinant hierarchy.

Animals↗
PubMed1998

Presentation of antigenic peptides by products of the major histocompatibility complex.

Molecules encoded by the major histocompatibility complex (MHC) are polymorphic integral membrane proteins adapted to the presentation of peptide fragments of foreign antigens to antigen-specific T-cells. The diversity of infectious agents to which an immune response must be mounted poses a unique problem for receptor-ligand interactions; how can proteins whose polymorphism is necessarily limited bind an array of peptides almost infinite in its complexity? Both MHC class I and class II determinants have achieved this goal by harnessing a limited number of peptide side chains to anchor the epitope in place while exploiting conserved features of peptide structure, independent of their primary sequence. While class I molecules interact predominantly with the N- and C-termini of peptides, class II determinants form an extensive hydrogen bonding network along the length of the peptide backbone. Such a strategy ensures high-affinity binding, while selectively exposing the unique features of each ligand for recognition by the T-cell receptor.

Animals↗
PubMed1997

Regulation of IL-18 (IFN-gamma-inducing factor) gene expression.

IL-18 (also known as IFN-gamma-inducing factor), although structurally unrelated to IL-12, shares with it the role of activating NK cells and polarizing T cells toward Th1 cell function. To understand how the IL-18 gene (and consequently Th1 function) is regulated, we have determined the gene structure and investigated the mechanisms of transcriptional control and cell type expression. The mouse IL-18 gene comprises seven exons distributed over 26 kb. Exons 1 and 2 of this gene are 5'-noncoding exons. Promoter activity was detected upstream of these noncoding exons in two distinct regions. Both promoters are TATA-less and not G+C rich. The promoter activity located upstream of exon 2 was shown to act constitutively, while the activity located upstream of exon 1 was up-regulated in activated macrophage and T cell lines. IL-18 gene expression may be regulated in a wide range of cell types by the activities of these two distinct promoters. IL-18 is known to be synthesized as a precursor, pro-IL-18, and its maturation is controlled by IL-1beta-converting enzyme (ICE). We observed concordant expression of IL-18 and ICE mRNAs in a wide range of cell types, unlike the more restricted expression of IL-12 p40 mRNA. The widespread IL-18 mRNA distribution and the special relationship with ICE lead us to the hypothesis that IL-18 expression may be coupled with apoptotic processes involving activation of ICE or ICE-like proteinase.

Animals↗
PubMed1997

Altered peptide ligands: prospects for immune intervention in autoimmune disease.

It has long been accepted that the response of T cells to a protein antigen is strongly influenced by parameters governing processing and presentation of the immunodominant epitopes. Recent evidence has suggested, however, that subtle changes in the nature of the ligand itself may also affect the outcome of T-cell receptor (TCR) ligation, necessitating a re-evaluation of previously accepted paradigms of T-cell activation. In particular, activation may no longer be regarded as an all-or-nothing event, but appears to involve both quantitative and qualitative dimensions. This revolution in our understanding has emanated from the recent discovery that analogues of immunogenic peptides, so-called altered peptide ligands (APLs), may elicit a subset of normal activation events, or profoundly influence responses to the wild-type epitope. As such, the potential offered by APLs for modifying the outcome of deleterious immune responses involved in autoimmunity has not passed unnoticed. Indeed, the design and exploitation of novel reagents based on the structure of autoantigenic epitopes has enjoyed some measure of success in the treatment of experimental models of autoimmune disease and holds promise for their exploitation within the clinical arena.

Animals↗
PubMed1996

Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein.

Following induction of experimental encephalomyelitis with a T-cell clone, L10C1, that is specific for the myelin basic protein epitope p87-99, the inflammatory infiltrate in the central nervous system contains a diverse collection of T cells with heterogeneous receptors. We show here that when clone L10C1 is tolerized in vivo with an analogue of p87-99, established paralysis is reversed, inflammatory infiltrates regress, and the heterogeneous T-cell infiltrate disappears from the brain, with only the T-cell clones that incited disease remaining in the original lesions. We found that antibody raised against interleukin-4 reversed the tolerance induced by the altered peptide ligand. Treatment with this altered peptide ligand selectively silences pathogenic T cells and actively signals for the efflux of other T cells recruited to the site of disease as a result of the production of interleukin-4 and the reduction of tumour-necrosis factor-alpha in the lesion.

Amino Acid Sequence↗
PubMed1996

Lowering the tone: mechanisms of immunodominance among epitopes with low affinity for MHC.

Past studies of immunodominance among T-cell epitopes have focused on peptides with high affinity for their restriction element, assuming epitopes of low affinity to be immunologically irrelevant. Here, Paul Fairchild and David Wraith challenge this assumption by reviewing evidence that such peptides may contribute to T-cell repertoire selection and autoimmune disease, and suggest approaches to immunotherapy based on exploitation of these peptides.

Animals↗
PubMed1996

The nature of cryptic epitopes within the self-antigen myelin basic protein.

Mechanisms that allow potentially autoreactive T cells to escape central tolerance and persist in the peripheral lymphoid organs of healthy individuals are poorly defined. It has been proposed that such cells are specific for epitopes which normally are not well presented to the immune system or, in other words, are cryptic. We have used synthetic peptides to define potential T cell epitopes within the N-terminal portion of myelin basic protein (MBP). These were defined in terms of their relative affinity for the MHC-restriction element I-Au and their ability to activate T cells in mice of the H-2(u) haplotype. Three epitopes were identified, one of which corresponded to the known dominant N-terminal epitope (Ac1-9). The other two epitopes (9-20 and 5-20) bound to their MHC-restriction element with relatively high affinity but were cryptic, as defined by the poor response to these epitopes following immunization with intact MBP. Even the longer of these two epitopes did not induce autoimmune encephalomyelitis in H-2(u) mice. These results demonstrate that antigen processing can control both the induction of and effector function of autoreactive T cells, and is therefore a principal mechanism involved in limiting the autoreactive T cell repertoire.

Amino Acid Sequence↗
PubMed1995

Low avidity recognition of self-antigen by T cells permits escape from central tolerance.

The immunodominant epitope of myelin basic protein, Ac1-9, is encephalitogenic in H-2u mice. We have previously demonstrated that this epitope displays low affinity for I-Au and have suggested that the avidity of T cell recognition in the thymus may be compromised, enabling autoreactive T cells to escape self-tolerance. We have addressed this hypothesis directly by constructing transgenic mice expressing an encephalitogenic T cell receptor (TCR). Parenteral administration of Ac1-9 had no discernable impact on developing thymocytes. In contrast, peptide analogs displaying far higher affinity for I-Au, provoked deletion of CD4+ CD8+ cells and transient down-regulation of the TCR by mature CD4+ CD8- thymocytes. The use of analogs of intermediate affinity permitted a margin of error to be defined for the induction of tolerance and confirmed that the affinity of Ac1-9 lies well below the critical threshold.

Animals↗
PubMed1995

Developmental changes predispose the fetal thymus to positive selection of CD4+CD8- T cells.

Selection of a competent T-cell repertoire is dependent on complex interactions between immature thymocytes and components of the thymic stroma. These events may be preserved in vitro by excising developing thymus rudiments and maintaining them under carefully controlled conditions in fetal thymus organ cultures (FTOC). Using this approach, we have shown that the ability of C57B1/6 thymi to sustain positive selection of mature CD4+CD8- cells is profoundly influenced by the day of gestation on which they are excised: while thymocytes from day 14 rudiments fail to progress beyond the CD4+CD8+ stage of the developmental pathway, day 15 and day 16 thymi support the differentiation of CD4+CD8- thymocytes. Importantly, day 16 thymocytes transferred to day 14 deoxyguanosine-treated rudiments are likewise arrested at the CD4+CD8+ stage, suggesting that the thymic microenvironment of day 14 rudiments, rather than the state of differentiation of the thymocytes they contain, is responsible for the block in positive selection. Our studies of the stromal elements of day 14 rudiments have, however, revealed no obvious deficiencies in the cell types represented, or their expression of class II major histocompatibility complex (MHC) determinants. Furthermore, we have been unable to circumvent the blockage in positive selection by the addition of certain cytokines expressed late during gestation. These results suggest that subtle changes occurring at day 15 of ontogeny render the thymic microenvironment capable of positive selection.

Animals↗
PubMed1993

An autoantigenic T cell epitope forms unstable complexes with class II MHC: a novel route for escape from tolerance induction.

The peptide rAc1-11 represents the dominant T cell epitope of rat myelin basic protein (MBP) in mice of the H-2u haplotype. Residue 4 has been shown previously to govern binding of the peptide to the class II molecule, I-Au. We have constructed peptide analogues bearing amino acid substitutions at position 4 and have assessed their ability to stimulate an antigen-specific T cell hybridoma when presented by viable antigen presenting cells (APC). Complexes between I-Au and one such analogue, rAc1-11[4A], were rapidly lost from the surface of live APC displaying a half-life (t 1/2) of approximately 10 min. Neither shedding of intact complexes from the cell surface, nor their internalization and recycling through an acidic intracellular compartment were found to account for their loss. The possible dissociation of rAc1-11[4A] from the peptide binding cleft was therefore addressed by comparing the t 1/2 of complexes between I-Au and peptide analogues of higher affinity. The tyrosine-substituted analogue, rAc1-11[4Y], remained stably bound to I-Au for at least 4 h, thereby displaying a t 1/2 far in excess of that evident for rAc1-11[4A]. Significantly, the wild type peptide, rAc1-11, bound so transiently that functional complexes could not be detected on the surface of peptide-pulsed APC. The physiological relevance of these findings was confirmed by extending our studies to an analysis of the homologous epitope of murine MBP; evidence that this epitope likewise displays minimal affinity for I-Au suggests a novel strategy for the escape from tolerance induction by encephalitogenic T cells.

Amino Acid Sequence↗