Cross-presentation of tumour antigens by human induced pluripotent stem cell-derived CD141(+)XCR1+ dendritic cells.

Monocyte-derived dendritic cells (moDC) have been widely used in cancer immunotherapy but show significant donor-to-donor variability and low capacity for the cross-presentation of tumour-associated antigens (TAA) to CD8(+) T cells, greatly limiting the success of this approach. Given recent developments in induced pluripotency and the relative ease with which induced pluripotent stem (iPS) cell lines may be generated from individuals, we have succeeded in differentiating dendritic cells (DC) from human leukocyte antigen (HLA)-A(*)0201(+) iPS cells (iPS cell-derived DC (ipDC)), using protocols compliant with their subsequent clinical application. Unlike moDC, a subset of ipDC was found to coexpress CD141 and XCR1 that have been shown previously to define the human equivalent of mouse CD8α(+) DC, in which the capacity for cross-presentation has been shown to reside. Accordingly, ipDC were able to cross-present the TAA, Melan A, to a CD8(+) T-cell clone and stimulate primary Melan A-specific responses among naïve T cells from an HLA-A(*)0201(+) donor. Given that CD141(+)XCR1(+) DC are present in peripheral blood in trace numbers that preclude their clinical application, the ability to generate a potentially unlimited source from iPS cells offers the possibility of harnessing their capacity for cross-priming of cytotoxic T lymphocytes for the induction of tumour-specific immune responses.

Investigation of the effect of a countywide protected learning time scheme on prescribing rates of ramipril: interrupted time series study.

BACKGROUND: Protected learning time (PLT) schemes have been set up in primary care across the UK. There is little published evidence of their effectiveness. OBJECTIVE: To investigate the effect of a PLT intervention for general practice to increase prescribing of ramipril for prevention of cardiovascular outcomes. DESIGN: Quasi-experimental, interrupted time series. SETTING: Lincolnshire, UK. METHODS: Prescribing data were analysed one year before and after the education for change in rate of increase of prescribing of ramipril, whether change in prescribing was related to postulated explanatory variables and to determine intervention costs. MAIN OUTCOME: The primary outcome was the rate of change of ramipril (10 mg) prescription items 12 months after compared with before the educational intervention. Secondary outcomes included cost. RESULTS: Ramipril prescribing at therapeutic dosage increased significantly (odds ratio 1.50, 95% CI 1.07-1.93) following education by 52,345 items (31,132 items at 10 mg) at a cost of pound 292k to pound 460k depending on formulation. This occurred despite a background of secular change. Most practices were represented by GPs, nurses or both during the education. Single-handed GPs were less likely to attend. Practices showed considerable variation in response to the educational intervention. The only predictor of whether practices increased in prescribing rate after the education was whether a practice nurse had undertaken specific diabetes training. Total list size, dispensing, training or single-handed status and GP attendance did not predict a change in prescribing. CONCLUSION: PLT schemes can contribute to beneficial changes in prescribing across a large geographical area.

Regulatory T cells in transplantation tolerance.

Our ability to harness tolerance mechanisms will have a major impact in organ transplantation if it becomes possible to minimize drug maintenance, or even wean off immunosuppressive drugs. An improved understanding of the biology of regulatory T cells will make it possible to replace current induction regimens with those favouring the vaccination and selection of T cells that prevent graft rejection. Once tolerance is established, the continuous supply of graft antigens should sustain T cell mediated regulation as the dominant mechanism preventing graft rejection.

Description and simulation of an active imaging technique utilizing two speckle fields: root reconstructors.

Quasi-monochromatic light will form laser speckle upon reflection from a rough object. This laser speckle provides information about the shape of the illuminated object. Further information can be obtained if two colors of coherent light are used, provided that the colors are sufficiently close in wavelength that the interference is also measurable. It is shown that no more than two intensities of two speckle patterns and their interference are required to produce an unambiguous band-limited image of an object, to within an overall spatial translation of the image, in the absence of measurement errors and in the case where all roots of both fields and their complex conjugates are distinct. This result is proven with a root-matching technique, which treats the electric fields as polynomials in the pupil plane, the coefficients of which form the desired complex object. Several root-matching algorithms are developed and tested. These algorithms are generally slow and sensitive to noise. So motivated, several other techniques are applied to the problem, including phase retrieval, expectation maximization, and probability maximization in a sequel paper [J. Opt. Soc. Am. A 19, 458 (2002)]. The phase-retrieval and expectation-maximization techniques proved to be most effective for reconstructions of complex objects larger than 10 pixels across.

Description and simulation of an active imaging technique utilizing two speckle fields: iterative reconstructors.

Quasi-monochromatic light will form laser speckle upon reflection from a rough object. This laser speckle provides information about the shape of the illuminated object. In a prior paper [J. Opt. Soc. Am. A 19, 444 (2002)], it was shown that two intensities of two speckle patterns and their interference are sufficient to produce an unambiguous (except for object translation) band-limited image of the object, based on a root-matching technique described therein, in the absence of measurement error and in the case of distinct roots of the field polynomials and their complex conjugates. On the other hand, algorithms based on the root-matching technique are found to be slow and sensitive to noise. So motivated, several other techniques are applied to the problem, including phase retrieval, expectation maximization, and statistical maximization. The phase-retrieval and expectation-maximization techniques proved to be most effective for reconstructions of complex objects larger than 10 pixels across, and high-quality images were formed by using three independent sets of two-field data (three frames of two-wavelength data), each comprising two speckle intensity patterns and their interference. Two additional results of note are reported. First, the expectation-maximization algorithm produced relatively good images when three or more frames each of only one speckle intensity pattern (data at just one wavelength) were used and second, the phase-retrieval algorithm when only the object autocorrelation was used also produced relatively good images for the chosen test object.

Therapeutic aspects of tolerance.

The immune system is naturally unresponsive to 'self' antigens. Improved knowledge of mechanisms underlying self tolerance is giving rise to a new generation of immunosuppressive agents, that can exploit these mechanisms and so reduce the nature and level of medication that needs to be given long-term to control diseases where the immune system does harm.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.