Antigen-specific T cell responses to BK polyomavirus antigens identify functional anti-viral immunity and may help to guide immunosuppression following renal transplantation.

Infection with the polyoma virus BK (BKV) is a major cause of morbidity following renal transplantation. Limited understanding of the anti-viral immune response has prevented the design of a strategy that balances treatment with the preservation of graft function. The proven utility of interferon-gamma enzyme-linked immunospot (ELISPOT) assays to measure T cell responses in immunocompetent hosts was the basis for trying to develop a rational approach to the management of BKV following renal transplantation. In a sample of transplant recipients and healthy controls, comparisons were made between T cell responses to the complete panel of BKV antigens, the Epstein-Barr virus (EBV) antigens, BZLF1 and EBNA1, and the mitogen phytohaemagglutinin (PHA). Correlations between responses to individual antigens and immunosuppressive regimens were also analysed. Antigen-specific T cell responses were a specific indicator of recent or ongoing recovery from BKV infection (P < 0·05), with responses to different BKV antigens being highly heterogeneous. Significant BKV immunity was undetectable in transplant patients with persistent viral replication or no history of BKV reactivation. Responses to EBV antigens and mitogen were reduced in patients with BKV reactivation, but these differences were not statistically significant. The T cell response to BKV antigens is a useful and specific guide to recovery from BKV reactivation in renal transplant recipients, provided that the full range of antigenic responses is measured.

Vitiligo pathogenesis: autoimmune disease, genetic defect, excessive reactive oxygen species, calcium imbalance, or what else?

The pathobiology of vitiligo has been hotly disputed for as long as one remembers, and has been a magnet for endless speculation. Evidently, the different schools of thought--ranging, e.g. from the concept that vitiligo essentially is a free-radical disorder to that of vitiligo being a primary autoimmune disease--imply very different consequences for the best therapeutic strategies that one should adopt. As a more effective therapy for this common, often disfiguring pigmentary disorder is direly needed, we must strive harder to settle the pathogenesis debate definitively--on the basis of sound experimental evidence, rather than by a war of dogmatic theories. Recognizing, however, that it is theories which tend to guide our experimental designs and choice of study parameters, the various pathogenesis theories on the market deserve to be critically, yet unemotionally re-evaluated. This Controversies feature invites you to do so, and to ask yourself: is there something important or worthwhile exploring in other pathogenesis scenarios than those already favoured by you that may help you improve your own study design, next time you have a fresh look at vitiligo? Vitiligo provides a superb model for the study of many fundamental problems in skin biology and pathology. Therefore, even if it later turns out that, as far as your own vitiligo pathogenesis concept is concerned, you have barked-up the wrong tree most of the time, chances are that you shall anyway have generated priceless new insights into skin function along the way.

Splenic T zone development is B cell dependent.

The factors regulating growth and patterning of the spleen are poorly defined. We demonstrate here that spleens from B cell-deficient mice have 10-fold reduced expression of the T zone chemokine, CCL21, a threefold reduction in T cell and dendritic cell (DC) numbers, and reduced expression of the T zone stromal marker, gp38. Using cell transfer and receptor blocking approaches, we provide evidence that B cells play a critical role in the early postnatal development of the splenic T zone. This process involves B cell expression of lymphotoxin (LT)alpha1beta2, a cytokine that is required for expression of CCL21 and gp38. Introduction of a B cell specific LTalpha transgene on to the LTalpha-deficient background restored splenic CCL21 and gp38 expression, DC numbers, and T zone size. This work also demonstrates that the role of B cells in T zone development is distinct from the effect of B cells on splenic T cell numbers, which does not require LTalpha1beta2. Therefore, B cells influence spleen T zone development by providing: (a) signals that promote T cell accumulation, and: (b) signals, including LTalpha1beta2, that promote stromal cell development and DC accumulation. Defects in these parameters may contribute to the immune defects associated with B cell deficiency in mice and humans.

Intact B cell tolerance in the absence of the first component of the classical complement pathway.

A critical role for complement in the regulation of self tolerance has been proposed to explain the strong association between complement deficiency and autoimmunity. To elucidate the role of the classical pathway of complement in the maintenance of B cell tolerance, C1q-deficient (C1qa-/-) mice were bred with anti-hen egg lysozyme (HEL) immunoglobulin (Ig(HEL)) and soluble HEL (sHEL) transgenic mice. B cell tolerance was intact in C1qa-/- mice. In vivo, double-transgenic (Ig(HEL)/sHEL) C1qa-/- and wild-type control mice down-regulated surface immunoglobulin expression on splenocytes and equivalent numbers of HEL-binding B cells accumulated in the periphery. Maturation of B cells, evidenced by CD21 expression, was retarded to the same extent and at a similar time point. The frequency of anti-HEL-producing plasma cells and serum levels of anti-HEL immunoglobulin were comparably reduced in control and C1qa-/- double-transgenic mice compared to control Ig(HEL) and C1qa-/- Ig(HEL) mice. Furthermore, splenocytes from double-transgenic C1qa-/- or wild-type mice did not modulate intracellular calcium levels after stimulation with HEL in vitro. These data demonstrate that a stable form of B cell anergy persists in the periphery of C1qa-/- mice, suggesting that activation of the classical pathway by C1q is not essential for the maintenance of B cell tolerance in this transgenic model.

Single epitope multiple staining to detect ultralow frequency B cells.

Here we describe a method for detecting ultralow frequency target cells from within a high background of irrelevant cells by a novel method, single epitope multiple staining (SEMS). Samples of murine splenocytes were seeded with a low number of splenocytes from mice transgenic for a hen eggwhite lysozyme (HEL)-specific immunoglobulin (Ig). These samples were stained with two reagents specific for the same epitope expressed by the transgenic B cells, which had been conjugated to two different detectable labels (FITC and biotin). This dual staining of a single epitope allowed us to reduce the background due both to non-specific binding of reagents and to probabilistic distribution of the cells. We also were able to detect the cells based on knowing only one thing about them, namely, their antigen specificity. The SEMS method allowed us to reproducibly detect transgenic cells at frequencies below one cell in one million cells. SEMS could be used to increase the sensitivity of numerous fluorescence-based applications in addition to the detection and isolation of antigen-specific lymphocytes, including the detection and highly specific isolation of genetically modified cells, transformed cells, stem cells, fetal cells, or infectious organisms.

Role of Syk in B-cell development and antigen-receptor signaling.

Antigen receptors (BCRs) on developing B lymphocytes play two opposing roles-promoting survival of cells that may later bind a foreign antigen and inhibiting survival of cells that bind too strongly to self-antigens. It is not known how these opposing outcomes are signaled by BCRs on immature B cells. Here we analyze the effect of a null mutation in the Syk tyrosine kinase on maturing B cells displaying a transgene-encoded BCR that binds hen egg lysozyme (HEL). In the absence of HEL antigen, HEL-specific BCRs are expressed normally on the surface of Syk-deficient immature B-lineage cells, but this fails to promote maturation beyond the earliest stages of B-lineage commitment. Binding of HEL antigen, nevertheless, triggers phosphorylation of CD79alpha/beta BCR subunits and modulation of receptors from the surface in Syk-deficient cells, but it cannot induce an intracellular calcium response. Continuous binding of low- or high-avidity forms of HEL, expressed as self-antigens, fails to restore the signal needed for maturation. Compared with the effects in the same system of null mutations in other BCR signaling elements, such as CD45 and Lyn kinase, these results indicate that Syk is essential for transmitting a signal that initiates the program of B-lymphocyte maturation.

B cell antigen receptor signalling in the balance of tolerance and immunity.

The quantity and quality of signals from the B cell antigen receptor (BCR) drives the positive and negative selection of B lymphocytes and establishes the balance of tolerance and immunity. Experiments using immunoglobulin transgenic mice and mutations in key BCR signalling components have given insight into how the antigen receptor is tuned and how thresholds for qualitatively different outcomes are established and maintained. This research also describes how genetic variants can shift the balance between autoimmunity and tolerance.

Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and selection.

A B lymphocyte hyperactivity syndrome resembling systemic lupus erythematosus characterizes mice lacking the src-family kinase Lyn. Lyn is not required to initiate B cell antigen receptor (BCR) signaling but is an essential inhibitory component. lyn-/- B cells have a delayed but increased calcium flux and exaggerated negative selection responses in the presence of antigen and spontaneous hyperactivity in the absence of antigen. As in invertebrates, genetic effects of loci with only one functional allele can be used to analyze signaling networks in mice, demonstrating that negative regulation of the BCR is a complex quantitative trait in which Lyn, the coreceptor CD22, and the tyrosine phosphatase SHP-1 are each limiting elements. The biochemical basis of this complex trait involves a pathway requiring Lyn to phosphorylate CD22 and recruit SHP-1 to the CD22/BCR complex.

The regulation of self-reactive B cells.

Self-reactive B cells are eliminated in a series of checkpoints that are triggered by antigen binding. Recent reports have shown that in addition to the processes of elimination at the immature B-cell stage, B-cell anergy and regulation of T-cell help, self-reactive cells are also controlled by follicular competition, Fas-mediated elimination by T cells and censoring in the germinal centres. Each checkpoint operates at a threshold that reflects the need to maintain immune diversity at the same time as suppressing autoimmune disease. Analysis of the motheaten mutation has given a direct demonstration of how such thresholds can be modulated by genetic effects.

Linkage disequilibrium mapping of a type 1 diabetes susceptibility gene (IDDM7) to chromosome 2q31-q33.

The role of human chromosome 2 in type 1 diabetes was evaluated by analysing linkage and linkage disequilibrium at 21 microsatellite marker loci, using 348 affected sibpair families and 107 simplex families. The microsatellite D2S152 was linked to, and associated with, disease in families from three different populations. Our evidence localizes a new diabetes susceptibility gene, IDDM7, to within two centiMorgans of D2S152. This places it in a region of chromosome 2q that shows conserved synteny with the region of mouse chromosome 1 containing the murine type 1 diabetes gene, Idd5. These results demonstrate the utility of polymorphic microsatellites for linkage disequilibrium mapping of genes for complex diseases.

Polygenic control of autoimmune diabetes in nonobese diabetic mice.

Partial exclusion mapping of the nonobese (NOD) diabetic mouse genome has shown linkage of diabetes to at least five different chromosomes. We have now excluded almost all of the genome for the presence of susceptibility genes with fully recessive effects and have obtained evidence of linkage of ten distinct loci to diabetes or the prediabetic lesion, insulitis, indicative of a polygenic mode of inheritance. The relative importance of these loci and their interactions have been assessed using a new application of multiple polychotomous regression methods. A candidate disease gene, interleukin-2 (Il-2), which is closely linked to insulitis and diabetes, is shown to have a different sequence in NOD, including an insertion and a deletion of tandem repeat sequences which encode amino acid repeats in the mature protein.

Genetics of a multifactorial disease: autoimmune type 1 diabetes mellitus.

1. Three non-major histocompatibility complex genes, Idd-3, Idd-4 and Idd-5, that influence the onset of autoimmune type 1 diabetes in the non-obese diabetic mouse have been located on chromosomes 3, 11 and 1. 2. In undertaking this study, a new map of the mouse genome has been created from polymerase chain reaction-analysable mouse microsatellite markers. 3. The homologues of these genes may reside on human chromosomes 1 or 4 (Idd-3), 17 (Idd-4) and 2q (Idd-5).

Mouse microsatellites from a flow-sorted 4:6 Robertsonian chromosome.

Twenty microsatellites were generated from a previously characterized lambda gt10 library containing C57BL/6J mouse DNA from a flow-sorted 4:6 Robertsonian chromosome. These sequences were analyzed for size variation between different strains of mice with the polymerase chain reaction (PCR) and mapped by use of either strain distribution patterns (SDPs) in recombinant inbred (RI) strains, or intra- and interspecific backcrosses. Eighty-five percent of the sequences showed allelic variations between different inbred strains of mice and the wild mouse, Mus spretus, and 70% were variant between inbred strains. Eight (62%) of the 13 repeats that have been mapped lie on Chromosomes (Chr) 4 and 6. This approach is an effective way of generating informative markers on specific chromosomes.

Type 1 diabetes in mice is linked to the interleukin-1 receptor and Lsh/Ity/Bcg genes on chromosome 1.

Human type 1 (insulin-dependent) diabetes is a common auto-immune disease of the insulin-producing beta cells of the pancreas which is caused by both genetic and environmental factors. Several features of the genetics and immunopathology of diabetes in nonobese diabetic (NOD) mice are shared with the human disease. Of the three diabetes-susceptibility genes, Idd-1 -3 and -4 that have been mapped in mice to date, only in the case of Idd-1 is there any evidence for the identity of the gene product: allelic variation within the murine immune response I-A beta gene and its human homologue HLA-DQB1 correlates with susceptibility, implying that I-A beta is a component of Idd-1. We report here the mapping of Idd-5 to the proximal region of mouse chromosome 1. This region contains at least two candidate susceptibility genes, the interleukin-1 receptor gene and Lsh/Ity/Bcg, which encodes resistance to bacterial and parasitic infections and affects the function of macrophages.

The generation of a library of PCR-analyzed microsatellite variants for genetic mapping of the mouse genome.

Forty-three sequences containing simple sequence repeats or microsatellites were generated from an M13 library of total genomic mouse DNA. These sequences were analyzed for size variation using the polymerase chain reaction and gel electrophoresis without the need for radiolabeling. Seventy-two percent of the sequences showed allelic size variations between different inbred strains of mouse and the wild mouse, Mus spretus; and 53% showed variation between inbred strains. Thirty-seven percent were variant between B6/J and DBA/2J, and 81% of these were resolved using minigel agarose electrophoresis alone. This approach is a useful way of generating the large number of variants that are needed to create high resolution maps of the mouse genome.

Genetic analysis of autoimmune type 1 diabetes mellitus in mice.

Two genes, Idd-3 and Idd-4, that influence the onset of autoimmune type 1 diabetes in the nonobese diabetic mouse have been located on chromosomes 3 and 11, outside the chromosome 17 major histocompatibility complex. A genetic map of the mouse genome, analysed using the polymerase chain reaction, has been assembled specifically for the study. On the basis of comparative maps of the mouse and human genomes, the homologue of Idd-3 may reside on human chromosomes 1 or 4 and Idd-4 on chromosome 17.